Pseudomonas aeruginosa PcrV binding single variable domain antibodies

专利摘要:
Polypeptides are provided that are capable of significantly inhibiting and/or neutralizing
公开号:AU2013224851A1
申请号:U2013224851
申请日:2013-03-04
公开日:2014-08-28
发明作者:Evelyn De Tavernier；Bruno Dombrecht；Guy Hermans；Ann Union
申请人:Ablynx NV；
IPC主号:C07K16-12

专利说明:
WO 2013/128031 PCT/EP2013/054262 1 PSEUDOMONAS AERUGINOSA PCRV BINDING SINGLE VARIABLE DOMAIN ANTIBODIES The present Tnvention re ates to polypeptides that bind the PcrV protein of Pseudoinonas 5 aeruginosa. More specifical y, the present invention relates to multiparatopic polypeptides (also referred to he rei n as "multiparotopc polypeptide(s) of the invention" )that hind PcrV and neutralize P. oeruginosoa The invention further relates to monovalent polypeptides (also referred to herein as 'monovalent polypeptides of t he invention. 10 The invention also relates to nucleic acids encoding such polypeptides (also referred to herein as "nucleic acids) of the invention" t~o methods for preparing such polypeptides; to host celIs expressing or capab e of expressing such polypeptides; to compositions, and in particular to pharmaceutical compositions, that cornprise such polypeptides, nucleic acids and/or host cels; and to uses of polypeptildes, nucleic acids, host cells and/or compositions, in particular for prophylactic and/or therapeutic purposes, such as the prophylactic and/or therapeutic purposes mentioned herein. Other aspects, embodiments, advantages and applications of the invention will becorne clear from the further description herein. BACKGROUND ART 20 Pseudamanas aeruginosa is an environmental Gram-negative bacterium, associated with a broad spectrum of infections in humans. It has a very large genome which is remarkably flexible rnetabolically, explaining why it can be found in very diverse environments. This opportunistic pathogen can cause acute lung injury and mortality through the delivery of exotoxins by the type Ill secretion systern (TTSS). The Type Ill Secretion System (TTSS) of P. oeruginosa is a cornplex multi-protein structure crossing 25he camp ete cell wal It is aspecialised hollow needle-like molecular structure secreting only TTSS proteins and pathogenicity related toxins Many different proteins form the TTSS, both on the bacterial cytoplasmic sde and externally. ExternaIly, only the single 'barrel homopolymeric forming protein and the 'need e tip' protein are accessible to antibodies, The needle protein PcrV is thought to form a ring type structure on the tip oft te needle. The TTSS cornplex can inject various exotoxins, produced by the 30 bacterium, directly into the cytoplasm of host cells. The involvement of this translocation apparatus in pathogenesis may not be limited to the transport of exotoxins, as indeed rnutants expressing TTSS but not the toxins are cytotoxic as well (Lee et WO 2013/128031 PCT/EP2013/054262 2 at infect. Imrnun. 73: 1695-1705, 2005). The trans ocation pore itself is sufficient to cause the death of host cells, either directly through pore-rnediated increases in membrane permeability, or indirectly through the activation of broad cellular defence responses The TT55 virulence mechanism on the bacteria's external surface enables P. oeruginosa to evade human immrune defences by killing white 5 blood cells and epithelal cells and triggering tissue-damaging inflammation. Under norrnal circumstances, the baterium is perfectly harrnless. lHowever, under certain circumstances the bacterium can colonize hosts with a weakened immune system. It is recognized as a major cause of nosocomial bacteremia and infections associated with invasive devices, mechanical ventilation, burn wounds, or surgery in the imrnunocompromis ed and the irnmunocornpetent patients 10 (Giamarel ou and KaneI akopoulou Crit. Care Clin. 24: 261-278, 2008), such as bone marrow transplant patients (Ve asco et al. Clin. Microbiol. Infect. 10: 542-549, 2004). P. oeruginoso typically causes nosocornial infections of the pulmonary tract, urinary tract, (burn) wounds and also sepsis. in Cystic Fibrosis (CF) patients, Pi oeruginosa infection follows well-established pattern of recurrent pulmonary infection in early childhood leading to the establishment of chronic infection in 1o older CF patients, where it is a major contributing factor in the progressive decline in lung function and disease exacerbations leading to respiratory failure (FitzSirnmons J1. Pediatr. 122: 1-9, 1993; Kerem et aL. J, Pediatr. 116: 714-719, 1990; Lyczak et al, Clin. Microb. Rev. 15: 194-222, 2002). Once chronic P. oeruginosa pulmonary infection is established, eradication of the organism appears impossible using P. oerugmnosa ha seeral different manifestations in the setting of chronic obstructive pulmonary disease (COPD). The organism is acolonizer that is cleared quickly, causes acute exacerbations and also may cause hroni infections n a subset of adults with COPD (Murphy Curr. Opn. Pulm, Med. 15: 138 142, 200 A good overview on the current treatrment of P. aeruginosa pneumonia s given by Giamarellou and 25 Kanellakopoulou (Crit. Care Clin. 24: 261-278, 2008), Malcolm and Heirn (Curr, Opin. Pharmacol 9: 558 565, 2009), El Solh and Alhajhusain (J. AntirnicrobiaI Chemotherapy 64: 229-238, 2009) and Roux and Ricard ( nfectious Disorders -Drug Targets 11: 389-394, 2011) Current treatment for patients stilI relies on antibiotics. Antibiotics of the four rnajor structural cIasses are in use against P oeruginosa infection (Giamarellou and Kanellakopoulou Crit. Care Clin. 24: 261-278, 2008). importantly, once P. oeruginosa 3o colonisation has been etablished it cannot be successfully cleared using antibiotics due to biofilmn formation. Biofilm limnts the access of certain antibiotics to the deeper Iayers of the filrn (diffusion limiting). More importantly, the deeper layers of biofilm contain rnany P. aeruginosa bacteni which are WO 2013/128031 PCT/EP2013/054262 3 live but virtually completely inactive for lack of nutrient access. Antibiotics of various classes act on cell division or highly active metabolic pathways, and are thus unable to kill these dorrnant bacteria. Once therapy is tapered back or withdrawn, these cells rapidly re-colonise the patient. Furthermore, P. aeruginosa has the ability to evade new antimicrobial therapies and develop 5 resistance, being on one hand intrinsically resistant to many drugs, on the other hand rapidly acquiring resistance via anurnber of mechanisms (Malcorn and Hemlr Curr. Opin. Pharrnacol 9: 558-565, 2009), Because of the versatility and the large siz of P. aeruginosa genome, various resistance mechanisms can be present simultaneously, causing cross-resistance to several a ntipseudomonal agents (Giamarel ou and Kanellakopoulou Crit. Care ClIn. 24: 261-278, 2008). Novel variants on the samn basic antibiotic 10 structures are in development and may alleviate current resistance to s orne extent, but are very likely to give rise to novel resistance once in widespread clinical use. No novel cIasses of antibiotics are known to be in clinical development. Because the development of new classes of antibiotics has lagged far behind our growing need for such drugs, we now face apost-antibiotic era with imited capacity to combat these 15 Topical adrninistration of existing antibiotics (e.g., aerosol administration of tobramycin or colistin) has been used to deliver higher local concentrations of antibiotics without exposing the patient to high systemic levels which may be toxic to the patient (Luyt et al Curr. Opnr. Infect. Dis. 22: 154-158, 2009). However, continued concerns are raised about its efficacy and potential emergence of resistance aswell (El Solh and Alhajhusain J. Antimicrobial Chemotherapy 64: 229-238, 2009; Roux and Ricard Infectious 20 Diorders Drug Targets 1 389-394, 2011 With the pipeline of new antimicrobial agents running dry, treatment of P. oeruginosa continues to rely on the theoretical advantages of combination therapy and the revival of old drugs previously abandoned because of serious toxicity, like polymyxins (Giamarellou and Kanellakopoulou Crit, Care Clin. 24: 261-278, 2008; El Solh and Alhajhusamn J. Antirnicrobial Chemotherapy 64: 229-238, 2009). However, 25 resistance to such treatment is rapidly emerging with very worrisome latest resistance rates, and the appearance of Pseudomonas strains with rnultidrug-resistant, or even pan-resistant, phenotypes (Malcolm and Heim Curr. Opin. Pharmacol, 9: 558-565 2009). Based on the reported resistance surveillance data, it is evident that the current therapeutic approach for P. aeruginosa infections is approaching its lirnits (Giamarellou and Kanellakopou ou Crit, Care C in. 24. 261-278, 2008) 30 There are currently no non-antibiotc based treatments on th e market. However, there are a nurnber of drug candidates in development.
WO 2013/128031 PCT/EP2013/054262 4 Varous monoclonal antibodies, mostly directed to P. ceruginosa flagelin or strain-specific LPS have been described. Most did not reach clinical stage. One LPS-reactive 1gM (Kenta (Berna/Crucell) is lted as in active Phaselil development However, the serotype specificity of this antibody underscores the needafo a ikssay to determine h seomype of the infectious agent in the ho pia etting and a the development of antibodies specific for other clinic-relevant serotypes (Roux and Ricard Infectious Disorders - Drug Targets 11: 389-394, 2011). A mouse monoclonal anti-PcrV antibody, monoclonal antibody (Mab) 166, with potent neutralizing activity in mouse and rat models of Pseudomonas infection had been described by Frank et al (1. Infect Dis, 186 64-73, 2002) and Faure et al (J. Immune based Therapies and Vaccines 1: 2, 2003). WO 10 2009/073631 A2 and Baer et al (Infection and irnmunity 77 1083-1090) describes several engineered hurnan antibody Fab fragments (amongst which Fab1A8) specific for P. oeruginoso PcrV protein and which compete with MAb 166 for binding to the sarne epitope on PcrV. These Fbs show potent neutralization activity against the P. aeruginosao Type Ili secretion system. KB001 (KaloBios US) is a Humaneered"' anti-PcrV PEGylated antibody Fab' fragment (Anti-PcrV Program Fact Sheet, KaloBios) that 15 showed potent Type lIi Secretion System (TTSS) neutralising activity in celar cytotoxicity assays. KBO01 is being developed for the prevention of Pa ventilator-associated pneumonia (VAP) and for the treatment of CF Prelirninary evidence of activity and safety has been demonstrated in both indications in Phase 1/2 trials conducted by KaloBios It still remains to be determined, however, whether or not escape mutants will develop to this monospecific monoclonal antibody once administered to patients, 20 Taken together, the increased incidence in certain types of infections, the increased use of invasive devices in the hospital as wel as the increased frequency of multi-resistant Pseudomonas strains have clearly let to a shortage of treatment options for nosocomial Pseudomonos infections. Despite the above efforts, management of P. aeruginos ainfection represents a difficult therapeutic chalenge for critical care physicians (EI Solh and Alhajhusain i AntimicrobialI Chemotherapy 64: 229-238, 2009), For patients 25with multi-drug resistant strains, very few clinical options rernan. It is therefore considered imperative to discover and develop novel anti-Pseudomonos drugs to fill a dangerous void in the anti-bacterial armamentarium of the linician (Malcolrn and Heim Curr. Opin, Pharmacol 9: 558-565, 2009) 30 The present invention provides polypeptides with improved prophy actic, therapeutic and/or pharmacological properties, in addition to other advantageous properties (such as, for example, irnproved ease of preparation, good stability, and/or reduced costs of goods), compared to the prior art WO 2013/128031 PCT/EP2013/054262 5 arnino acid sequences and antibodies. More particularly, the present invention provides rnu tivalent polypeptides comprising two or more immrunoglobulin single variable domains that show improved properties for neutralizing PcrV compared to the PcrV neutralizing molecules described in the prior art. The inventors surprisingly observed that biparatopic polypeptides comprising two different PcrV-binding c immunoglobulin single variable dornains showed a significant increase in PcrV-neutralizing efficacy as compared to the PcrV neutralizing capacity of the rnonovaLent PcrV-binding building blocks alone. For this purpose, the present invention in addition aso makes available a number of highly advantageous imrnunoglobulin single variable domains (i.e, monovalent polypeptides)t that specifically bind PcrV and/or that are capable of significantly inhibiting or neutralizing PcrV'. These PcrV-binding 1o immunoglobulin single variable dornains and polypeptides comprising th e same form further aspcts of the invention. Accordingly, the present invention provides polypeptides comprising or essentially consisting of two or rnore irnmunoglobu in single variable domains that specifically b'nd to the PcrV protein of P. aeruginosa! (herein referred to as "PcrV") Such polypeptides are also referred to herein as "multivalent 15 polypeptide(s) of the invention" The two or more immnunoglobulin single variable domains may optionally be linked via one or rnore peptidic linkers. Preferably, the rnultivalent polypeptide comprises two or more immunoglobulin single variable domains directed against PcrV, wherein the "first" immunoglobulin single variable dorna'n directed against PcrV and the 'second" immunoglobulin single variable domain directed against PcrV havey 20 different paratope. Such polypeptides are aiso referred to herein as'multiparatopcpolypeptide(s) of the invention". Accordingly, the present invention relates to a po ypeptide comprising or consisting of two or more immunoglobulin single variable dornain: that are directed against PcrV, wherein the "first" immunoglobulin single variable domain directed against PcrV and the 'second' immunoglobulin single variable doaan directed against Pcrv have different paratopes. Such polypeptides comprise or consist of 25two or more immunoglobulin single variable dornains that are directed against different epitopes on PcrV. More specifically, such polypeptides cornprise at least one "first' immunoglobulin single variable dornain that is directed against a first epitope on PcrV and at least one 'second" immunoglobulin single variable dornain that is directed against second epitope on PcrV different from the first epitope on PcrV. Preferably, these multiparatopic polypeptides of the invention are biparatopic or triparatopic 30 poly pe ptides (also refe rred to herei n as"bparatopc polypeptide(s) of the invention" a nd "triparatopi polypeptide's) of the invention"), as further defined herein.
WO 2013/128031 PCT/EP2013/054262 6 Multiparatopic (such as the biparatopic or triparatopic) polypeptides as described herein, showed improved properties for neutralizing PcrV compared to the PcrV neutralizing molecu es described in the prior art. The multiparatopic (such as biparatopic or trilparatopic) polypeptide of the invention is capable of neutrallzing PcrV with 100% efficacy in a cytotoxicity assay, such as e.. a cytotoxicity assay with 8 P3X63 cell as target at an MOI of 12. Apart from this and/or in addition, the multiparatopic (such as biparatopic orftriparatopc) polypeptide of the invention i capable of neutralizing PcrV with an 1C50 of 300 5.0x10* M or lower in a cytotoxicity assay (such as e.g. a cytotoxicity assay with P3X63 cells target at an MGi of 12. Apart from this and/or in addition, the multiparatopic {such as biparatopic or triparatopic) polypeptide of the invention has a decrease in potency after 24 hours in the presence of P. aeruginosa co elastase (3 ug/ug polypeptide) of maximal 5 fold (e.g., 5 fold, 3 fold, 2 fold or lower). Apart frorn this and/or in addition, the multiparatopic (such as biparatopic or triparatopic) polypeptide of the invention ha a derease in potency after 24 hours in the presence of human neutrophil elastase (1-2 ug/ug polypeptide) of maximal 15 fold (e.g. 10 fold, 5 fold, 3 fold, 2 fold or lower). Preferred multiparatopic (such as biparatopic or triparatopic) polypeptides of the invention 15 ormprise or essentially consist of two or more mrnunoglobulin single variab e domains wherein at least one immunoglobulin single variable domain consists of 4 framework regions (FIR1 to FR4, respectively) and 3 cornplementarity determining regions (CDR1 to CDR3, respectively) in which (see Table A-6): - CDRi is chosen from the group consisting of: a) the amino acid sequences of SE ID1 NOs: 20-37; 20 b) arnino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 20-37; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequin es of SEQ ID NOs: 20-37; a nd/or 25 - CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NOs: 38-56; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 38-56; f) amino acid sequ ences that have 3, 2, or 1 arnino acid difference with at least one of the amino acid so sequences of SEQ ID NOs: 38-56; a nd/or - CDR3 ischosen from the group consisting of: WO 2013/128031 PCT/EP2013/054262 7 g) the amino acid sequences of SEQ ID NOs: 7-75; h) amino acid sequences that have at least 80% amino acid identity with at least one of the arnino acid sequence of EQ ID NO 57-75; ) arnino acid sequences that have 3, 2, or 1 amino acid difference with at east one of the amino acid 5 sequences of SEQ ID NOs: 57-75. More particularly, the multiparatopic (such a- biparatopic or triparatopic) polypeptide of the invention comprise or essential y consist of two or rnore imrnunog obulin single variable dornains, wherein at least one of the imrnunoglobu in single variab e domains consits of 4 framework regions (FR1 to FR4, respectively) and 3 cornplernentarity determining regions (CDR1 to CDR3, respectively), in which: 10 CDR1 is chosen frorn the group consisting of: a) the amino acid sequences of SEQ D NO : 20-3 b) amino aid sequences that have at least 80% arnino acid identity with at least one of the amino acid sequences of SE Q I NO : 20-37 c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid 15 sequences of SEQ ID NO : 20-37]; and - CDR2 is chosen frorn the group consisting of: d) the arnino acid sequences of SEQ ID NOs: 38-56; e) amino acid sequences that have at least 80% amnno acid identity with at least one of the mrino 20 acid enN f) amino acid sequences that have 3, 2, or amno am id difeence wit a les one ofth amino ci sequences of EQID NOs 38-S6 and - CDR3 is chosen frorn the group consisting of: 25 g) the amino acid sequences of SEQ ID NOs: 57-75; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 57-75; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 57-75. so Preferred multiparatopic (such as biparatopic or triparatopic) polypeptides of the invention comprise or essential y consist of two or more immunoglobulin single variable domains wherein, in at least one of the immunoglobulin single variable domains, the CDR sequences have at least 70% amino WO 2013/128031 PCT/EP2013/054262 8 acid identity, preferably at least 80% amino acid identity, rnore preferab y at least 90% amino acid identity, such as 95% amino acid identity or more, or even essentially 100% amino acid identity with the CDR squences of at least one of the immunoglobulin single variab e domains with SEQ ID NOs: 1-19 5 In a preferred aspect, the multiparatopic (such as biparatopic or triparatopic) polypeptides of the invention comprise ore esentially consist of two or more immunoglobulin single variable dornains, wherein at least one of the immunoglobulin single variable dornains cross-blocks the binding to PcrV of at Ieast one of the immunoglobulin single variable domains with SEQ ID NOs: 1-19 and/or is cross blocked frorn binding to PcrV by at least one of the immunoglobulin single variable domains with SEQ ID 10 NOs: 1-19. In a preferred aspect, each of the two or more immunoglobulin single e variable domains present in the multiparatopic (such as biparatopic or triparatopic) polypeptides of the invention are as defined above. Exarnples of rnultiparatopic (such as biparatopic or triparatopic) polypeptides of the invention are SE DN118-1 1 (Table A-S) is In a preferred aspect, each of the two or rnore immunoglobulin single variable domains oft te multiparatopic (such as biparatopic or triparatopic) polypeptide of the invention, that is directed against PcrV belongs to a different epitope bin. Accordingly, the present invention relates to a polypeptide comprising or essentially consisting of two or more immunoglobulin sing e variable domains directed against PcrV, wherein each of the two or rnore immrunoglobu in single variable domains that are directed -o against PcrV belong to a ifferent epitope bin. Immunoglobulin single variable domains that belong to a different epitope bin, by definition do not cross-compete with each other for binding the target, PcrV. Accordingly, the present invention reltes to a polypeptide comprising or essentially consisting of two or more immunoglobulin single variable dornains against PcrV, wherein the first imrnunoglobu in sing e variable domain does not cross-block the binding to PcrV of the second immunoglobulin single variable 25domain and/or wherein the first immunoglobulin single variable is not cross-blocked from binding to PcrV by the second immunoglobulin single variable domain, Preferred combination of immunoglobulin single varnable domains present in the multiparatopic (such as biparatopic or triparatopi) polypeptide of the invention may encompass any of the following: - the first immunoglobulin single variab e dornain cross-b ocks the binding to PcrV of at least one 30 of the immunoglobulin single variable domains with SEQ ID NOs: 3-10 [epitope bn1] and/or is cross-b ocked from binding to PcrV by at least one of the immunoglobulin sing e variable domains with SEQT ID NOs: 3-10 [epitope binl~ and the second immunog obulin single variable WO 2013/128031 PCT/EP2013/054262 9 domain cross-blocks the binding to PcrV of at least one of the immunog obulin single variable domains with SEQ ID N~s: 1-2 [epitope bin2) and/or is cross-blocked from binding to PcrV by at least one of immunog obulin single variable dornains with SEQ ID NOs: 1-2 [epitope bin2] - the first immunoglobulin single variable dornain cross-blocks the binding to PcrV of at least one 5 of the immunoglobu in single variable domains with SEQ ID NOs: 3-10 [epitope bin1] and/or i cross-blocked from binding to PcrV by at least one of the immunoglobulin single variable domains with SEQ ID NOs: 3-10 [epitope bin1]; and the second irmmunoglobulin single variable domain cross-blocks the binding to PcrV of at least one of the immunog obullin sing e variable dornains with SEQ ID NOs: 11-12 epitope bin3l and/or is cross-blocked from binding to PcrV by 10 at least one of the irnmunoglobulin single variable domains with SEQ ID NOs 11-12 [epitope bin3]{; - the first irnrunoglobulin single variable dornain cross-blocks the binding to PcrV of at least one of the immunoglobulin single variable domains with SEQ||D NOs: 1-2 [epitope bin2] and/or is cross-blocked from binding to PcrV by at least one of the immunoglobulin single variable 15 dornans with SEQ ID NOs: 1-2 [epitope bin2]; and the second immunoglobulin single variable dornain cross-blocks the binding to PcrV of at least one of the imnrunoglobu in single variable domains with SEQ ID NOs: 3-10 [epitope bin1] and/or is cross-blocked from binding to PcrV byat least one of the immunoglobulin single variable dornains with SEQ ID NOs: 3-10 [epitope bin1} - the first immunoglobulin single variable domain eross-blocks the binding to PcrV of at least one 25 of the immrunog obulin single variable dornains with SEQ ID NOs 1-2 [epitope bin2] and/or is cross-blocked from binding to PcrV by at le a one of the immunog obulin single variable domains with SEQ ID NOs: 1-2 {epitope bin2}; and the second immunoglobulin single variable domain cross-blocks the binding to PcrV of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 11-12|[epitope bin3] and/or is cross-blocked from binding to PcrV by 25 at least one of the immunoglobulin single variable domains with SEQ ID NOs: 11-12 [epitope bn3] - the first immunoglobulin single variable domain cross-blocks the binding to PcrV of at Ieast one of the mrmunoglobulin sing e variable domains with SEQ ID NOs: 11-12 [epitope bin3} and/or is cross-blocked from binding to PcrV by at least one of immunoglobulin sngle variable domains so with SEQ ID NOs: 11-12 [epitope bin3]; and the second immunoglobulin single variable domain cross-b ocks the binding to PcrV of at least one of the immunoglobulin single variable dornains WO 2013/128031 PCT/EP2013/054262 10 with SEQ ID NOs 3-10 [epitope bin1] and/or is cross-blocked from binding to PcrV by at least one of the immunoglobusin single variable dornains with SEQ ID NOs:3-10 [epitope bin1]; or -the first immunoglobulin single variable domain cross-bloclks the binding to PcrV of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 11-12 [epitope bin3] and/or is S cross-blocked from binding toP PrV by at least one of the irnmunoglobu in single variable domains with SEQ ID NOs 11-12 [epitope bin3] and the second immrunoglobulin sing e variab e domain cross-blocks the binding to PcrV of at least one of the immunoglobulin sing e variable domains with SEQ ID NOs: 1-2 [epitope bin2] and/or is cross-blocked from binding to PcrV by at least one of the immunoglobulin single variab e dornairns with SEQ ID NOs: 1-2 [epitope bin2). 10 Preferred multiparatopic (such as biparatopic or triparatopic) polypeptides of the invention may comprise or essentialy consist of one of the following combinations of imrnunog obulin single variable dornains: 15 o a polypeptide that binds full length PcrV (SEQ ID NO: 159) and that shows reduced binding (30 90% compared to full length PcrV) or no binding to chimera 4 (SEQ ID NO: 202) and chimera (SEQ ID NO: 204 o a polypeptide in which the CDR sequences have at least 70% amino acid identity preferably at least 80% amino aid identity, more preferably at least 90% amino acid identity such as 95% 20 amino acid identity or more or even essentially 100% arino acd identity with the CDR sequences of at least one of the immunoglobulin single variab e dornains with SEQ ID NOs: 3-10; o a polypeptide that cross-blocks the binding to P rV of at least one of the immunoglobulin single variable domains with SEQ 1D NOs: 3-10 and/or that is cross-blocked from binding to PerV by at least one of the immunoglobulin single variable domains with SEQ 0D NOs: 3-10; and 23 o a polypeptidethatisan ofSEQIDNOs: -10; and the second immunoglobu in single varnable domain is a polypeptide that belongs to epitope bin 2 and is selected from any one of: oa polypeptide that binds to full length PcrV (SEQ ID NO: 159) and that shows reduced binding (30-90% cornpared to full length PcrV) or no binding to chirnera 7 (SEQ ID NO: 205); so o a polypeptide, in which the CDR sequences have at least 70% arnino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% mrino acid identity or more or even essentially 100% amino acid identity with the CDR WO 2013/128031 PCT/EP2013/054262 11 2; oapolypeptide that cross-block s the binding to PcrV of at east one of the immunoglobulin single variable domains with SEQ ID NO: 1 and 2 and/or that is cross-blocked frorn binding to PcrV by 5 at least one of the immunoglobulin single variable domains with SEQ ID NOs: land 2; and o a o yeptde hat s ay o SE IDNOs: land 2; o a polypeptide that binds full length PcrV (SEQ ID NO: 159) and that shows reduced binding (30 to 90% cornpared to fuR Iength PcrV) or no binding to chimera 4 (SEQ ID NO: 202) and chimera 6 (EIDNO: 204); o a polypeptide in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% arnino acid identity, such as 95% amino ci! identity or more or even essentialy 100% ar o a id identity with the CDR 15 sequences of at least one of the immunoglobulin single variable domains with SEQ lID NOs: 3-10; o a po ypeptide that cross-block the binding to PcrV of at least one of the immunoglobu in single variable domains with SEQ ID NOs: 3-10 and/or that is cross-blocked from binding to PcrV by at least one of the immunoglobulin single variab e domains with SEQ ID NOs: 3-10; and o apolypeptide that is any of SEQ ID NOs 3-10; 20 and the second immunoglobulin single variable domain is a polypeptide that belongs to epitope bin 3 and is selected from any one of: o a polypeptide that binds full Iength PcrV (SEQ ID NO: 159) and that shows reduced binding (30 90% compared to full length PcrV) or no binding to chimera 2 (EQ ID NO: 200); o a polypeptide in which the CDR sequences have at east 70% amino acid identity, preferably at 25 leaIst 80% amino acid identity, more preferably at least 90% amino acd identity, such as 95% arnino acid identity or more or even essentially 100% amino ad identity with the CDR sequences of at least one of the iornunoglobulin single variable domains with SEQ ID NOs: 11 and 12; o a polypeptide that cross-blocks the binding to PcrV of at least one of the irmmunog obulin single so variable domains with SEQ ID NOs: 11 and 12 and/or that iscross-blocked from binding to PcrV by at least one of the mrmunoglobulin single variab e dom a wit SE NO:1 and1an o a polypeptide that is any of SEQ ID NOs: 11 and 12; WO 2013/128031 PCT/EP2013/054262 12 -the first immunoglobulin single variable dornain is a poynptd ta belog oe~ bn2adi selected from any one of: o a polypeptide that binds to fui length PerV (SEQ ID NO: 159) and that shows reduced binding (30-90% compared to full length PcrV) or no binding to chimera 7 (SEQ ID NO: 205) 5 o a polypeptide, in which the CDR sequences have at least 70% amino acid 'dentity, preferably at least 80% amino acid identity, more prefer ably at least 90% amino acid identity, such as 95% amino acid identity or more or een ess entially 100% arnino acid identity with the CDR sequences of at least one of the immunoglobulin single variab e domains with SEQ ID NOs: 1 and 10 o a polypeptide that cross-blocks the binding to PcrV of at least one of the imrn unoglobulin single variable dornains with SEQ ID NOs: 1 and 2 and/or that is cross-blocked from binding to PcrV by at least one of the immunoglobulin single variable domains with SEQ ID NOs: 1 and 2; and o a N n 1s and is selected from any one of: o a polypeptide that b inds full length PcrV (SEQ ID NO: 159) and that shows reduced binding (30 90% compared to full length PcrV) or no binding to chimera 4 (SE 0D NO: 202) and chimera 6 (SQIDN:00) o a po ypeptide in which the CDR sequences have at least 70% arnno acid identity, preferably at 20 least 80% amino acid identity, more preferably at least 90% arnino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 3-10; o a polypeptide that cross-blocks the binding to PcrV of at least one of the immunoglobulin single variable domains with SE 0 D NOs: 3-10 and/or that is cross-blocked from binding to PcrV by at 25 ~least one of the immunoglobulin single varnable dornains with SEQ ID NOs: 3-10; and o a polypeptide that is any of SEQ ID NOs: 3-10; the first immunoglobulin single variable domain is a polypeptide that belongs to epitope bin 2 and is selected from any one of: o a polypeptide that binds to full length PcrV (SEQ ID NO: 159) and that shows reduced binding so (30-90% compared to full length PcrV) or no binding to chime era 7 (SEQ ID NO: 205); o a polypeptide, in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity more preferably at least 90% anino acid identity, such as 95% WO 2013/128031 PCT/EP2013/054262 13 arnino acid identity or rnare or even essentially 100% amino acid identity with the CDR sequences of at least one of the immunoglobulin single variable domains with SEQ D NOs: 1and 2; o a polypeptide that cross-blocks the binding to PcrV of at least one of the immunog obulin single 5 variable dornains with SEQ ID NOs: 1 and 2 and/or that is cross-blocked frorn binding to PcrV by at least one of the mmunoglobulin singe varnble domain with SEQ ID NO and 2 and o a polypeptide that s any of SEQ ID NOs: l and 2; 10 o apolypeptide that binds full length PcrV (SEQ ID NO: 159) and that shows reduced binding (30 90% cornpared to ful I ength PcrV) or no binding to chimera 2 (SEQ ID NO: 200); o a polypeptide in which the CDR sequences have at least 70% arnino acid identity preferab y at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% arnino acid identity with the CDR 15sequences at least one of the immunoglobu in single variable dora'ns with SEQ ID NOs: 11 and 12; o a polypeptide that cross-blocks the binding to PcrV of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 11 and 12 and/ar that is cross-blacked from binding to PcrV by at least one of the immunoglobulin single variab e domains with SEQ ID NOs: 11 and 12; and 20 a a palypeptide that is any of SEQ ID NOs: 11 and 1 the first immunoglobu lin single variable dornain is a polypeptide that belongs to epitope bin 3 and i selected from any one of: o a palypeptide that binds full length PcrV (SEQ ID NO: 159) and that shaws reduced binding (30 90% compared ta full length PcrV) or no binding to chirnera 2 (SEQ ID NO: 200); 25 a polypeptide in which the CDR sequences have at least 70% mrina acid identity, preferably at least 80% arnino acid identity, mare preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% arnino acid identity with the CDR sequences of at least one of the immunoglobulin single variable domains with SEQ ID NOs; 11 and 12; so a a polypeptide that crass- bocks the binding to PcrV of at least one of the irnmunag obulin single variable domains with SEQ IlD NOS: 11 and 12 and/or that is cross-blocked frorn binding to PcrV by at least one of the immunoglobulinn single variable damains with SEQ ID NOs: 11 and 12; and WO 2013/128031 PCT/EP2013/054262 14 o a polypeptide that isany of SEQ 0D NOs: 11 and 12; and the second immunoglobulin single variable domain is a poly ptide ta belong to epoe bmin and is selected frorn any one of: o a polypeptide that binds ful length PcrV (SEQ ED NO: 159) and that shows reduced binding (30 5 ~90% compared to full ength PcrV) or no binding to chimera 4 (SEQ ID NO: 202) and chirnera6 (SEQID NO:204); o a polypeptide in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amnno acid identity, such as 95% amino acid identity or more or even essentially 100% amno acid identity with the CDR 10 sequences of at least one of the immunog obu in single varnable dornains with SEQ ID NOs 3-10; o a polypeptide that cross-b ocks the binding to PcrV of at least one of the immunoglobulin singe variab e domains with SEQ 0D NOs: 3-10 and/or that is cross-blocked from binding to PcrV by at least one of the immunoglobulin sing e variable domnans with SEQ ID NOs: 3-10; and o a polypeptide that is any of SEQ ID NOs: 3-10; or 15 -the first imrnunoglobuhn single variable dornain is po ypeptide that belongs to epitope bin 3 and is selected from any one of: o a polypeptide that binds fu I length PcrV (SEQ ID NO: 159) and that shows reduced binding (30 90% cornpared to full length PcrV) or no binding to chirnera 2 (SEQ ID NO: 200); o polypeptide in which the ODR sequences. have at east 70% arnino acid identity, preferably at 20 least 80% amino acid identity, more preferably at least 90% amino cdd identity such as 95% arnino acid identity or more or even essential 100% arnino acid identity with the CDR sequences of at least one of the immunoglobulin sing e variable dornains with SEQ ID NOs: 11 and 12; o a polypeptide that cross-blocks the binding to PcrV of at least one of the immunoglobulin single 25 variable domains with SEQ ID NOs: 11 and 12 and/or that is cross-blocked frorn binding to PcrV byat least one ofthe immunoglobulin single variable domains withS SQID NOs: 11 nd 12- and o a polypeptide that is any of SEQ ID NOs: 11 and 12; and the second immunoglobulin sing e variable domain is a po ypeptide that belongs to epitope bin 2 and is selected from any o ne of: 30 o pa poypeptide that binds to full length PcrV (SEQ ID NO: 159) and that shows reduced bing (30-90% compared to full length PcrV) or no binding to hirnera 7 (SEQ ID NO: 205); WO 2013/128031 PCT/EP2013/054262 15 o polypeptide, in which the CDR sequences have at least 70% amino acid identity preferably at least 80% arnino acid identity, more preferably at least 90% arnino acid identity, such a. 95% amino acid identity or more or even essentially 100% amino acid identity with the CDR sequences of at least one of the immunoglobulinn single varnable domains with SEQ. D NOs: 1 and 2 2 o a polypeptide that cross-blocks the binding to PcrV of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 1 and 2 and/or that is cross-blocked from binding to PcrV by at least one of the immunog obulin single variable domains with SEQ ID NOs: 1 and 2; and o a poypmptid that is a of SE ID NTs 1 and 2. 10 Preferred poiypeptides of the invention are selected from any of SEQ ID NOs: 124-141 (Table A-5). In preferred aspect, the invention relates to a polypeptide as defined above, wherein the first immunoglobu in sing e variable domain is SEQ ID NO: 12. In another preferred aspect, the invention relates to a po ypeptide wherein the second irnrunoglobulin single variable domain is selected frorn any of SEQ ID NOs: 1 and 10. In another preferred aspect, the invention relates to a polypeptide which is isselecte.d from any of SEQ ID NOs: 129 and 134. In another preferred aspect, the invention relates to a polypeptide, wherein the second imrnunoglobulin single variable dornain is SEQ ID NO: 1. In another preferred aspect, the invention relates to a polypeptide, wherein the first immunoglobulin single variable domain is selected frorn any of SEQ ID NOs: 3 and 12. In another preferred aspect, the invention relates 20 In further aspect, each of the two or more mrmunoglobulin single variable domains of the rnultiparatopic (such as biparatopic or triparatopic) polypeptide of the invention, that are directed against PcrV belong to the same epitope bin. Accordingly, the present invention also relates to a polypeptide comprising or essential y consisting of two or more immunog obulin single variab e dornains directed against PcrV, wherein each oftthe two or more immunoglobulin single variable domains that are directed against PcrV belong to the same epitope bin. Imrnunoglobulin single variable domains that belong to the same epitope bin, by definition cross-cornpete with each other for binding the target, PcrV. Accordingly, the present invention relates to polypeptide comprising or essentially consisting of two or more immunoglobulin single variab e domains directed against PcrV, wherein the first immunoglobulin single variable dornain cross-blocks the binding to PcrV of the second immunoglobulin single variable 30 domain and/or wherein the first immunog obulin single variab e is cross-blocked from binding to PcrV b the second immunoglobulin single variable domain.
WO 2013/128031 PCT/EP2013/054262 16 Preferred combinations of immunoglobulin single variable domains present in such multiparatopic (such as biparatopic or triparatopic) polypeptide of the invention rnay encarmpass any of the following: - the frst and the second immunoglobulin single variable domains cross-block the binding to PcrV of at least one of immunoglobulin single variable dornains with SEQ ID NOs: 3-10 [epitope bin1] 5 and/or the first and the second irmmunoglobulin single variable domains are cross-blocked from binding to PcrV by at least one af the immunoglobulin single variable domains with SEQ ID NOs 31 [epitope bi - the first and the second irmmunoglobu in single variable damans cross-block the binding to PcrV of at least one of the immunoglobulin single variable dor ns with SEQ ID NOs: 1-2 [epitope 1o bin2) and/or the first and the second immrunoglobulin sing e variable domains are cross-bloclked frorn binding to PcrV by at least one of the irnmunoglobulin single variable damains with SEQ ID NOs: 1- aepitope bnr2 or - the first and the second immunoglobulin single variable domains cross-block the binding to PcrV of at least one of the immunoglobulin sngle variable dornains with SEQ ID NOs: 11-12 [epitope 15 bin3] and/art te first arnd the second d irnmunoglobulin sing e variable dornains are cross-blocked frarm binding to PcrV by at least one of the irnmunoglobulin single variab e domains with SEQ ID NOs: 11-12 [epitope bin3]. Such preferred multiparatopic (uch as biparatopic or triparatopic) polypeptides of the invention rnay comprise or essential y consist of one of the following combinations of irnrmunog obulin single 20 vabl dons: -the first and the second immunoglobulin single varlab e domains are polypeptides that belong to epitope bin 1 and are selected frorn any one of: o a polypeptide that binds full length PcrV (SEQ ID NO: 159) and that shows reduced binding (30 90% as compared to iul Iength P rV or no binding ao cirnera 4 (SEQ ID NO: 202) and cnimera 6 25 SEDNO: 204 o a polypeptide in which the CDR sequences have ate lest 70% amino acid identity, preferably at least 80% arnino acid identity, more preferably at least 90% amino acid identity, such as 95% amino arid identity or m ore or even essentially 100% arnino acid identity with the CDR sequences of at least one of the immunoglobu in single variable domains with SEQ ID NOs: 3-10; 30 a polypeptide that cross-blocks the binding to PcrV of at least one of the imnrunoglobulin single variable domains with SEQ ID NOs: 3-10 and/or that is cross-blocked fram bincing to PcrV by at least one of the irnmunoglobu in single variable drains with SEQ ID NOs: 3-10; and WO 2013/128031 PCT/EP2013/054262 17 o ap epitope bin 2and are seced from any one of: o a polypeptide that blinds to full length PcrV (SEQ IlD NO: 159) and that shows reduced binding 5 (30-90% as compared to full length PcrV) or no binding to chimera 7 (SEQ ID NO: 205); o a polypeptide, in which the CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, rnore preferably at least 90% amino acid identity, such as 95% arnino acid identity or more or even essentially 100% amino acid identity with the OR sequences of at least one of the irnmunoglobu in single variable dornains with SEQ ID NOs: 1 and 10 o a polypeptide that cross-blocks the binding to PcrV of at least one of the imrnunog obulin single variable domains with SEQ ID NOs: 1 and 2 and/or that is cross-blocked from binding to PcrV by at least one of the immunoglobulin single variable dornains with SEQ ID NOs: 1 and 2; and o a polypeptide that is any of SEQ ID NOs: 1 and 2; or 15 - the first and the second immunog obulin sing e variab e dornains are polypeptides that belong to epitope bin 3 and are selected from any one of: o apolypeptide that binds full length PcrV (SEQ ID NO: 159) and that shows reduced binding (30 90% as compared to full length PcrV) or no blinding to chirnera 2 (SEQ|ID NO: 200); o a polypeptide in which the CDR sequences have at leat 70% amino acid identity, preferably at 20 least 80% amino acid identity, rnore preferably at eas 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% mrino acid identity with the CIDR sequences of at least one of the immunoglobulin single variable dornains with SEQ ID NOs: 11 o a polypeptide that cross-blocks the binding to PcrV of at least one of the iornunoglobulin single 20 varneble domains with SEQ ID NOs: 11 and 12 and/or that is cross-blocked from binding to PcrV by at least one of the immunoglobulin single variable domains with SEQ ID NOs: 11 and 12; and o a polypeptide that is any of SEQ ID NOs: 11 and 12. In a preferred aspect, the invention relates to a polypeptide as defined above, which is selected frm any of SEQ ID NOs: 118-123 (Table A-5). 30 In another preferred aspect, the invention relte to e a p dfin dmboe wheei at leastone immunoglobulin single variable dornan is SEQ ID NO: 3. In another preferred apect, the invention relates to polypeptide as defined above, which is selected from any of SEQ ID NOs: 118, 120 WO 2013/128031 PCT/EP2013/054262 18 and 121. In another preferred spect, the invention relates to a polypeptide as defined above, wherein at least one immunoglobulin single variable domain is SEQ lID NO: 1. In another preferred aspect, the invention relates to a polypeptide as defined above, which is selected from any of SEQ ID NOs: 122 and 123 s The two or more immunog obulin single variable dornains present in the polypeptide of the invention may consist of a light chain variable domain squence (e.g., a VLesequence) or of a heavy chain variable dornain sequence (e.g., a Vesequence). They rnay consist of a heavy chain variable domain sequence that is derived from conventional four-chain antibody or of a eavy chain variable dornain squence that is derived from heavy chain antibody. They may consist of a domain antibody (or an amino to acid that is suitable for use as a domain antibody), of a single domain antibody (or an amino acid that is suitable for use as a single dornain antibody), of a dAb" (or an arnino acd that is suitable for use as a dAb) or of a Nanobody (including but not limited to a Ves). In a preferred aspect, the two or more immunoglobulin single variable domains consist of a partially or fully humanized Nanobody or a partially 15 The multivalent, such as multiparatopic, polypeptides of the invention can generally be provided (and in particular, purposefully designed for specific biological action) by suitably linking (optionally via suitable linkers) or combining two or rnore (monovalent) immunoglobulin single variable domains (or by suitably linking or combining nucleotide sequences encoding such (monovalent) immunoglobulin single variable domans to provide a nu cleic acid that encodes the desi red multivalent construct, and then 20 suitably expressing said multivalent construct) Thus, it is clear that the invention not only rnalkes avalable the mu tivalent, preferably multiparatopic, polypeptides described herein, but also provides by making available the monova ent polypeptides described herein - the skilled person with a range of different "bindng domains" or "binding units"' that can be used as "building bocks" to provide a range of different rnultivalent, preferably multiparatopic (and in particular, biparatopic and triparatopic) 25 polypeptides (which may have different binding affinities avidities, speciificities, potencies and/or efficacies through the use of suitable "building blocks as described herein. Consequently, the various irnmunog obulin single variable dornains and/or monovalent polypeptides of the invention (and/or nucleotide sequences and/or nucleic acids encoding the same) and their use of as "building blocks" in or for preparation of multivalent and/or multiparatopic polypeptides 30 (or nucleotide sequences/nucleic aids encodingthe same) form an important aspect of the invention.
WO 2013/128031 PCT/EP2013/054262 19 Aco digy in a futhe asp ct the inveono aso reate to a polypeptide (also referred to herein as "monovalent polypeptide(s) of the invention" that cormprises at least one stretch of arnino acid residues that chosen from the group consisting of: -CDR1 sequences: 5 a) SEQ ID NOs: 20-37; b) stretches of mrino acid sequences that have at least 80% amino aci idenfty wit nt lest one of the amino acid sequences of SEQ ID NOs: 20-37; c) stretches of amino acid sequences that have 3, 2, or 1 anoa aci df enewttlatoeoth armino acid sequences of SEQ ID NOs: 20-37; 10 and/or CDR2 sequences: d) SEQ IDNOs 38-56 e) stretch of amino acid sieqnene with at least one of the amino acid sequen ces of SEQ ID NOs: 38-56; f) stretches of amino acid sequences that have amino acid if e with at least one of the amino cid sequences of SEQ ID NOs: 38-56; and/or -CDR3 sequences: g) SEQ ID NOs: 57-75; 20 h) stretches of amino acid sequences that have at least 80% amno acid identity with at least one of the amnmo acd sequences of SEQ ID NOs: 57-75; i) stretches of amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs 57-75. Monovalent polypeptides comprising one or more of the above specified stretches of amino acid 25 residues show irnproved properties such as e~g. improved binding characteristics (suitably measured and/or expressed as a Kr-value (actual or apparent), Kevalue (actual or apparent), a k 0 1 ,rate and/or a kefrate, or alternative y as an I|C 50 value, as further described herein), improved affinity and/or improved avidity for PerV and/or improved efficacy and/or potency for neutralizing PcrV. For exarnple, in aT SS-dependent cytotoxicity assay with P3X63 cells asthe target at an average so MvOl of 2,8, the monovalent polypeptides of the invention may have 1C50 values between 1 nM and 10000 nM, between 5 nM and 1000 nM, preferab y between 5 nM and 500 nM, more preferably between 5 nM and 200 nM, such as between 5 nM and S0 nM r less WO 2013/128031 PCT/EP2013/054262 20 A part frorn this and/or in addition, in such a TTSS-dependent cytotoxicity assay, the monovalent polypeptides of the 'nvention may have an efficacy (% inhibition; see Example 4 4) of 50% or more, preferably 90% or more, such as 100%. In a preferred aspect, the monovalent polypeptides of the invention have the structure FR1-CDR1 5 R2-CDR2-FR3-CDR3-FR4, in which CDR1, CD|R2 and CDR3 are as defined herein for the rnonovalent polypeptides of the invention, and FR1, FR2, FR3 and FR4 are framework sequences. Accordingly, the present invention also relates to a monova ent polypeptide that essentially consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, 10 CDRi is chosen from the group consisting of: a)the arnino acid sequences of SEQ ID NOs 20-37; b) mrino acid sequences that have at leat 80% amino aci ientt iha es nrfth ioai sequences of SEQ ID NOs: 20-37; c) amino acid sequences that have 3, 2, or 1 arnino acid difference with at least one of the amino acid 15 sequences of SEQ ID NOs: 20-37; and/or - CDR2 is chosen frorn the group consisting of: d) the amino acid sequences of SEQ 0D NOs: 38-56 e) arnino acid sequences that have at least 80% amino acid identity with at least one of the amino acid 0 sequences of SEQ ID NOs: 38 f)amino acid sequences that have 3,2, or 1 mrino acid difference with at least one of the amino acid sequences ofS SQID NOs: 38-S6 and/or -CDR3 is chosen from the group consisting of: 25 g) the amino acid sequences of SEQ ID NOs: 57-75; h) amino acid sequences that have at least 80% amino acd identity with at least one of the arnino acid sequences of SEQ|ID NOs: 57-75; a) amino acid sequences that have 3,2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 57-75. 30 Preferred monovalent polypeptides essentially consist of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: - CDRis chosen from the group consisting of: WO 2013/128031 PCT/EP2013/054262 21 sequel of SEQ ID NO: 20-S7 c) amino acid sequences that have 3, 2, or aano acd a equen ces of SEQ ID NOs 20-37; and -CDR2 is chosen from the group consisting of: d) the amino acid sequence of SEQ ID NOs: 38-56 e) amino acid sequences that have at least 80% arnino acid identity with at least one of the amino acid sequences of SEQ I|D NOs: 38-56; f) amino acid sequences that have 3, 2, or amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 38-56; and - CDR3 ~is chse frmtegopcnitnr 15 g) the amino acid sequences of SEQ ID NOs: 57-75; h) amino acid sequences that have at least 80% amino acid identity with at least one of the mrino acid sequences of SEQ ID NOs: 57-75; i) minor acid sequences that have 3, 2, or 1 minor acid difference wth at least one of the amno acid sequence of SEQ ID NOs: 57-75, 20 In a preferred monovalent polypeptide of the invention, the CDR sequences have at least 70% mrino acid identity, preferably at least 80% arnino cid identity, rnore preferably at least 90% amino acid dentity, such as 95% amino acid identity or more or even essentially 100% arnino acid identity with the. CDR sequences of at least one of the immunoglobulin single variable dornains with SEQ||D NOs: 1-19. The invention also relates to rnonovalent polypeptides drected agaInst PcrV, that cross-block the 20 idi to PcrV of at least one of the immunoglobulin single variable dornains with SEQ ID NOs: 1-19 and/or that are cross-blocked frorn binding to PcrV by at least one of the immunoglobulin single variable domains with SEQ ID NOs: 1-19. Preferred rmonovalent polypeptides of the invention are selected from any of SEQ ID NOs: 1-19. The present inventors furthermore observed that imrnunoglobulns belonging to certain epitope for the preparation of rmultiparatopie such as e.g. biparatopic or triparatopic polypeptides Preferred irnmunoglobulins belong to epitope bins 1, 2 or 3 (as further defined herein).
WO 2013/128031 PCT/EP2013/054262 22 Accordingly, in a further aspect, the present invention relates to an immunoglobulin that belongs to epitope bin 1 and that has one or more of the following features: * it cross-blocks the binding to PcrV of at Ileast one of the immunog obulin single variable domains with SEQ ID NOs: -10; o * it is cross-blocked frorn binding to PcrV by at least one of the immrunoglobulin single variable domnins wth SEQ D NOs: -10 o it binds full length PcrV (SEQ IlD NO: 159) whiLe showing reduced (30-90% as compared to full length PcrV) or no (lower than 30% as compared to fu I length PcrV) binding to chimera 4 (SEQ ID NO: 202) and chimera 6 (SEQ ID NO: 204); 10o it consists of 4 framework regions (FR1 to FR4, respectively) and 3 cormplementarity determining regions (CURl to CDR3, respective y), in which: -CURl is chosen frorn the group consisting of: a) the amino acid sequences of SEQ ID NOs: 22-28; b) arnino acid sequences that have at least 80% mrino acid identity with at least one of the amino 15 acid sequences of SEQ ID NOs: 22-28; c) arnino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the arnino acid sequences of SEQ ID NOs: 22-28; and -CDR2 is chosen from the group consisting of: 20d) the amino acid sequences of SEQ ID NOs: 40-47; e) arnino acid sequences that have at least 80% arnino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 40-47; f) arnino acid sequences that have 3, 2, or amino acid difference with at least one of the amino 25 and - CDR3 is chosen from the group consisting of: g) the arnino acid sequences of SEQ ID NOs: 59-66 h) amino acid sequences that have at least 80% arnino acid identity with at least one of the amirno acd sequences of SEQ ID NDs 59-66 30 WO 2013/128031 PCT/EP2013/054262 23 * its CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid entity rnore preferably at least 90% amino acid identity, such as 95% amino acid identity or more, or even essentially 100% ino acid identity with the CDR sequences of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 3-10; or 25 it is selected from any of SEQ ID NO 3-10. In another aspect, the present invention relates to an immunoglobulin that belongs to epitope bin 2 and that has one or more of he flowing features: * it cross-blocks the binding to PcrV of at least one of the iornunoglobulin single variabLe domains to e it is cross-blocked from binding to PcrV by at least one of the immunoglobulin single variable 205) 1* it consists of 4 framework regions (FR1. to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDR3, respectively), in which: - CDR11s chosen from the group consisting of a) the amino acid sequences of SEQ ID NOs: 20-21; b) amino acid sequences that have at least 80% amino acid identity with at least one of the arnino 20 acid sequences of SEQ ID NOs: 20-21; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amno acid sequences of SEQ ID NOs: 20-2 1; and - CD|R2 is chosen from the group consisting of: 25 d) the amino o acid sequences of SQ ID NOs: 38-39; e) nino acid sequences that have at least 80% amino acid identity with at least one of the arino acid sequences of SEQ ID NOs: 38-39; f) amino acid sequences that have 3, 2., or 1 amino acid difference with at last one of the amino acid sequences of SEQ ID NOs: 38-39; so and - CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NOs: 57-58; WO 2013/128031 PCT/EP2013/054262 24 h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SE IlD NOs: 57-58; i) mrino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the arnino acid sequences of SEQ ID NOs: 57-58; 5 its CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, rnore. preferably at least 90% arnino acid identity, such as 95% arnino acid identity or more or even essentially 100% amino acid identy with the CDR sequences of at least one of the immrunoglobulin sing e variable dornains with SEQ ID NOs 1 and 2; or 10 In another aspect, the present invention relates to an immrunoglobulin that belongs to epitope bin 3 and that has one or rnore of the following features: * it c ross-bloc s t he binding to PcrV of at least one of the irnmunog obulin single variable domain with SEQ ID NOs: 11 and 12; * it is cross-block ed frorn binding toPr by a t one of ti mmuo obnm sigl aable 15domamns with S SQ ID NOs 11: and 12 * it binds to ful length PcrV (SEQ ID NO: 1S9), while it shows reduced (30-90% compared to full length PcrV) or no (below 30% as compared to full length PcrV) binding to chirnera 2 (SEQ ID NO: 2000 * it consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining 20 regions (CDIl to CDR3, respectively), in which: -ClDR1 is chosen from the group consisting of a) the amino acid sequences of SEQ ID NOs: 29-30; b) amino acid sequences that have at least 80% arnino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 29-30; 25 c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 29-30; and - CDR2 is chosen frorn the group consisting of: d) the amino acid sequences of SEQ ID NOs: 48-49 so e) amino acid sequenices that have at least 80% amno acid identity with at least one of the amino acid sequences of SEQ ID NOs: 48-49; WO 2013/128031 PCT/EP2013/054262 25 f) arnino aid sequences that have 3, 2, or 1 miaci i rnewt es n h mn acid sequences of SEQ ID NOs: 48-49; -CDR3 is choser from the group consisting of: 5 g) the amino acid sequences of SQ ID NOs: 67-68; h) amino acid sequences that have at least 80% armino acid identity with at Ieast one of the amnno acid sequence of SEQ 1D NOs 67-68; i)amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 67-68; o * its CDR sequences have at least 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity, such as 95% arnino acied identity or more or even essentially 100% amino acid identity with the CDR sequences of at leas t one of the immunoglobusin single variable domains with SEQ 0D NOs: 11 and 12; it is selected from any of SEQ ID NOs: 11 and 12 15 The monovalent polypeptides of the invention may essentiaI y consist of an imrnunog obulln single variabl domain selected from a light chain variable domain sequence (e.g., a V-sequence) and from a heavy chain variable domain sequence (e.g., a Srs equence). The monovalent polypeptides of the invention may essentially consists of an immrunoglobulin single variable dornain selected from a heavy chain variable dornain sequence that is derived from a conventionaI four-chain antibody and from a 2o heavy chain variable domain sequence that is derived from heavy chain antibody. The mon ovalent polypeptides of the invention rnay essentially consists of an immunoglobulin single variable domain selected frornm domain antibody (or an amino acid that is suitable for use as a domain antibody) a single domain antibody (or an amino acid that is suitable for use as a ing e domain antibody), a "dAb" (Or an mrino acid that is suitab e for uses a dAb) or a Nanobody (including but not limited to a Vs ), In a 25 preferred aspect, the monovalent polypeptide of the invention essentially consists of partialy or fully humanized Nanobody. such as a partialy or fully humanied VHH As described above, the invention a so relates to the use of a monovalent polypeptide as described herein in preparing a ultivalent, preferably multiparatopic po ypeptide of the invention. Accordingly, the present invention relates to the use of a monovalent polypeptide of the invention as a binding The invention further relates to a polypeptides (also referred to herein as a "polypeptide(s) of the invention") that comprises or essentiaIly consists of one or more monova ent polypeptide or one or m ore WO 2013/128031 PCT/EP2013/054262 26 multivalent, preferably multiparatopic, po ypeptide of the invention, and optionally further comprises one or more other groups, residues, mo eties or binding units, optional y linked via one or rnore peptidic Vinkers. As will become clear to the skilled person frorn the further disclosure herein, such further groups, residues, moieties, binding units or arnino acid sequences rnay or may not provide further functionaity 5 to the monovalent or multivalent, preferably multiparatopic, polypeptide of the invention and may or may not rnodify the properties of the monovalent or rnultivalent polypeptilde of the invention. The invention also relates to nucleic acids or nucleotide sequences that encode a polypeptide of the invention. Such a nucleic acid will also be referred to herein as "nud eic acid(s) of the invention" and may for exarnple be in the forrn of genetic constru c, further described herein. Accordingly, the 10 present invention also relates to a nucdeic acid or nucdeotide sequence that is in the form of a genetic construct, Nucleic acids encoding a monovalent polypeptide of the invention can be linked to obtamn a nucdeic acid encoding amrultivalent, preferably multiparatopic, polypeptide of the invention. Accordingly, the present invention also relates to the use of a nucleic acid or nucleotide sequence that encodes a 15 monovalent polypeptide of the invention for the preparation of genetic construct that encodes a multivalent, preferably rnultiparatopic, polypeptide of the invention. The invention further relates to a host or host celI that expresses (or that under suitable circumstances is capable of expressing) a polypeptide of the invention; and/or that contains a nucleic acid of the invention. Sorne preferred but nonlimiting examples of such hosts or host cel s will becorne 3 de from th further desalption herei The invention further relates to a composition containing or comprising at lea one polypeptid of the invention and/or at least one nucleic acid of the invention, and optionally one or more further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a composition may for exarnple be a pharmaceutical composition (as described herein) or a veterinary composition. Sorne preferred but nonrlirniting exarnples of such compositions will become clear frorn the further description herein. The invention further relates to rnethods for preparing polypeptides, nucle acds, host cells, and compositions described herein. The invention further relates to applications and uses of the polypeptides, nudeic acids, host cells, 3o and compositions described herein well as to rnethods for the prevention and/or treatment of P. aeruginosa infections. Sorne preferred but non-limiting appliiations and uses will become clear from the further descrption herein.
WO 2013/128031 PCT/EP2013/054262 27 As such, polypeptides and compositions of the present invention can be used for the prevention and/or treatment of P. aerugin~osa infections. Patient groups susceptible to PF aeruginosa infections will be clear to the skilled person and for example include (without being imiting) ventilator-associated pneumona yA rn victims, ehana ventilated patient Cystic FirossF patient, 5hernatopoletic cell transplantation patients, bone narrow transplant patients, patients undergoing surgery, patients with chronic obstructive pulmonary disease (COPD), patients with bron niectasis patients with sepsis and patients with cancer-associated neutropenia. Accordingly, the present invention also relates to a method for the prevention and/or treatment of 10 ventilated patients, Cystic Fbrosis (CF) patients hernatopoietic cell transplantation patients, bone marrow transp ant patients, surgery, chronic obstructive pulmonary disease (COPD), bronciectasis, sepsis, cancer-associated neutropenia, said method comprising administering, to subject in need theivnin 15 The inventon also relates to the use of a polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention and/or treatment of P. aeruginosa infections in least one of venti ator-associated pneurnona (VAP), burn vctirns, mechanical ventilated patients, Cystic Fibrosis CF) ptients hematopoietic cel transplantation patients bone marrow transplant patientE surgery chronic obstructive pulmonary disease (COPD), bronchiectasis, sepss, caaocat e neopeni 20 and/or for use in one or more of the methods described herein, The invention also relates to a polypeptide of the invention or a composition of the invention for prevention and/or treatment of P. aeruginosa infections in least one of ventilator-associated pneumonia (VAP) burn victims, mechanical ventilated patients, Cystic Fibrosis (CF) patients hematopoietic cell transplantation patients. bone marrow transplant patients, surgery. chronic obstructive pulmonary 25 disease COPD), bronchiectasis, sepsis, cancer-associated neutropenia. Other applications and uses of the polypeptides and compositions of th ineonm wi become clear to the skiled person from the further disclosure herein, FIGUlRE LEGENDS so Figures 1A-1D: Analysis of monovalent ant-cVN an obode inctotoxicity aa with 3 el st trget described in Example 5.
WO 2013/128031 PCT/EP2013/054262 28 Figures 2A-2B: Analysis of bivaient/bsparatopic anti-PerV Nanobodies in cyttoxcit asay wit P3X63 cells as target as described in Exarnple 7. Figure 3: Schernatic representation of molecules used for epitope rmapping. The PcrV-LcrV chirnera design was based on primary sequence and structural (particularly secondary structure) information. aSeven different chirneric molecules were designed by introducing seven fragments of LcrV (transparent bars) of length between 17 and 47 arnino acid residues in replacement of the structurally corresponding counterparts of PerV (black bars). A Pcry fragment (arnino acids 144-257) described by Frank et at (The JournaI of infectious diseases 186: 64-73, 2002 and US 6,827,935) wa aso generated. Numbers above and below the bars indicate PcrV or LcrV armino acid residue nurnbers, respectively. The amino acid to sequence of the different constructs is given in Table A-7. Figures 4A-4B: Survival curves obtained in acute Pi aeruginosa infection mouse rnodel after inoculation with Nanobodies 39, 360 and 376 7 to 8 C57B1/6 mice per group were intranasally challenged with aprernix of either Nanobody, Fab 13,37 or buffer alone premixed with Pseudomonas oeruginosa. The mice were monitored for survival durn 96 hours or 125 hours. 15 Figures 5A-5C: Lung inflammation parameters and bacteria burden, 3 to 5 C5781/6 mice per group were intranasally challenged with a prernix of either Nanobody Fab 13.37 or buffer alone prernixed with Pseudomonais aeruginosa. An additional group received Tobramycin at 10mg/|kg i p. to serve positive control All the mice were sacrificed at 24 hours post-infection following which rmyeloperoxidase activity (A), bacterial burden (B) and percentage lung weights to total body weight (C) were assessed. Results are 2 depicted as mean±SEM. Statistics were performed using a one-way ANOVA with a post-hoc Bonferroni s multiple comparison test. P-values <0.05 were considered statistically significant. Fure 6: Relative weight lo ss 7 to 8 C57|BW/ mice per group were intranasally challenged with a prernix of either Nanobody, Fab 13.37 or buffer alone premixed with Pseudomonas aeruginoso. The rnice were monitored for 5 as and body weight wer remorded ail t 1pmo Reult are depact s 25 mean±5EM. Figure 7. OD500 values of the Nan obody samples before and after nebulisation. The polypeptides 339, 360, 354 and 376 (in D-PBS with and without Tween80) were nebulized by the AKITA" APIXNEB nebula zer system (Activaero) The nebulisation experiment was performed in duplicate, 500 U of sample wanblJootnosyvaamehnbl rwt mmmrne h rslwscletdi o~~~~~~~~~ 10wigtshote n hn nlsd WO 2013/128031 PCT/EP2013/054262 29 Un ess indicated or defined otherwise, aH terms used have their usua rneaning in the art, which awil be clear to the skilled person. Reference is for exarnp e rnade to the standard handbooks, such as Sarnbrook et at (Molecular Cloning: A Laboratory Mainual (2nd-Ed.) Vols, 1-3, Cold Sprin g Harbor Laboratory Press, 1989), F. Ausubel et al (urrent protocols in molecular biology, Green Publishing and Wiley lnterscience, New York, 1987), Lewin (Genes|||| John Wi ey & Sons, New York N.Y., 1985), Old et aL. (Principles of Gene Manipulation: An lntroduction to Genetic Engineering (2nd edition) University of 1> California Press, Berlkeley, CA, 1981); Roitt et al (Irnmunoiogy (6th. Ed.) Mosby/Elsevier, Edinburgh, 2001), Roitt et at. (Roitt's Essential Immunology (10~ Ed.) Blackwel Publishing, UK, 2001), and Jane.way et at (Immunobiology (6th Ed. Garland Science Publishing/Churchill Livingstone, New York, 2005), as well sotegnera backgon ar ied herein. Unless indicated otherwise, all methods, steps, techniques and manipulations that are not 15 specifically described in detail can be performed and have been performed in manner known per se, as will be clear to the skilled person. Reference is for exam ple again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; as well as to for exarnple the following reviews Presta (Adv. Drug Deliv. Rev. 58 (5-6): 640-563 2006), Levin and Weiss (Mol Biosyst. 2(1): 49-57, 2006) Irving et al. (1 Immunol Methods 248(1-2): 31-45, 2001), Schmitz et al 2o (Placenta 21 SuppL. A: 3106-12, 2000), Gonzales et at (Tumour BioL. 26(1): 31-43, 2005), which describe techniques for protein engine eering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins The term 'sequence" as used herein (for example in terms ilke "irmmunoglobulin sequence", "antibody sequence", 'variable domain sequence', "Va s sequence" or "protein sequence") should 25general y be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the sarne, unless the context requires a nore imited interpretation, Arnino acid residues will be indicated according to the standard three-letter or one-letter amino acid code Reference is made to Table A-2 on page 48 of WO 08/020079. A nucleic acid or amino acid is considered to be "(in) (essentialy) isolated (form)' - for exarnple, so cornpared to the reaction mediurn or cultivation rnedium front which it has been obtained - when it has been separated ron, at least one other component wit h which it is usually associated in said sourceorm rnedim suchn a other nucleic acid, another protein/ polypeptide, another biological component or WO 2013/128031 PCT/EP2013/054262 30 macromolecule or at least one contaminant, impurity or minor component, In particu ar, nuc eic acid or amino acid is considered" "essentially) isolated" when it has been purified at least 2-fold, in particular at east 10-fold, more in particular at least 100->fold, and up to 1000-fold or more. A nucleic acid or amino acid that is "in (essentially) isolated form" is preferably essentially homogeneous, as determined using a c5 suitable technique, such as suitable chromatographical technique, such as polyacrylamide-gel electrophoresis. When a nucleotide sequence or amino acid sequence is said to "comprise" another nucleotide sequence or amino acid sequence, respectively, or to "essentially consist of" another nucleotide sequence or amino acid sequence, this may rnean that the latter nucleotide sequence or arnino acid 1o sequence has been incorporated into the first mentioned nucleotide sequence or armino acid sequence, respectively, but rore usualy this generally reans that the first mentioned nucleotide sequence or arnno acid sequence comprises within its sequence. a stretch of nucleotides or arnno acid residues respectively, that has the same nucleotide sequence or arnino acid sequence, respectively, as the latter sequence, irrespective of how the first mentioned sequence has actually been generated or obtained 15(which may for example be by any suitable method described herein). By means of a non-limiting exarnple, when a polypeptide of the invention i s sid to comprise an immunoglobulin single variable domnan, this may mean that said immunoglobulin single variable dornain sequence has been incorporated into the sequence of the polypeptide of the invention, but more usually this generally means that the polypeptide of the invention contains within its sequence the sequence of the 20 immunoglobulin single variable domains irrespective of how said polypeptide of the invention has been generated or obtained. Also, when a nucleic acd or nucleotide sequence is sid to comprise another nucleotide sequ ence, the first mentioned nucleic acid or nucleotide sequence is preferably such that, when it is expressed into an expression product (e.g. a polypeptide), the mrino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, that the latter 25 nucleotide sequence is in the same reading frame as the first mentioned, larger nucleic acd or By "essentially consist of" is rneant that the inmmunoglobulin singe varliable domain used in the method of the invention either is exactly the same as the polypeptide of the invention or corresponds to the po ypeptide of the invention which has a lirnited number of amino acid residues, such as 1-20 arnno 30 acid residues, for exarnple 1-10 amno acid residues rnd preferab y 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 arnino acid residues, added at the amino terminaI end, at the carboxy terminal end, or at both the amino terminal end and the carboxy terminal end of the immunoglobulin single variable domain.
WO 2013/128031 PCT/EP2013/054262 31 For the purposes of comparing two or more nucleotide sequences, the percentage of "sequence identity" between first nucleotide sequence and second nucleotide sequence may be calculated by dividing [the nurnber of nucleotide in the first nucleotide sequence that ae identical to the nucleotide at the corresponding positions in the second nlucleotide sequence) by [the total number of nucleotides in s the first nucleotide sequence] and multiplyng by [100%}, in which each deletion, insertion, substitution or addition of a nucleotide in the second nucleotide sequence - compared to the first nucleotide sequence - is considered as difference at a ingle nucleotide (position) Alternatively, the degree of sequence identity between two or rnore nucleotide sequences may be calculated using a known computer algorithm for sequence alignment such as NCBi Blast v2.0, using standard settings. Some other to techniques, computer algorithms and settings for determining the degree of sequence identity are for exarnple described in WO 04/037999, EP 0967284, EP 1085089, WO 00/55318, WO 00/78972, WO 98/49185 and GB 2357768. Usually, for the purpose e of determining the percentage of' "equence identity" between two nucleotide sequences in accordance with the calculation method outlined hereinabove, the nucleotide sequence with the greatest number of nucleotides will be taken as the is "first" nucleotide sequence, and the other nucleotide sequence wil be taken as the 'second" nucleotide sequence. For the purposes of comparing two or mare arnino acid sequences, the percentage of "sequence identity" between a first amino acid sequence and second amino acid sequence sas referred to herein as "arnino acid identity") may be calculated by dividing [the number of arnino acid residues in the first 20 arnino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequencel by [the total nurnber of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an mrino acid residue in the second armino acid sequence -compared to the first arnino acid squence-s considered as a difference at a single amino acid residue (position), ie, asan "arnino acid difference' as 2r defined herein. Alternatively, the degree of sequence identity between two amino acid sequences rnay be calculated using a known computer algorithmn such as those mentioned above for determining the degree of sequence identity for nucleotide sequences, again using standard settings, Usually, for the purpose of deterrnininig the percentage of 'sequence identity" between two amino acid sequences in accordance with the calculation method outlined hereinabove, the arnino acid sequence with the so greatest number of amnno acid residues will be taken as the "first' amino acid sequence, and the other amino acid sequence will be taken as the "second' arnino acid sequence.
WO 2013/128031 PCT/EP2013/054262 32 Also, in determining the degree of sequence identity between two amino acid sequences the skilled person may take into account so-called "conservative" amino acid substitutions, which can generally be described as arnino acid substitutions in which ar mrino acid residue is replaced with another anmno acid residue of similar chemical structure and which has Ittle or essentially no influence 5 on the function, activity or other biological properties of the polypeptide, Such conservative amino acid substitutions are well known in the art, for exarnple from WO 04/037999, Gb 335768, WO 98/49185, WO 00/46383 and WO 01/09300; and (preferred) types and/or combinations of such substitutions may be selected on the basis of the pertinent teachings from WO 04/037999 aswell as WO 98/49185 and from to Such conservative substitutions preferably are substitutions in which one arnino acid within the following groups (a) - (e) 'iso ubtituted by another amino acid residue within the same group: (a) srnall aliphatic, nonpolar or slightly poiar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged amide-: Asp Asn, Gu and Gin; i polar positvel charged reidue.. His Arg and Lys '(d) large aliphatic, nonpolar residues: Met, Leu, lIe, Val and Cys 'and ()arornatic residues 15 Phe, Tyr and Trp. Particularly preferred conservative substitutions are as follows: A a into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into G u; Cys into Ser; Gin l'nto Asn; Glu into Asp' GIy into Ala or into Pro' His into Asn or into Gin' lie into Leu or into Val' Leu into lie or into Val; Lys into Arg, into GIn or into G u; Met 'nto Leu, into Tyr or into lie; Phe into Met, into Leu or into Tyr' Ser into Thr; Thr into Ser; Trp into Tyr' Tyr into 1rp, and/or Phe mn- Vat le oi eu 20 Any amino acid substitutions apphied to the polypept'des des r'bed here nmay also be based on the analysis of the frequencies of arnino acid variations between homologous proteins of different species deve oped by Schulz et at ("Princ'ies of Prote'n Structure", Springer-Verlag, 1978), on the analyses of structure forrning potentials developed by Chou and Fasrman (Biochernistry 13: 211, 1974; Adi En ymol , 47: 4.9, 1978), and on the anai of hydr ophobicity patterns in proteins developed 25 by Eisenberg et at (Proc. Natt. Acad Sci. USA 81: 140-144, 1984), Kyte and Doolittle (1 Molec. BioL, 157: 105-.132, 1981), and Goldrnan et al (Ann. Rev. Biophys. Chern. 15: 321-353, 1986), all incorporated herein in their entirety by reference. Information on the prirnary, secondary and tertiary structure of Nanobodies is given in the description herein and in the general background art cited above. Also, for this purpose, the crystal structure of a V, dornain from a lama is for exarnple g'ven by Desmyter et at, so (Nature Structural biology, 3: 803, 1996), Spinell et al (Natural Structural bio ogy, 3: 752-757, 1996) and Decanniere et aL. (Structure, 7 (4) .361, 1999). Further information about some of the amino acid WO 2013/128031 PCT/EP2013/054262 33 residues that n onventional Vs domains form the VH/Vt interface and potential ca i ubstn on these positions can be found in the prior art cited above. Amino acid sequences and nucleic acid sequences are said to be "exactly the same" if they have 100% sequence icdentity (as defined herein) over their entire length. 5 ~When comparing two amino acid sequences, the terrn 'arnino acid difference" refers to an insertion, deletion or substitution of a sin gle amino acid residue on a position of the first sequence cormpared to the second sequence; it being understood that two arnino acid sequences can contain one, two or rnore such amino acid differences More particular, in the amino acid sequences and/or polypeptides of the present invention, the terrn "arnno acid difference' refers to an insertion, deletion 1o or substitution of a singe amino acid rsidue on a position of the CDR sequence specified in c), f) ori) compared to the CDR sequence of respectively a), d) or g); it being understood that the CDR sequence of c), f) and i) can contain one, two or maximal three such amino acid differences cornpared to the CDR The ' amino a- id differenc e' an be any one, two or maximaI three substitution ,deletions or 15insertions, or any combination thereof, that either improve the properties of the polypeptide of the invention or that at least do not detract too much frorn the desired properties or from the balance or combination of desired properties of the polypeptide of the invention, n this respect, the resu ting polypeptide of the invention should at least bind PcrV with the sarne about the same, or a higher af nt compared to the polypeptide comprisin gthe one or rnore CDR sequence without the one, two or 2o maxirnal three substitute tions, deletions or insertions, said affinity as rneasured by surface plasmon resonance, In this respect, the amino acid sequence according to c), f) and/or i) may be an amino acid sequence that is derived frorn an amino acid sequence according to a), d) and/or g) respectively by means of affinity maturation using one or more techniques of affinity maturation known per so. 25 For example. and depending on the host organism used to express the polypeptide of the invention, such deletions and/or substitution s may be designed in such a way that one or more sites for post-translational modification (such as one or rnore glycosy ation sites) are rernoved, as will be within the abiit of the person sI lled n the art The terrns "epitope' and "antigenic determinant", which can be used interchangeably, refer to the 30 part of a macromolecule, such as polypeptide or protein that is recognized by antigen-binding molecules, such as immunoglobulins, conventional antibodies, immunoglobulin single variable domains and/or polypeptides of the invention, and rnore particularly by the antigen-binding site of said WO 2013/128031 PCT/EP2013/054262 34 molecules. Epitopes define the rninirnum binding site for a munooblnadth rerstth target of specificity of an irnmunog obulin. The part of an antigen-binding molecule (such asan immunoglobulin, a convention a ntibody, an immrunoglobulin single variable domainm and/or a polypeptide of the invention) that recognizes the epitope is called a "paratope" A polypeptide (such as an immunoglobulin, an antibody, an immunoglobulin single variab e domain, a polypeptide of the invention, or generally an antigen binding molecule or a fragment thereof) that can "bind to" or "specifically bind to", that "has affinity for" and/or that "has specificity for" a certain epitope, antigen or protein (or for at least one part, fragrnent or epitope thereof) is said to be 1o "against" or "drected against" said epitope, antigen or protein or is a "binding" molecule with respect to such epitope, antigen or protein, or is said to be "anti"-epitope, 'anti"-antigen or "anti"-protein (eg~, anti '-PcrV The term "specificity" has the meaning given to it in paragraph n) on pages 53-56 of WO 08/020079; and mentioned therein refers to the numberof different types of antigens or antigenic 15- determinants to which a particular antigen-binding molecule or antigen-binding protein (such as an immunoglobulin single variable dornan and/or a polypeptide of the invention) can bind. The specificity of an antigen-binding protein can be determined based on affinity and/or avidity, as described on pages 53-56 of WO 08/020079 (incorporated herein by reference) which also describes some preferred techniques for rmeasuring binding between an antigen-binding molecule (such as an immunoglobulin 20 single variable dornain and/or polypeptide of the invention) and the pertinent antigen. Typically, antigen binding proteins (such as the iornunoglobulin single variable domains and/or polypeptides of the invention) will bind to their antigen with a dissociation constant (Ks) of 10-> to 10 moles/liter or less, and preferably 10O to 10O moles/liter or less and more preferably 104 to 10'4 moles/liter (ie. with an~ association constant (KA) of 105 to 10" liter/ moles or more, and preferably 10' to 10' liter/moles or 25 more and more preferably 10s to 10" liter/moles) Any KD value greater than 104 mol/ ter (or any K/ value lower than 104 M') liters/mol is generally considered to indicate non-specific binding. Preferably, a rnonovalent polypeptide of the invention will bind to the desired antigen with an affnity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as e. between 10 and 5 nM or less. Specific binding of an antigen-binding protein to an antigen or antigenic determinant can be 30 determined in any suitable rnanner known per se, including, for example, Scatchard analysis and/or competitive binding assays, such as radioimmunoassays (IRIA) enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other WO 2013/128031 PCT/EP2013/054262 35 techniques mentioned herein.As wil beclear tothe skied person, and as described on pages 53-56 of WO 08/020079, the dissociation constant may be the actual or apparent dissociation constant. Methods for determining the dissociation constant wil be clear to the skilled person, and for example include the techniques mentioned on pages 53-56 of WO 08/020079, SAn imrnunoglobu in sing e variable dornain arnd/or polypeptide is said to be "specific for" a first target or ant gen compared to a second target or antigen when it binds to the first antigen with an affinity (as described above, and suitably expressed as a K 5 value, K4 value, Ka rate and/or K(. rate) that is at least 10 times, such as at least 100 times, and preferably at east 1000 tires, and up to 10000 irnes or rnore better than the affinity with which the irnmunoglobu in single variable dornain and/or 10 p0lypeptide binds to the second target or antigen. For exarnple, the immunoglobulin single variable domain and/or polypeptide rmay bind to the first target or antigen with a Ko value that is at least 10 times less, such as at least 100 times less, and preferably at least 1000 tires less, such as 10000 trnes less or even less than that, than the Ko with which said immunog obulin single variab e domain and/or polypeptide binds to the second target or antigen. Preferably, when an imrnunog obu in single variable 15 dornain and/or polypeptide is "specific for' a first target or antigen compared to a econd target or antigen, it is directed against (as defined herein) said first target or antigen , but not directed against said second target or antigen. The termn "cross)-block", "(cross)-blocked", "(cross)-blocking" 'competitive binding", "(cross) cornpete", "(cross)-competing 'and "(cross)-competition" are used interchangeably herein to mean the 20 ability of an imunoglobulin, antibody, immunoglobulin sngle variable dornain, polypeptide or other binding agent to interfere with the binding of other immunoglobu ins, antibodies, immunog obulin single variable domains, polypeptides or binding agents to a given target. The extent to which an imrnunoglobulin, antibody, immrunoglobulin single varnable domain, polypeptde or other binding agent i able to interfere with the binding of another tot the target, and therefore whether it can be said to cross 25 block according to the invention, can be determined using competition binding assays. One particularly suitable quantitative cross-blocking assay uses a Biacore instrument which can rneasu re the extent of interactions using surfce plasmon resonance technology. Another suitable quantitative cross-blocking assay uses an ELSA-based approach to rreasure competition between immunoglobulin antibodies, immunoglobulin sngle variable domains, polypeptides or other binding agents in terrns of their bindng The following generally describes a suitable Biacore assay for determining whether an imrnunoglobulnn antibody, immunoglobulin single variable doman, po ypeptide or other binding agent WO 2013/128031 PCT/EP2013/054262 36 cross-blociks or is capable of cross-blocking according to the invention. It will be appreciated that the assay can be used with any of the immunoglobulins, antibodies, immunoglobulin single variable domains, polypeptide or other binding agents des cribed herein. The B'acore instrument (for exarnple the Biacore 3000)1i operated in line with the manufacturer's recommendations. Thus in one cross-blocking asay, 5the target protein (e.g. PerV) is coupled to a CM5 Biatore chip using standard amne coupling chemistry to generate a surface that is coated with the target. Typically 200-800 resonance units of the target would be coupled to the chip (n amount that gives easily measurable levels of binding but that is readily saturable by the concentrations of test reagent being used). Two test binding agents (terrned A* and B* to be assessed for their ability to cross- block eac other are rnixed at a one to one rnollar rtin of binding to sites in a suitable buffer to create the test mixture, When calculating the concentrations on a binding site basis the molecular weight of a binding agent is assumed to bet te total molecular weight of the binding agent divided by the number of target binding sites on that binding agent. The concentration of each binding agent in the test mix should be high enough to readily saturate the binding sites for that binding agent on the target mo ecules capture on the Biacore chip. The binding agents in the rnixture are at the 15sarne molar concentration (on a binding basis) and that concentration would typically be between 1.00 and 1,5 mncrornolar (on a binding site basis) Separate solutions containing A* alone and B* alone are also prepared. A* and B* in these solutions should be in the same buffer and at the same concentration as in the test mix. The test mixture is passed over the target-coated Biacore chip and the total amount of binding recorded The chip is then treated in such a way asto rernove the bound binding agents without 20 damaging the chip-bound target. Typically this is done by treating the chip with 30 mM HCI for 60 seconds. The solution of A* alone is then passed over the target-coated surface and the amount of binding recorded. The chip is again treated to remove a I of the bound binding agents without darnaging the chip-bound target. The solution of B* alone is then passed over the target-coated surface and the amount of binding recorded. The maxirnurn theoretical binding of the mixture of A* and B* is next calculated, and is the surm of the binding of each binding agent when passed over the target surface alone. if the actual recorded binding of the mixture is ess than this theoretical maximum then the two binding agents are said to cross-block each other. Thus, in general, across-blocking irnmun oglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent according to the invention is one which will bid to the target in the above Biacore cross-blocking assay such that durng~ sothe assay and in the presence of a second immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent the recorded binding is between 80% and 0.1% (e.g. 80% to 4%) of the rnaximum theoretical binding, specifically between 75% and 0.1% (e.g. 75% to 4%) of the WO 2013/128031 PCT/EP2013/054262 37 rnaximum theoretical binding, and more specifically between 70% and 0.1% (e-g. 70% to 4%) of maximurn theoretical binding (as just defined above) of the two mrmunoglobulins, antibodies, immunoglobu in single variable dornains, polypeptides or bincing agents in combination. The Biacore assay described above is primary assay used to determine if immuinoglobulins antibodies, o immrunoglobulin single variable domains, polypeptide or other binding agents cross-bock each other according to the invention. On rare occasions particular immunoglobulins, antibodies, imrnunog obulin single varnable domains, polypeptides or other binding agents rnay not bind to a target coupled via amne chemistry to a CM5 Biacore chip (this usually oc-curs when the relevant binding site on the target i masked or destroyed by the coupling to the chip). In such cases cross-blocking can be determined using a 10 tagged version of the target, for example aN-terminaI His-tagged version. In this particular format, an anti-His antibody would be coup ed to the Biacore chip and then the His-tagged target would be passed over the surface of the chip and captured by the anti-His antibody. The cross blocking analysis would be carried out essentially as described above, except that after each chip regeneration cycle, new His tagged target would be loaded back onto the anti-His antibody coated surface, In addition to the 1 exarnple given using N-terrninal His-tagged target, C-terrninal His-tagged target could alternatively be used- Furtherrnore, various other tags and tag binding protein combinations that are known in the art could be used for such a cross-blocking analysis (e.g. H A tag with anti-H A antibodies; FLAG tag with ai FLAG antibodies; biotin tag with streptavidin). The following generally describes an [LISA assay for determining whether an immunoglobulin, 20 antibody immunoglobulin single variable domain, polypeptide or other binding agent directed against target (e.g., PcrV) cross-blocks or is capable of cross-blocking as defined herein. It will be appreciated that the assay can be used with any of the imrnunoglobulins, antibodies, immunoglobulin single variable domains, polypeptides or other binding agents described herein. The general principal of the assay is to have an immunoglobulin, antibody, immunoglobulin single variable dornain, polypeptide or binding 2 agent that is directed against the target coated onto the wells of an [LISA plate. An excess amount ofa second, potentially cross-blocking, anti[-target immunog obulin, antibody, immunoglobu in single variable domain, polypeptide or other binding agent is added in solution (Le. not bound to the ELISA plate). A ignited amount of the target is then added to the wells. The coated immunoglobulin, antibody immunoglobulin single variab e domain, polypeptide or other binding agent and the immunoglobulin, so antibody, immunoglobulin single variable domain, polypeptide or other binding agent in solution cornpete for binding of the limited nurnber of target o ecules T he plate is washed to remove excess target that has not been bound by the coated imrnunoglobulin, antibody immunoglobulin single variable WO 2013/128031 PCT/EP2013/054262 38 dornain, polypeptide or other binding agent and to also remove the second, solution phase irmmunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent as well as any complexes forrned between the second, solution phase immunoglobuln, antibody, immunoglobulin single variable domain, polypeptide or other binding agent and target. The arnount of 5 bound target is then measured using reagent that appropriate to detect the target. An immunoglobulin, antibody, immunoglobulin single variable dornain, polypeptide or other binding agent in solution that is able to cross-block the coated immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent will be able to cause a decrease in the number of target rno ecules that the coated imrnunog obulin, antibody, immunoglobulin single variable domain, 10 po ypeptide or other binding aent can bind relative to the number of target rmolecules that the coated immunoglobulin, antibody, immunoglobulin single variable dornain, polypeptide or other binding agent can bind in the absence of the second, solution phase, immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent, in the instance where the first immunoglobulin, antibody, immrunoglobulin single variable dornain, polypeptide or other binding agent, e~g., an Ab-X, is 15chosen to be the irnrobilized immunoglobulin, antibody, imrnunoglobulin single variable domain, polypeptide or other binding agent, it is coated onto the wells of the EUISA plate, after which the plates are blocked with suitable blocking solution to minimize non-specific binding of reagents that are subsequently added. An excess amount of the second immunoglobulin, antibody, irmmunoglobulin single variable dornain, po ypeptide or other binding agent, .e. Ab-Y, is then added to the ELUSA plate such that 2) the rnoles of Ab-Y target binding sites per well are at least 10 fold higher than the moles of Ab-X target binding sites that were used, per well, during the coating of the EUSA plate. Target is then added such that the moles of target added per well are at least 25-fold lower than the mo es of Ab-X target binding sites that were used for coating each well Following a suitable incubation period the EUISA plate is washed and r regent for detecting the target is added to rneasure the amount of target specifically 25 bound by the coated anti-target irmmunoglobulin, antibody, immunog obu in single vrable domain, polypeptide or other binding agent (in this case Ab-X). The background signal for the assay defined as the signal obtained in wells with the coated irmmunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent (in this case. Ab-X), second solution phase imrnunoglobulin single variable domain, polypeptide or other binding agent (in this case Ab-Y), target buffer only (i.e., so without target) and target detection reagents. The positive control signal for the assay is defined as the signal obtained in we Is with the coated immunoglobulin, antibody, immunoglobulin single variab e domain, polypeptide or other binding agent (in this case Ab-X), second so ution phase immunoglobulin, WO 2013/128031 PCT/EP2013/054262 39 antibody, irnmunoglobulin single variable domain, polypeptide or other binding agent buffer only (i~e~, without second so ution phase imrnunoglobulin, antibody, immunoglobulin single variable dornain, polypeptide or other binding agent), target and target detection reagents The EUSA assay may be run in such amranner so as to have the positive control signal be at least 6tirnes the background signal. To Savoid any artefacts (e~g. significantly different affinities between Ab-X and Ab-Y for the target) resulting from the choice of which immunoglobulin, antibody, immunoglobulin single variab e domain, polypeptide or other binding agent to use asthe coating immunoglobuin, antibody, irmunoglobu in single variable domain, polypeptide or other binding agent and which to use as the second competitoro) immunoglobulin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent, 10 the cross-blocking assa rnay to be run in two formats: 1) forrnat 1 is where Ab-X is the immunoglobulin, antibody, immunoglobulinn single variable dornain, polypeptide or other binding agent that is coated onto the EUISA plate and Ab-Y is the competitor immunoglobulin, antibody, imrnunoglobulin single variable domain, polypeptide or other binding agent that is in solution and 2) format 2 is where Ab-Y is the immunoglobutin, antibody, immunoglobulin single variable domain, polypeptide or other binding agent 15that is coated onto the ELIA plate and AbN is6 the compe itor immunog obuln, antibody immunogliobulin single variable domain, polypeptide or other binding agent that is in solution. Ab-X and Ab-Y are defined scross-block'ng if, either in format 1 or in format 2, the solution phase anti-target immunoglobulin, antibody, imnrunoglobu in single variable domain, polypeptide or other binding agent is able to cause a reduction of between 60% and 100%, specifica y between 70% ad 100%, and more 20 specifica!ly between 80% and 100%, of the target detection signaI (i., the amount of target bound by the coated immunoglobulin, antibody, immrunoglobulin single variable domaini, polypeptide or other binding agent) as compared to the target detection signal obtained in the absence of the solution phase anti- target immunoglobulin, antibody immunoglobulin sing e variable dornain, polypeptide or other binding agent (/eA the positive control wels). 25 Other rnethods for determining whether an immunoglobulin, antibody immunoglobulin single variable domain, polypeptide or other binding agent directed against a target cross-blocks, is capable of cross-b ocking, competitively binds or is cross-competitive as defined herein are described eg. in Xiao Chi Jia et aL(Journal of Imrnunco gica lMethods 288: 91-98, 2004) Milleret at. ( ourn al of Immunological Methods 365: 118-125, 2011 and/or the methods described herein (see eg. Example 30 "pitope binning" refers to the use of competitive binding assays or cross-blocking assays to identify pairs of immunoglobulns, antibodies, immunoglobulin single variable domains, polypeptides, or WO 2013/128031 PCT/EP2013/054262 40 other binding agents that are, or are not, capable of binding the target (e., PcrV) simultaneously thereby identifying immunoglobulins, antibodies, immunog obulir single variable domains polypeptides or other binding agents that bind to the same, or overlapping epitopes on the target. An "epitope bin" as used in the present specification therefore is a farnily of irnmunoglobulins, Antibodies, immunoglobulin single variable domains, polypeptides, or other binding agents having the same or overlapping binding spec&ficity. As described above, the sorting of the immunoglobulin, antibodies, immunoglobulin single variable domains, polypeptides, or other binding agents into epitope bins is based on cross-competition (cross-blocking) of the immunoglobulins, antibodies, irnmunog obulin single variable domains, polypeptides, or other binding agents for antigen binding. The cross-competition 1o (cross-blocking) assay analyzes the simultaneous binding (pairing) of the immunog obulins, antibodies immunoglobulin single variable domains, polypeptides or other binding agents to the antigen and groups together irnmunoglobulins, anti bodies immunoglobulin single variable dornains, polypeptides, or other binding agents with similar pairing profiles Immunoglobulins, antibodies, immrunoglobulin single variab e dornains, polypeptides or other binding agents with similar profiles (i.e. belonging to the sarne epitope 15 bin) may bind to the same, closely related and/or overlapping epitopes. An amino acid sequence is said to be cross-reactive" or two different antigens or ntigenic determinants (such as e,., serum albumin frorn two different species of mammal, such as e. human serum albumin and cyno serurn albumin, such as ep. PcrV from different strains of P. oeruginosa) if it is specific for (as defined herein) both these different antigens or antigenic determinants. 20 The term "PcrV" a- used herein refers to the needle protein PcrV press ent in the Type IlI Secretion System (TTSS) of Pseudomonas aerug/nosa (P. oeruginosa)'. The terrn "potency' of a polypeptide of the invention, as used herein, is function of the arnount of polypeptide of the invention required for its specific effect to occur. It is measured simply as the inverse of the IQso for that polypeptide. it refers to the capacity of said polypeptide of the invention to 2' neutralize P, aeuinosa; such as to modulate, inhibit and/or prevent infectivity of P. oeruginosa, to modulate, inhibit and/or prevent colonization of tie host by P. oeruginosa, to modulate inhibit and/or prevent TTSS virulence rnechanisrns of P. aeruginose, to modulate, inhibit and/or prevent injection by P. aeruginosa into the host cell of various exotoxins, to rmodulate, inhibit and/or prevent pore-mediated increases in host ce I membrane permeability induced by P. aeruginosa, to rnodulate, inhibit and/or prevent activation of broad cellular defence responses induced by P. aeruginoso and/or to rnodulate, inhibit and/or prevent triggering of tissue-damaging inflammation induced by P. aeruginosa. The potency may be measured by any suitable assay known in the art or described herein, such as ep., an in vitro WO 2013/128031 PCT/EP2013/054262 41 cytotoxicit asy (eg. as described by Fan et a. The Journal of infectious diseases 186: 64 73, 2002; Vanceetalnfectionand Imnity 73106 13,2005; ElISohbetat.AmJ Respr Grt Car eMed 18: Incntrast, th "efcacy of the polypeptide of the invention measures the maximum strength o the effect iself at atuatng poypeptid dconcentratos Efcyindicate the maximum response ahievablefo the popeptide ofthe inentin Itrefers to the ability ofap peptide tprduce the 10 The "halieof a polypeptide ofthe inentioncan generally bedefined as desced in prgah o) on page 57 of WO 08/020079 and as mentioned therein refers to the tirne taken for the serurn haflieofapppieo h nvetn can be deemie n a manne knw pee ucha by 15 pharmacokineti analyst Suitabl techniqueswl be cear to the person skilled in the atand myfr sndar handbooksasuc asdenneth et aG(hemia Stabilty of Pharmaceuticals AHandbook fo 2 haracitsJohn Wiley & Sonsnc, 1986) and M Gibaldi and 0 Perron (Eharniacokinetis". Marcel Dekker 2ndev Edition 1982) The term ncreei hal life" o"iceased half-life" arlo as dfned ipaarponag 57ofW 08/2079 and in priuarefe toanraei thet/ et iher wit or without an inre ithe tl2-lha adotheAGo boh. Unless indicated otherwise, the terrn imunoglobuln -whethe used herein to refer to a heavy 2 ca anibod ort onventonal 4-hai antibody-i used aageneralterm tincludebot the full siz antibody the indivdua chisteef wls alparts, doais fr~agmnents theeof includingn but notu mted to antigenbinding domingo f aets suc as domain orV/ donmains, respectively The ter "domn" o polpeptde orprten caused heei rees tafoded prtei s&trutr 0 wic asteabltyt eti istetaytucueieedetl ofterssft e ein Geeally WO 2013/128031 PCT/EP2013/054262 42 rernoved or transferred to other proteins woutls ffntino heande ofteprti and/o of the dornain. (uch as e.g., a chain of aconyentional 4-chain antibody or of a hevy chain antibody) or to a 5 polypeptide that essentially consits of such a globular region. Immunog obulin domains are characterized in that they retain the immunoglobulin fod characteristic of antibody molecules, which consists of a two-layer sandwich of about seven antiparalle beta-strands arranged in two beta-sheets, The terrn "irnmunoglobulin variable domain" as used herein mean n irnmunoglobulin domain to essentially consisting of four "ramework regions" which are referred to in the art and herein below as "ramework region 1"or "FR1" as frameworkk region 2" or "FR2"; as "framework region 3" or 'FR3" and as "framework region 4' or "FR4", respective y; which framework regions are interrupted by three "cornplernentarity determining regions" or "CDRs", which are referred to in the art and herein below as "complementarity determining region 1" or "CDR1"; as 'complementarity determining region 2" or 1 "CDR2"; and as complementarityy determining region 3" or "CDR3", respective y. Thus, the general structure or sequence of an imrnunog obuliin variable domain can be indicated as folows: FR1 - CDR1. FR2 - CDR2 - FR3 - CDR3 - FR4, It is the mrmunoglobulin variable domain s) that confer specificity to an antibody for the antigen by carrying the antigen-binding site The terrn "immunoglobulin single variable dornain", interchangeab y used with 'single variable 20 domain", defines molecules wherein the antigen binding site is present on, and formed by, a sngle immunoglobulin domrnn This sets immunoglobulin single variable domains apart from "conventional' imrnunoglobulins or th eir fragments, wherein two immunoglobulin domains, in particular two variable domains, interact to forrn an antigen binding site. Typicaly, in conventional immunoglobu ins, heavy chamn variable domnan (VH) and a light chain variable domain (VL) interact to form an antigen binding 25 site. In this case, the complementarity determining regions (CDRs) of both VH and VL wIl contribute to the antigen biding site, ie. a total of 6CDRs will be involved in antigen binding site formation. In view oft te above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an lgG, IgMA IgA, lgD or igE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as disuiphide linked Fv or a sc v fragment, or a diabody (all known in the art) 30 derived frorn such conventional 4-chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in thesec caes, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains WO 2013/128031 PCT/EP2013/054262 43 such as light and heavy chain variable domains, ie. by aVH- V! pair of immunoglobulin domamns. which jointly bind to an epitope of the respective ant'gen. In contrast, irmmunoglobulin single variable dornains are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobusin variable domain. The binding site of an immrunog obulin single variable dornain is formed by a single VH/VH H or VL domain. He.nce, the antigen binding site of an immunoglobu in single variab e dornain is forrned by no more than three CDRs. As such, the single variable domain may be a light chain variable dornain sequence (.g., aVL.
sequence) or a suitable fragmrnn thereof; or heavy chain variable dornain sequence (e.g. VHI sequence or VHH sequence) or a suitable fragrnent thereof; as long as it is capable of forming single 1o antigen binding unit (h-e., a functional antigen binding unit that essentIly consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit) In one embodiment of the invention, the immunoglobulin single variable domans are heavy chain variable domain sequences (e.g., a VH-sequence); more specifically, the immunoglobulin single variable 15dornains can be heavy chain variable domain sequences that are derived from a conventional four-chain antibody or heavy chain variable domain sequences that are derived from a heavy chain antibody, For example, the mrmunoglobulin single variable dornain may be a (single) domain antibody (or an arnino acid that is suitable for use as a(single) dornain antibody), a "dAb" or dAb (or an amino acid that i suitab e for use as a dAb) or a Nanobody (s defined herein, and including but not limited to a VHH); 20 other single v able domains, or am suitable fragrnent of any one thereof. In particular, the irmmunoglobulin single variable domain may be a Nanobody*~ (as defined herein) or a suitable fragrnent thereof [Note: Nanobody", Nanobodies* and Nanoclone* are registered trademarks of Ablynx N.V.] For a general description of Nanobodies reference is made to the further description below weI as to the prior art cited heren, suc h a e g described in WO 08/020079 (page 25 16). "HH domains, aiso known as VHHs, V H domains VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin (variable) domain of "heavy chain antibodies" (.e, of "antibodies devoid of light chains"; Harners-Casterman et at. Nature 363-:446-448, 1993) The terrn "VHH domain' has been chosen in order to distinguish these variable dornains frorn the so heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domnans" or"VIH domains") and frorn the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "V 1 domains" or "VL. domains"). For a WO 2013/128031 PCT/EP2013/054262 44 further description of VHH's and Nanobodies, reference is madle to the review article by Muyldermans (Reviews in Molecular Biotechnology 74: 277-302, 2001), as well as to the following patent applications, which are mentioned as general background art: WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrie Uiniversiteit Brussel; WO 94/25591, WO 99/37681, W0 00/40968, Wa 00/43507, WO 00/65057, 5 WO 01/40310, WO 01/44301, EP 1134231 and WO 02/48193 of Unilever; WO 97/49805, WO 01/21817 WO 03/035694 WO 03/054016 and WO 03/055527 of the Vlaams Instituut voor Biotechnologie (VIB); WO 03/050531 of Algonomics N.V. and Ablynx N.V; WO 01/90190 by the National Research Coun ci of Canada W0 03/025020 (= [P 1433793) by the institute of Antibodies;a well as WO 04/041867, WO 04/041862, WO 04/041865, WO 04/041863, Wa 04/062551, WO 05/044858, Wa o6/40153, Wa 10 06/079372, WO 06/122786, WO 06/122787 and WO 06/122825, by Ablynx N.V. and the further published patent applications by Ab ynx N.V. Reference is aiso made to the further prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41-43 of the International application W0 06/040153, which lIst and references are incorporated herein by reference. As described in these references, Nanobodies (in particular VHH sequences and partially humanized 15 Nanobodies) can in particular be characterized by the presence of one or more "Hallrmark residues" n one ormrore of the framework sequencesA further description of the Nanobodies, including humanization and/or camelzation of Nanobodies, as we I as other modifications, parts or fragments, derivatives or "Nanobody fusions", rnultivalent constructs (including some non- imiting examples of liniker sequences) and different modifications to increase the halif- ife of the Nanobodies and their preparations can be found eg. in WO 08/101985 and WO 08/142164. For a further general description of Nanobodies, reference is made to the prior art cited herein, such as .. described in Wa 08/020079 (page 16) "Domain antibodies", also known as "Dab's, "Dornain Antibodies", and "dAbs" (the terms "Domain Antibodies' and 'dAbs" being used as trademarks by the GlaxoSrnithKline group of companies) have 2s been described in epg, [P 0368684, Ward et at (Nature 341: 544-546, 1989), Holt et at (Tends in |Biotechnology 21: 484-490, 2003) and WO 03/002609 as well as for example WO 04/068820, WO 06/030220, WO 06/003388 and other published patent applications of Dornantis Ltd. Dornain antibodies essentially correspond to the VH or VL donains of non-camelid mammralians, in particular hurnan 4-chain antibodies In order to bind an epitope as single antigen binding domain, a~. without being paired with 30 a VL or VH dornain, respective y, specific selection for such antigen binding properties is required, eg. by using libraries of hurnan single VH or VL domain sequences. Domain antibodies have, like VHHs, a WO 2013/128031 PCT/EP2013/054262 45 molecular weight of approximately 13 to approxirmately 16 kDa and, if derived from fully hurnan sequences do not require humanization for e.g. therapeutical use in humans. it should aso be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, single variable domains can be derived frorn certain species s of shark (for example the so-called "IgNAR domains", see for exarnple WO 05/18629), Thus, in the meaning of the present invention, the term "immunoglobutin single variable domain" or "single variab e domain" comprises polypeptides which are derived from a non-human source, preferably acameid, preferably acameld heavy chain antibody They may be humanized, as previously described. Moreover the terrn comprises po ypeptides derived from non-came id sources, e.g. mouse or to human, which have been "camelized", as e.g, described in Davies and Riechrnann (FEBS 339; 285-290, 1994; Biotechnolt 13: 475-479, 1995; Prot, Eng. 9: 531-537, 1996) and Riechmann and Muy derrmans ( Irnmunot. Methods 231: 25-38, 1999). The amino acid residues of a VHH domain are numbered according to the general numbering for Vs domains given by Kabat et al ("Sequence of proteins of imrnunological interest", US Pub~c Health 15Services, NIH Bethesda, MD, Publication No. 91), as app ied to VHH domains from Came ids, as shown e ., in Figure 2 of Riechmann and Muyldermans (. ImmunoL Methods 231: 25-38, 1999). Alternative methods for numbering the amino acid residues of Vs domains, which methods can also be applied in an analogous manner to VHH domains, are known in the art. However, in the present description, claims and figures, the numbering according to Kabat applied to VHH domains as described above will be It should be noted that - as is well known in the art for VH dornains and for VHH domains - the total nurnber of amino acid residues in each of the CDRs may vary and rnay not correspond to the total number of amino acid residues indicated by the K abat numbering (that is one or more positions according to the Kabat numbering rnay not be occupied in the actual sequence, or the actual sequence 25 may contain rnore amino acid residues than the number allowed for by the Kabat numbering) This means that, general, the numbering according to Kabat rmay or may not correspond to the actual numbering of the amnno acid residues in the actual sequence. The totaI nurnber of amino acid residues in aVH domain and a VHH domain will usuaIly be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaher and longer sequences may also be suitable for the Determination of CDR regions may also be done according to different rnethods. ln the CDR determination according to Kabat, FR1 of aVHH comprises the amino acid residues at positions 1-30, WO 2013/128031 PCT/EP2013/054262 46 CDR1 of aVH-H comprises the arnino acid residues at positions 31-35, FR2 of a VHH comprises the amino acids at positions 36-49, CDR2 of a VHH comprises the amino acid residues at positons 50-65 FR3 ofa VHH cornprise the arnino acid residue at positions 66-94, CDR3 of a VHH comprises the arnino acid residues at positions 95-102, and FR4 of a VHH comprises the amino acid residues at positions 103-113. 5In the present application, however, CDR sequences were determined according to Kontermann and Dabel (ds., Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51, 2010) According to this method, FR1 comprises the arnino acid residues at positions 1-25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the arnino acids at positions 36-49, CDR2 comprises the mrino acid residues at positons 50-58, FR3 comprises the amino acid residues at to positions 59-94, CDR3 comprises the arnino acid residues at positions 95-102, and FR4 comprises the arnino acid residues at positions 103-113, Immunoglobulin single variab e domains such as Domain antibodies and Nanobodies (including VHH dornains) can be subjected to humanization. In particu ar, humanized immrunoglobulin single variable domains, such as Nanobodies includingg VHIH domains) may be immunoglobulin single variable 1s domains that are as generally defined for in the previous paragraphs, but in which at least one amino acid residue is present (and in particular, in at least one of the framework residues) that is and/or that corresponds to a humanzing substitution (as defined herein) Potentially useful humanizing substitutions can be ascrtained by comparing the sequence of the framework regions of a naturally occurring VH< sequence with the corresponding framework sequence of one or rnore closely related human VH 20 sequences, after which one or more of the potentially useful hurnanizing substitutions (or combinations thereof) thus determined ca be introduced into said Vs sequence (in any manner known per se, a further described herein) and the resulting humanized Va, sequences can be tested for affinity for the target, for stability, for ease and level of expression, and/or for other desired properties. in this way, by means of a limited degree of tria a nd error, other suitable humanizing substitutions (or suitable 2 combinations thereof) can be determined by the skilled person based on the disclosure herein. Also, based on the foregoing, (the framework regions of) an immunoglobulin single variable dornain, such as a Nanobody (including VHH domains) may be partially humanized or fully humanized. Immunoglobulin sin ge variable domains such as Domain antibodies and Nanobodies (including VHH dornains and humanized VHH domains), can also be subjected to affinity maturation by introducing 30 one or more alterations in the amino acid sequence of one or rnore CDRs, which a terations result in an improved affinity of the resulting imrnunoglobulin single variable domain for its respective antigen, as compared to the respective parent rnolecule. Affinity-matured immunoglobulin single variable domain WO 2013/128031 PCT/EP2013/054262 47 molecules of the invention may be prepared by methods known in the art, for example, as described by Marks et at (Biotechnology 10:779-783, 1992)1, Barbas, et al (Proc. Nat. Acad. Sci USA 91: 3809-3813, 1994), Shier et al (Gene 169: 147-155, 1995), Yelton et at. (imrunol 155: 1994-2004, 1995), Jackson et al (J. immunol. 154: 3310-9, 1995), Hawkins et at (L Iviol BioL. 226: 889 896 1992), Johnson and 5Hawkins (Affinity maturation of antibodies using phage display, Oxford University Press, 1996). The proces of designing/selecting and/or preparing a polypeptide, starting frorn an immunoglobutin single variable domain such as aDornain antibody or aNanobody, is also referred to herein as "ormatting" said immunoglobulin single variable domain; and an immunoglobulin single variable domain that is made part of a polypeptide is said to be "formatted" or to be "in the format of" 1o said polypeptide. Exarn pes of ways in which an inunog obulin sing e variable dornain can be formatted and exarnples of such forrnats will be clear to the skilled person based on the discdosure herein; and such formatted immrunoglobulin single variable domain form further aspect of the invention. For example, and without limitation, one or more immunoglobulin sing e variable domains rmay be used as a "binding unit" "binding dornain" or "building block" (these terms are used interchangeable) for 15the preparation of a polypeptide, which may optionally contain one or more further immunoglobulin single variable domains that can serve as a binding unit (i.e., against the same or another epitope on PcrV and/or against one. or more other antigens, proteins or targets than PerV). Monova lent polypeptides comprise or essential y consist of only one binding unit (such as e.g., imrmunoglobulin single variable domains). Polypeptides that cornprise two or more binding units (such as 20 e., immunoglobulin single variable domains) will also be referred to herein as " multivalent" polypeptides, and the binding units/irnmunog obulin single variable domains present in such polypeptides will also be referred to herein as being in a"multivalent format" For exam ple a "bivalent" polypeptide rmay comprise two imrnunoglobu in single variable domains, optionally Inked via a linker sequence, whereas ' triva ent' polypeptide may comprises three immunoglobulin single variable 25 domains, optionalv linked via two linker sequences; etc. In a multivalent polypeptide, the two or more immunoglobuln single variab e domans may be the sarne or different, and may be directed against the same antigen or antigenic determinant (for example against the same part(s) or epitope(s) or against different parts or epitopes) or may alternatively be directed against different antigens or antigenic determinants; or any suitable combination thereof. 30 Polypeptdes that contain at least two binding units (such as e. irnmunoglobutin single variable domains) in which ati est one binding unit is directed against a first antigen (i.e PcrV) and at least one binding unit is directed against a second antigen (i., different from PcrV) will also be referred to as WO 2013/128031 PCT/EP2013/054262 48 amultispecific"~ polypeptides, and the binding units (such as e.g., immunoglobulin single variable domains) present in such polypeptides will also be referred to herein as being in a "multispecific format". Thus, for example, a "bispecific" polypeptide of the invention is a polypeptide that comprises at least one imnmunoglobulin single variable domain directed against a first antigen (le, PcrV) and at least one further 5 immnunoglobulin single variable domain directed against a second antigen (iae., different from PcrV), whereas a "trispecific" polypeptide of the invention is a polypeptide that comprises at least one immunoglobulin single variable domain directed against a first antigen (ihe, PcrV), at least one further immunoglobulin single variable domain direct ed against a second antigen (h e, different from PcrV) and at least one further immunoglobulin single variable domain directed against a third antigen (he, is different from both Pcaind thesecond antienetc "Mliaratopicipolypeptides"',suh as e, bipraopi poypeptides" or "triparatopi polypeptides comprise oreenilyconss f two or moe dining units thateach have different paratope (as will be frther described hrise chape on mulivaln polypepies of the invention) is Monovalent polypeptides of the invention The present invention provides stretches of arrino acid residues (SEQ ID NOs: 2037, SEQ ID NOs: 3&5Eand SEQ ID NOs 57-75; able A- that ar particularly suited or binig to PcrV. These stretches inenini priclr nsuha a tha thyfr pato)teantienbinding site of he polypeptide 20 f heinvnton Tee strethso amn acid rsidue have been generaed a COR sequences of hev chinatiode o Hseuecs ha er aised agais PcV Ths stetches of aioacid residues aeaso referred to here as "CPsequences) of te inento"(eas "CURi sene s)the inention" "CDRsequence(s) othe invention"and "CR3 sequence~s of te invention',respectvey) Ishoud however be noted that there inention in ts broadest sense isnot irmited to aspecifi 25 strutua roe orfuntio that these stretchso amn acd residue ma hav inapoypeptide ofthe inetin as long as these stretches of amin ad residues allow thepolyepe of th inetion to r m re frher amno aci sequences such tha the entire polypepid fom a idng don ad/o WO 2013/128031 PCT/EP2013/054262 49 binding unit that is capable of binding to PcrV, It should however also be noted that the presence of only one such CDR sequence in a monovaent polypeptide of the invention may by itself already be sufficient to provide the rnonova ent polypeptide of the invention the capacity of binding to PcrV; reference is for example again made to the so-called "Expedite fragrments" described in WO 03/050531, a) Thsenap ii)btnn mif asec the mooaetpoppiefftem to a b) stretches of arino acid sequences that have at east 80% amino acid identity with at least one of 10 the arnino acid sequences of SEQ 0D NOs: 20-37; c) stretches of amino acid sequences that have 3,2, or 1arnino acd difference with at least one of the amino acid sequences of SEQ ID NOs: 20-37; a nd/or 15 d) SEQ iD NOs: 38-5 e) stretches of amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 38-56; f) stretches of amino acid sequences that hve 3, 2 or 1 amino cd different with at least one of the - CDR3 sequences g) SEQ ID NO: 5 7-75 h) stretches of amino aid sequences that have at least 80% amno acid identity with at least one of the amino acid sequences of SEQ 0D NOs 57-75; 25 i) stretches of amino acid sequences that have 3, 2, or 1 amino acid dfference with at least one of the amino acd sequences of SEQ ID NOs: 755 Monovalent polypeptides comprising one or more of the above specified stretches of amino acid residues show improved properties such as e.g., improved binding characteristics (suitably measured and/or expressed as aKr-value (actual or apparent) a KA-value (actual or apparent), a k,,-rate and/ora 30 kg-rate, or alternatively as an ICso value, a- further described herein), improved affinity and/or irnproved avidity for PcrV and/or improved efficacy and/or potency for neutralizing PcrV.
WO 2013/128031 PCT/EP2013/054262 50 More in particular, the rnonovalent polypeptides of the invention comparing one or more of the above specified stretches of amino acid residues can bind to protein PcrV with an affinity (suitably measured and/or expressed as a K 5 -value (actual or apparent), a Kr-value (actual or apparent), a ker-rate and/or a~ rate, or alternatively as an 1C 5 o value, as further described herein) preferably such that they: 5-~ bind to PcrV with dissociation constant (Kj) of 1000 nM to 1 nM or less, preferably 100 nM to 1 n|M or less, more preferably 15 nM to 1 nM or even 10 nM to 1 nM or less; and/or such that they: - bind to PcrV with a ko-rate of between 104 M 's" to about 10 M a pree ably betw wen 10 M s and 107 M~'s, more preferably about 106|M s1 or more; io ado such that the - bind to PcrV with akg rate between 10 s 4 (t=0.69 s) and 10 s (providing a near irreversible complex with ato, of multiple days), preferably between 104 so and 104 s4, or lower. Sorne preferred|IC o values for binding of the monovalent polypeptides of the invention to PcrV wi I 15 Assays to determine the IC o include binding in ELISA or, rnore preferably, cytotoxicity assays such as the TTSS-dependent cytotoxicity assay described by Frank et al (The Journal of infectious disea s 186: 64-73, 2002), Vance et at, (nfection and Immunity 73: 1706-1713, 2005), El Solh et al (Am. I Respir. Crit. Care Med. 178: 513-519, 2008) modifications of these assays such as e.g. described in Example 4.4, or acytotoxicity assay with human lung epithe al cells (A549 ce s) as described in Exarnple 7.2, and 20 mdfctosteef For example, in a TTSS-dependent cyotoxicity assay with P3X6 ' ells as the target atan average MOI of 2.8, the rnonovalent polypeptides of the invention may have ICso values between 1 nM and 10000 nM, between 5 nM and 1000 nM, preferably between 5 nM and 500 nM, more preferably between S nM and 200 nM, such as between S nM and 50 nM orles 25 In such a TTSS-dependent cytotoxicity assay, the monovalent polypeptides of the invention may have an efficacy (% inhibition; see Example 4.4) of 50% or more, preferably 90% or more, such as 100%. ln particular, amonovalent polypeptide of the invention rmay be amon ovalent polypeptide that comprises one antigen binding site, wherein said antigen binding site comprises at least on. stretch of amino acid residues that is chosen frorn the group consisting of the CDRi sequences, CDR2 sequences so and CDR3 sequences as described above (or any suitable combination thereof). In a preferred aspect, however, the monovalent polypeptide of the invention comprises more than one, such as two or more stretches of amino acid residues chosen from the group consisting of the CDR1 sequences of the WO 2013/128031 PCT/EP2013/054262 51 invention, the CDR2 sequences of the invention and/or the CDR3 sequences of the invention. Preferably, the monovalent polypeptide of the invention comprises three stretches of arnino acd residues chosen from the group consisting of the CDR1 sequences of the invention, the CDR2 sequences of the invention and the CDR3 sequences of the invention, respectively. The combinations of CDR's that are mentioned 5 herein as being preferred for the monovallent polypeptildes of the invention are listed in Table A-6 It should be noted that the invention is not lirnited asto the origin of the monovalent polypeptide of the invention (or of the nucleic acid of the invention used to express it) nor as to the way tha the monovalent polypeptide or nucleic acid of the invention is (or ha been) generated or obtained. Thus, the monovalent polypeptides of the invention may be naturally occurring monovalent polypeptides 10 (frorn any suitable species) or synthetic or smi-synthetic monovalent polypeptides Furthermore, it will also be cdear to the skiled person that it is possible to "graft" one or rnore of the CDR's mentioned above onto other "scaffolds", including but not limited to human scaffolds or non immunoglobulin scaffolds. Suitable scaffolds and techniques for such CDR grafting will be lear to the ski led person and are well known in the art, see for example US 7,180,370, WO 01/27160, EP 0605522, 15EP 0460167, US 7,054,297, Nicaise et al. (Protein Science 13: 1882-1891, 2004), Ewert et al (Methods 34: 184-199, 2004), Kettleborough et aL. (Protein Eng. 4: 773-783, 1991), 0'Brien and Jones (Methods Mol Biot. 207: 81-100, 2003), Skerra (2. Mol Recognit. 13 1674187, 2000) and Saerens et al (2. Mol BioL, 32: 597-607, 2005) and the further references cited therein, For example, techniques known per se for grafting mouse or rat CDR's onto hurnan frameworks and scaffolds can be used in an analogous rnanner 20 to provide chimeric proteins comprising one or rnore of the CDR sequences defined herein *or the monovalent polypeptides of the invention and one or more human framework regions or sequences. Suitable scaffolds for presenting arnino acd sequences wi I be clear to the ski led person, and for example comprise, without limitation, to binding scaffolds based on or derived from irnmunoglobulins (i.e. other than the immunoglobulin sequences already described herein), protein scaffolds derived frorn 25 protein A dornains (such asAffibodies ), tendamistat, fibronectin, lipocalin, CTLA-4, T-cell receptors, designed anikyrin repeats, avimers and PDZ domains (!inz et at Nat. Biotech., 23: 1257, 2005), and binding moieties based on DNA or RNA including but not limited to iDNA or RNA aptarners (Ulrich et at, Comb. Chem. High Throughput Screen 9: 619-32, 2006) in said monovalent polypeptides of the invention, the CUR's may be linked to further arnino acid 30 sequences and/or may be linked to each other via amino acid sequences, in which said amino acid sequences are preferably framework sequences or are arnino acid sequences that act as framework sequences, or together forrn a scaffold for presenting the CDR's, WO 2013/128031 PCT/EP2013/054262 52 According to preferred, but non-limiting ernbocdiment, the rnonovalent polypeptides of the invention cornprise at least three CDR sequences [nked to at last two framework sequences in which preferably at least one of the three CDR sequences is a CDR3 sequence, with the other two CDR sequences being CDR1 or CDR2 sequences, and preferably being one CDR1 sequence and one CDR2 s sequence. According to one specifically preferred, but non-l[miting embodiment, the rmonovalent polypeptides of the invention have the structure FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, in which CDR1, CIDR2 arnd CDR3 are as defined herein for the monovalent polypeptides of the invention, and FR1, FR2, FR3 and FR4 are framework sequences. In such a monova ent polypeptide of the invention, the framework sequences may be any suitable framework sequences, and exarnples of suitable framework to sequences wIu be clear to th skil ed person, for exarnple on the basis the standard handbooks and the further disclosure and prior art mentioned herein. Accordingly, the present invention also relates to a rnonovalent polypeptide against PcrV which essentially consists of 4 framework regions (R1 to FR4, respectively) and 3 complementarity determining regions (CDR1 to CDIR3 respectively), in which: 15- CDR1 is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NOs: 20-37; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 20-37; c) amino acid sequences that yae 3, 2, or 1 mno a dffe nc with a east oneoth min a sequences of SEQ |ID NOs: 20-37; and/or - CDR2 is chosen from the group consisting of d) the amino acid sequences of SEQ ID NOs: 38-S6 e) amino acid sequences that have at least 80% amino acid identity with at least one of the arnino acid 25 sequences of SEQ ID NOs: 38-S6 f) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the arnino acid sequences of SEQ ID NOs: 38-56; and/or - CDR3 is chosen from the group consisting of: so g) the amino aid sequences of SEQ ID NOs: 57-75; h) arnino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 57-75; WO 2013/128031 PCT/EP2013/054262 53 I) amino acid sequences that have 3, 2, or aino c'd d n sequences of SEQ ID NOs: 57-75. rnonovalent polypeptide against PcrV, which consists of 4 framework regions (FR1 to Rrepcily a nd 3cormpementarity determining regions (CDR1 to CIDR3 respective y), in which: - CDR1 is chosen from the group consisting of: a) the arnino acd sequences of SEQ ID NO: 20-37; b) arnino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 20-37; 10 c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino aci sequences of SEQ ID NOs: 20-37; and - CDR2 is chosen from the group consisting of: d) the amino acid sequences o EQ ID NOs: 38-56 5 e) amino acid sequences that have at least 80% arnino acid identity with at least one of the arnino acid sequences of SEQ ID NOs: 38-56 f) arnino acid sequences that have 3, 2, or 1 armino acid difference with at least one of the amino aci sequences of SEQ ID NOs: 38-56; and 20- CDR3S is chosen from the group consisting of: g) the arino acid sequences of SEQ ID NOs: 5775; h) amino id sequence that have at least 80% amno acid identity with at least one of the minor acid sequences of SEQ ID NOs: 57-75; The invention also relates to a monova ent polypeptide in which the CDR sequences have at Ieast 70% amino acid identity, preferably at least 80% amino acid identity, more preferably at least 90% amino acid identity such as 95% mrino acid identity or rnore or even (essentially) 100% mrino acid identity with the CDR sequences of at least one of the amino acid sequences of SEQ ID NOs: 1-19. 30 In one speific but non-iting aspect, the monovalent polypeptide of the invention may be a monovalent polypeptide that comprises an immunoglobulin fold or amronovalent polypeptide that, under suitable conditions (such as physio ogical conditions) is capable of forming an immunoglobulin fold WO 2013/128031 PCT/EP2013/054262 54 (i.e., by folding) Referen ce is inter all inade to the review by Halaby et al (J Protein Eng. 12 563-71, 1999). Preferably, when properly folded so as to forrn an immunoglobulin fold, the stretches of arnino acid residues may be capable of properly forrning the antigen binding site for binding PcrV, Accordingly, the framework sequences are preferaby (a suitable combination of) irnmunoglobuln afrarnework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by sequence optimization such as humanization or came ization) For example, the framework sequences may be framework sequences derived from an immunoglobulin single variable domain such a light chain variable domain (eg a VWsequence) and/or frornm heavy chain variable domain (e.g., a VHsequence). In one particularly preferred aspect, the framework sequences are either io framework sequences that have been derived from aVaH-sequence (n which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences that have been invention isan immunoglobulin single variable domain such as a Domain antibody or an amino acid 15 sequence that i suitable for use as a domain antibody); is single dornain antibody (or an arnino acid that is suitable for use as single domain antibody) is a "dAb" (or an amino acid that is suitab e for use as a dAb); or is a Nanobody* (inc uding but not limited to Vas) Again, suitable framework sequences will be clear to the skilled person, for example on the b is thsandoar h an dbok and thne furhe disclosure and prior art mentioned herein. 20 In particular, the framework sequences present in the monovalent polypeptides of the invention may contain one or rnore of Ha Imarik residues (as defined in WO 08/020079 (Tables A-3 to A-8)), such that the monovalent polypeptide of the invention is Nanobody. Some preferred, but non-Imiting examples of (suitable combinations of) such framework sequences w I becorne clear from the further disclosure herein (see e.g., Table A-6). General y, Nanobodies (in particular Ve, sequences arnd partially 25 humanized Nanobodies) can in particular be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences (as e.g., further described in WO 08/020079, page 61, line 24 to page 98, line 3) More in particular, a Nanobody can be an immunoglobulin single variable domain and/or 30 FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 WO 2013/128031 PCT/EP2013/054262 55 in which FR1 to FR4 refer to framework regions to 4, respectively, and in which CDR1 oCR rfrt the corn plernentarity determining regions 1 to 3, respectively, and which: h) yae at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 1-19 (ee Table A-4), in which for the purposes of determining the degree of arnino aci 5 ~identity the amino acid residues that forrn the CDR sequences are disregarded. In this respect, reference is also made to Table A-6, which Iists the framework 1 sequences (SEQ ID NOs 76-80), framework 2 sequences (SEQ ID NOs: 81-93), framework 3 sequences (SEQ ID NOs: 94-112) and frarnework 4 sequences (SEQ ID NO: 113-117) of the immrunoglobulin single variable domains of SEQ ID NOs: 1-19 (see Table A-4); or to ii) combinations of framework sequence as depicted in Table A-6; and in which: iii) preferably one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are chosen from the Hallmark residues mentioned in Table A-3 to Table A-8 of WO 08/020079, 15 ~In a preferred aspect, the present invention provides an immunoglobulin single variable domain or monovalent polypeptide that is selected frorn any of SEQ 0D NOs: 1-19. The present invention also provides rnonovalent polypeptides that belong to the sarne epitope bin as any one of the immunogobulin single vriab dorainswith SEQlD NOs: 1-19.Accordingly the present invention also relates to monovalent polypeptides directed against PcrV, that cross-blocks the 20 binding to PcrV of at least one of the imrnunog obulin single variable domains with SEQ ID NOs: 1-19 and/or that are cross-blocked frorn binding to PcrV by at least one of the immunoglobulin single variable dornains with SQ ID NOs: 1- 9. Againa, such monovalent polypeptlides may be an immunoglobulin single variable domain derived in any suitable manner and frorn any suitable source, and may for example be naturally occurring VH 25 sequences (i., frornm suitable species of Camelid) or synthetic or semi-synthetic amino acid sequences, including but not limited to "humanized" (as defined herein) Nanobodies or VHH sequences, "camelized" (as defined herein) immunoglobulin sequences (and in particular camelized heavy chain variable dornain sequnces, as well as Nanobodies that ave been obtied by techniques such a- affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR so grafting, veneering, combining fragments derived from different irnmunog obulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing as further WO 2013/128031 PCT/EP2013/054262 56 described herein. Also, when an imrnunoglobu. in sing e variable domain comprises a Ve sequence, said immunoglobulin single variable dornain may be suitably humanized, as further described herein, so as to provide one or more further (partially or fully) humanized immrunoglobulin single variable dornains of the invention. Sirmilary, when an immunoglobulin single variable dornain comprises a synthetic or semi 5synthetic sequence (such as a partially humanized sequence)}, said immunoglobulin single variable dornain may optionally be further suitably hurmanized, again as described herein, again so as to provide one or more further (partially or fully) humanized imrnunoglobulin sing e variable dornains of the These monovalent polypeptides of the invention, and in particular the immunoglobulin single 1o variable domains comprising the CDR sequences of the invention are particu arny suited for use as building block or binding unit for the preparation of multivalent polypeptides. According y, the rnonovalent polypeptides of the invention that bind PcrV can be in essentially y isolated form (as defined herein), or they rnay forrn part of a protein or polypeptide, which rmay cornprse or essentially consist of one or rnore monovalent polypeptides that bid PcrV and which rmay optionally is further comprise one or more further amino acid sequences (all optionally linked via one or more suitable linkers). The present invention also relates to a protein or polypeptide that comprises or essentially consists of one or rnore rnonovalent polypeptides of the invention (or suitable fragments thereo The one or more monovalent polypeptides of the invention are thus used as a binding uni ory 20 building block in such a protein or polypeptide, so as to provide a monovalent, multivalent or multiparatopic polypeptide of the invention, respectively, a las described herein. The present invention thus also re ates to a polypeptide which is a monovalent construct comprising or essential y consisting of one monovalent po ypeptide of the invention. The present invention thus also relates to a polypeptide which is a multivalent polypeptide, such as e.g., a bivalent or trivalent polypeptide comprising or 25 essentially consisting of two or more monovalent polypeptides of the invention (for rnultivalent and multispecific polypeptides containing one or more VHIH domains and their preparation, reference is also made to Conrath et al. , Biol Chem, 276: 7346-7350, 2001, as well as to for example WO 96/34103, WO 99/2221 and WO 2010/115998) The present inventors furthermore found that immunoglobulins belonging to certain epitope bins are particularly suited for binding to PcrV, neutralization of P. oeruginosa and/or as binding unit for the WO 2013/128031 PCT/EP2013/054262 57 preparation of the rnultiparatopic, such as e~. biparatopic or triparopic, polypeptides of the invention. Preferred imrnunoglobulins include immrunoglobulins (such as heavy chain antibodies, conventional 4 chain antibodies (such as lgG, igM, IgA, IgD or igE moleculess, Fab fragments F(ab')2 fragments, Fv fragments such as disuiphide linked Fv or scFv fragments, or diabodies derived from such conventional 4 achain antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding dornains or fragments such as mrnunoglobulin single variable domains), monovalent polypeptides of the invention, or other binding agents) that belong to epitope bins 1 or 3 a further defied Accordingly, in a first aspect, the present invention relates to an immunoglobulin that belongs to 10epitope bin 1. Epitope bin 1 encompasses a family of imrmunoglobulins (including monovalent polypeptides of the invention) that have the same or over apping binding specificity based on cross com petition (cross-blocking) of the imrmunog obulins, More particularly, immunoglobulins belonging to epitope bin 1 cross-block the binding to PcrV of at least one of the immunoglobulin single variabe domrnn with SEQ ID NO: 3-10 and/or are cross-blocked from binding to PcrV by at least one of the im rmunoglobulin single variable dornains with SEQ ID NOs: 3-10, The imrnunoglobu ins belonging to epitope bin 1 are expected to bind to the sarne, closely related and/or overlapping epitopes. More particu arly, the irmmunoglobulins belonging to epitope bin 1 bind full length PcrV (SEQ ID NO: 159) while showing reduced (30-90% as compared to full length PcrV) or no (lower than 30% as compared to full length PcrV) binding to chimera 4 (SEQ ID NO: 202) and chimera 2o (S iD NO 204) Preferred immunoglobulins belonging to epitope bin 1 include monovalent polypeptides of the invention (as defined above) in which: - CDR1 is chosen fom the group consist of a) the amino acid sequences of SEQ I|D NOs: 22-28; 25 b) amino acid sequences that have at least 80% amnno acid identity with at least one of the amino acid sequences SEQ ID NOs: 22-28; c) arnino acid sequences that have 3, 2, or 1 arnino acid d ifference with at least one of the amino acid sequences of SEQ ID NOs: 22-28; and/or 30 - CDR2 is chosen from the group consisting of: d) the amino acid sequence of SEQ ID NOs: 40-47; WO 2013/128031 PCT/EP2013/054262 58 e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 40-47; f) amino acid sequ ences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 40-47; and/or - CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NOs: 59-6 h) amino acid sequences that have at least 80% arnino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 59-66; 0o ') amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 59-66 More partic uarty, monovalent polypeptides in which: -CDR1 is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NOs: 22-28; 15 b) amino acid sequences that have at least 80% arnino acid identity with at least one of the amino acild sequences of SEQ ID NOs: 22-28; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the mrino acid sequences of SEQ ID NOs: 22-28 and 20 - CDR2 is cos fo t rup onss of d) the amino acid sequences of SEQ ID NOs: 40-47; e) amino acid sequences that have at least 80% amino acid identity with at least one of the arnino acid sequences of SEQ ID NOs: 40-47; sequences of SEQ ID NOs: 40-47' and - CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ llD NOs: 59-66 h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino aid sosequences of SEQ ID NOs: 59-66; i) amino acid sequences that have 3, 2, or- 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 59-66 WO 2013/128031 PCT/EP2013/054262 59 More particularly th present invention relates to monovalent polypeptides of the invention that belong to epitope bin 1, in which the CDR sequences of said monovalent polypeptides have at least 70% arnino acid identity, preferably at least 80% arnino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more, or even (essentially) 100% amino acid identity with the 5 CDR sequences of at least one of the mrnunoglobufn single variable domains with SEQ 1D NOs: 3-10. Preferred monovalent polypeptides of the invention belonging to epitope bin 1 are se ected from any of SEQ ID N: 3 In another aspect, the present invention relates to an immunog obin that belongs to epitope bin 2. Epitope bin 2 encompasses a family of immrunoglobulins that have the sarne or overlapping binding 10 specificity, based on cross-competition (cross-blocking) of the immunoglobulns More particularly irnrunoglobulins belonging to epitope bin 2 cross-block the binding to PcrV of at leat one of the immunoglobulin single variable domains with SEQ ID NOs: 1 and 2 and/or are cross-blocked frorn bindng to PrV by at least one of th immunoglobulin single variable domains with SEQ D NOs: 1 and 2 The immunoglobulin belonging to epitope bin 2 are expected to bind to the mare, close y related 15 and/or overlapping epitopes. More particularly, the irnmunoglobulins belonging to epitope bin 2 bind to full length PcrV (SEQ IlD NO: 159), whIle they show reduced (30-90% as compared to full length PcrV) or no (below 30% as cornpared to full Iength PcrV) binding to chimera 7 (SEQ ID NO: 205). Preferred immrunoglobuhins belonging to epitope bin 2 nclude monovalent polypeptides of the invention defined above in which: 20- CDR1 is chosen frorn the group consisting of: a) the amino acid sequences of SEQ ID NOs: 20-21; b) amino acid sequences that have at least 80% amino acid identity with at least one of the arnino acid ence of SEQ N~s 20-21; c) amino acid sequences that have , or 1 amino a difere e it a le one of the amno ai 25 sequences of SEQ ID NO: 20-21; and/or -CDR2 is chosen frorn the group consisting of: d) the amino acid sequences of SEQ ID NOs: 38-39; e) amino acid sequences that have at least 80% arnino acid identity with at least one of the mrino acid so sequences of SEQ ID NOs: 38-39; f) mrino acid sequences that have 3, 2, or 1 arnino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 38-39; WO 2013/128031 PCT/EP2013/054262 60 and/cr -CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NOs: 57-58; b) amino cid sequences that have at lea-t 80% amino acid identity with at least one of the amino acid a sequences of SEQ ID NOs: 57-58; )amino cid sequences that have 3, 2, or 1 amino acid difference with at leas one of the amino acid sequences of SEQ ID NOs: 57-58. More particularly, rnonovalent polypeptidesn which: -CDR1 is chosen frorn the group consisting of: 10 a) the amino acid sequences of SEQ ID NOs: 20-21 b) amino acid sequence that have t east 80% amino acid identity with at least one of the amino cid sequences of SEQ ID NOs: 20-21 c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the arnino acid sequences of SEQ ID NOs: 20-21; and -CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NOs: 38-39; )aino acid sequences that have at least 80% arino acid identity with at leastone of the mno acid sequences of S0Q ID NOs: 38-39; 2o f) arnino acid sequence that have 3, 2, or 1 amino acid differenc-e with at least one of the anino acid sequences of SEQ ID NOs: 38-39; and -CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ lD NOs: 57-58; 25 h) arnno acid sequences that have at least 80% amnno acid identity with at least one of the amino acid sequences of SEQ ID NOs: 57-58; )amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 57-58. More particularly, the present invention relates to monovalent polypeptides of the invention that so belong to epitope bin 2, in which the CDR sequences of said monovalent polypeptide have at least 70% amino acid identity, preferably at least 80% amino acid identity, rnore preferably at least 90% amino acid WO 2013/128031 PCT/EP2013/054262 61 identity, such as95% amino acid identity or more or even essentiallyy) 100% amino acid identity with the CDR sequences of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 1 and 2. Preferred monovalent polypeptides of the invention belonging to epitope bin 2 are selected frorn any of SE NO 1 and2 5 In another aspect, the present invention relates to an irnmunog obulin that belongs to epitope bin 3. Epitope bin 3 encompasses a family of immunog obulins that have the same or overlapping binding immunoglobulin belonging to epitope bin 3 cross-bloc k the binding to PcrV of at least one of the immunoglobulin single yariable domain with SEQ ID NOs: 11 and 12 and/or are cross-blocked frorn to binding to PcrV by at least one of the immunoglobulin single variable domains with SEQ ID NOs: 11 and 12. The imrnunoglobulins belonging to epitope bin 3 are expected to bind to the same, closely related and/or overlapping epitopes More particularly, the immunoglobu ins belonging to epitope bin 3 bind to full length PcrV (SEQ ID NO: 159), while they show reduced (30-90% as compared to full Iength PerV) or 15 no (below 30% as. compared to full length PcrV) binding to chirnera 2 (SEQ ID NO: 200). Preferred immrunoglobulins belonging to epitope bin 3 include rmonovalent polypeptides of the invention (as defined above) in which: -CDR1 is chosen from the group consisting of: a)the amino acid sequences of SEQ ID NOs: 29-30; 20 b) amino acid sequences that have at least 80% amino acid identity with at least one of the arnino acid sequences of SEQ ID NOs: 29-30; c) amino acid sequences that have 3,2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 29-30; and/or 25 -CDR2 is chosen from the group consisting of: d) th e amino acid sequences of SEQ ID NOs: 48-49; e) amno acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 48-49; f) arnino acid sequences that have 3,2, or 1 mrino acid difference with at least one of the amino acid so sequences of SEQ ID NOs: 48-49; and/or - CDR3 is chosen from the group consisting of: WO 2013/128031 PCT/EP2013/054262 62 g) the amino acid sequences of SEQ ID NOs: 67-68; h) mino acid sequences that have at east 80% amino acid identity with at least one of the amino acid sequences of SQ ID NOs: 67- 8; i) amino acid sequences that have 3, 2, or 1 arnimno acd difference with at least one of the minor cid sequences of SEQ ID NOs: 67-68. More particularly, rnonovalent polypeptides in which: - CDRi is chosen from the group consisting of: a)the amino acid sequences of SEQ ID NOs: 29-30; b) arnino acid sequences that have at least 80% amino acid identity with at least one of the arnino acid 10 sequences of SEQ ID NOs: 29-30; c) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the arnino acid sequences of SEQ ID NOs: 29-30; and - CDR2 is chosen from the group consisting of: 15 d) the amino acid sequences of SEQ ID NOs: 48-49; e) amino acid sequences that have at least 80% arnino acid identity with at least one of the amino acid sequen ces of SEQ ID NOs: 48-49; f)amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 48-49; 20 and - CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NOs: 7-68; h) amino acid sequences that have at least 80% amino ac'd identit wit h at least one of the amino acid sequences of EQ ID NOs: 67-68 25 i) amino acid sequences hat have 3 o amno aci difiern ith a a e o nh an aci sequencms of SEQ ID NO : 67-68. More particularly, the present invention relates to rnonovalent polypeptides of the invention that belong to epitope bin 3, in which the CDR sequences of said monovalent polypeptide have at least 70% amino acid identity, preferably at least 80% amino acid identity , more preferably at least 90% amino acid so identity, such as 95% amino acid identity or more, or even (essentially) 100% amino acid identity with the CDIR sequences of at least one of the immunoglobulin sing e variable domains with SEQ ID NOs: 11 and 12.
WO 2013/128031 PCT/EP2013/054262 63 Preferred rnonovalent polypeptides of the invention belonging to epitope bin 3 are selected from SEQ ID NOs: 11 and 12. 5The invention further relates to a multivalent polypeptide (also referred to herein asa "multivalent polypeptide(s) of the invention") that comprises or (essentially) consists of two or more immunoglobulin single variable domains (or suitable fragments thereof directed against PcrV. The multilva ent polypeptide of the invention preferably is a rmultiparatopic polypeptide (also referred to herein as "multiparatopic polypeptide(s) of the invention" ),such as e~g., {a "biparotopic polypeptides) of the 1 invention" or "triparatopic polypeptide(s) of the invention", T he te rm "multiparatopic" (antige n-)bi ndi ng rnolecule or 'multiparotopic" polypeptide as used herein shal rnmean a polypeptide comprising at least two (Le. two or rnore) immunoglobu in single variable domains, wherein a "first" immunoglobulin sing e variab e domain is directed against PcrV and a "second" imnrunoglobu in single variable dornain is directed against PcrV, and wherein these 'first" and "second" immunoglobulin single variable domains 15 havey different paratope. Accordingly, the multiparatopic polypeptide comprises or consists of two or more irmmunoglobulin single variable domains that are directed against PcrV, wherein at least one 'first' immunoglobulin single variable domain is directed against a first epitope on PcrV and at least one 20 n preferred aspect, the polypeptide of the invention is abiparatopic polypeptide. The terrn "biparatopic" (antigen-)binding molecule or "biparatopic" polypeptide as used herein shall mean a polypeptide comprising a 'first' immunoglobulin single variable domain directed against Pcry and a "second" immunoglobulin single variabe domain directed against PcrV, wherein these 'first" and "second" irmmunoglobulin single variable domans have a different paratope. Accordingly, the 25 biparatopic polypeptide comprises or consists of two or rnore immunoglobulin single variable domains that are directed against PcrV, ' first" mrmunoglobulin single variable domain is directed against a first epitope on PcrV and a "second" immunoglobulin single variable domain is directed against second epitope on PcrV different from the first epitope on PcrV. The biparatopic polypeptides of the invention show improved properties such as e.g. irnproved 30 binding characteristics (suitably Imeasured and/or expressed as a Ko-value (actual or apparent), a Krvalue (actual or apparent), a k 0 grate and/or a kirate, or aIternatively as an lCso value, as further described WO 2013/128031 PCT/EP2013/054262 64 herein), improved afnite and/or m imroe a idt for P ndo inmoe effia an/o otencyfo neutralizing PcrV, More in part ula rIne biparatopi polypeptides of the invention can bind to PcrV with an affinity (suitabIy measured and/or expressed as a Krrvalue (actual or apparent), a KA-value (actual or apparent), a 5 kz'rate and/or a k krate, or alternatively as an ICo value, as further described heren preferably such - bind to PcrV with a dissociation constant (K 0 ) of 1000 nM to 1 nM or less preferably 100 nM to 1 nM or less, more preferably 15 nM to 1 nM or even 10 nM to 1 nM or less; 10 - bind to PcrV with a karate of between 10 M>'s4 to about 107 M 4 s , preferably between 105 Mas and 107 M s, more preferably about 104 M s" or more; and/or uhta h -5bind to PcrV with akof rate between 102 s' (t 1 =0.69 )and 10"* sl (providing near irreversible complex with a t 1 of multiple days), preferably between 10' sand 104 o or lower; 1Some preferred IC value for binding of th biparatopic polypeptides of the invention to PcrV will become clear frorn the further description and examples herein. Assys to determine the lCao include binding in ELISA or more preferably cytotoxicity assays such as the TTSS-dependent cytotoxicity assay described by Frank et at (The Journal of infectious diseases 186: 64-73, 2002), Vance et at. ( nfectior and Immunity 73: 1706-1713, 2005), EI Solh et al (Am, .1 Resp'r, Crit. 20 Care Med. 178: 513-519, 2008), modifications of this assay such as e.g. described in Example 4.4, or a cytotoxicity assay with human lung epithelI cells (A549 cells) as described in Example 7.2, and modifications thereof For example, in a TTSS-dependent cytotoxicity assay with P3X63 cells as the target at an MOI of 12, the biparatopic polypeptides of the invention may have IC 50 values between 0,01 nM and 50 nM, 25between 0.01 nM and 10 nM, preferably between 0.01 nM and 5 nM, more preferably between 0.01 nM and 1 nM, such as between 0.01 nM and 0.1 nM or less. Apart from this and/or in addition, in such a TTSS-dependent cytotoxicity assay, the biparatopic polypeptides of the invention have an efficacy (% inhibition; see Exampe 4.4) of 100%, Moreover, the biparatopic polypeptides of the invention were shown to be stab e and maintained After 24 hours in the presence of P. aeruginao elastase, the biparatopic polypeptides of the invention may have a decrease in potency of maxirnal 10 fold, preferably maxima 5 fo d, such as 3 fold, 2 WO 2013/128031 PCT/EP2013/054262 65 fold, 1 fold or lower, After 24 hours in the presence of human neutrophil Elastase, the biparatopic polypeptides of the invention may have a decrease in potency of maxirnal 100 fold, preferably maximal 30 fold, such as 15 fo d, 10 fold, 5 fold, 3 fold, 2 fold or lower. In another preferred aspect, the polypeptide of the invention is a triparatopic polypeptide. The 5 term "tripartopic" (ntigen-)binding rnolecule or "riparatopic" polypeptide as used herein shall mean a polypeptide comprising a first" immunoglobulin single variable domain directed against PcrV,a "second" immunoglobul n single variable domain directed against PcrV and a third" immunoglobulin single variable domain directed against PcrV, wherein these "first" "second and "third" immunog obulin single variable domains have a different paratope Accordingly, the triparatopic po ypeptide comprises or 10 consists of three or more immunoglobulin single variable domains that are directed against PcrV, wherein a"first" immunoglobulin single variable domain is directed against a first epitope on PcrV, a "second" immunoglobu in single variable domain directed against second epitope on PcrV different from the first epitope on PcrV, and a third" immunoglobulin single variable domain is directed against a 15 The two or more immunoglobulin single variabe dornains present in the rmu tiparatopic polypeptide of the invention may consist of alight chain variable domain sequence (e~g., a Vesequen ce) or of a heavy chain variable domain sequence (e~g., a Ve-sequence); they may consist of a heavy chain variable domain sequence that is dernved from a conventional four-chain antibody or of a heavy chain variable dornain sequence that is derived from heavy chain antibody. In preferred aspect, they consist 2o of a Domain antibody (or an amino acid that is suitable for u se as a dornain antibody), of a sing e dornain antibody (or an amino acid that is suitable for usea a single domain antibody), of a "dAb" (or an amino acid that is suitab e for use as adAb) or of N Nnobody (including but not limitedd to a Vs ). The two or more immunoglobulin single variable domains may consist of a partiall or fully humanized Nanobody or a partially or fully humanized VIHH. In a preferred aspect of the invention, the immunoglobulin single 25 variable domains encompassed in the rnultiparatopic polypeptide of the invention are one or more monovalent polypeptides of the inentions de imed herin In preferred aspect of the invention, the first immunoglobulin single varnable domain and the second immrunoglobulin single varnable dornain present in tie rnultiparatopic (preferably biparatopic or triparatopic) polypeptide of the invention do not (cross)-compete with each other for binding to PcrV 30 and. as such, belong to different epitope bin . Accordingly, the present invention relates to a multiparatopic (preferably biparatopic or triparatopic polypeptide comprising two or rnore immunoglobulin single variable domains wherein each immunoglobulin single variable domain belongs WO 2013/128031 PCT/EP2013/054262 66 to a different epitope bin. Accordingly, the first immunoglobulin single variable dornain of this preferred rnultiparatopic (preferably biparatopic or triparatopic) polypeptide of the invention does not cross-block the binding to PcrV of the second immunoglobulin single variable domain of this preferred multiparatopic (preferably biparatopic or triparatopic) polypeptide of the invention and/or the first immunoglobulin single variable is not cross-blocked from binding to PerV by the second immunoglobulin single variable dornain. Different epitope bins (ito 3) have been identified amongst the monova ent polypeptides of the invention (see Tab es B-4 and B-i0) Accordingly, the present invention relates to mu rutiparatopic polypeptide comprising two or more immunoglobulin single variable domains wherein each 1o immunoglobulin single variable dorn belongs to different epitope bin as defined hereint In a preferred aspect, following combination of two or more immunoglobulin single variable domains are envisaged in the multiparatopic (preferably biparatopic or triparatopic) polypeptide of the invention: "first" immunoglobulin single variable " second" immunoglobuhn single variable domain belongs to~ domain belongs to: Epitope bin 1 Epitope bin 2 Epitope bin i Epitope bin 3 Epitope bin 2 Epitope bin i Epitpe bn 2Epitope bin 3 Epitope b n 3 Epitope bin i Epitope b n 3 E pitope bin 2 15 Preferred immunoglobulin single variable domains for use in these multiparatop c, (preferably biparatopic or triparatopic) polypeptides of the invention are the monovalent polypeptides of the invention a dest ribed above) belonging to the respective epitope bins In another preferreda spect, the rmultiparatopic polypeptide of the invention is selected from aniy of SEQ ID NOs 124-141. Preferred combination of immunoglobulin single variable domains for use in the rnultiparatopic 2 (preferably biparatopic or triparatopic) polypeptides of the invention are: -irnmunoglobulin single variable dornains belonging to epitope bins i and 2; - immunoglobulin single variable dornains belonging to epitope bins 3 and i; -irnmunoglobulin single variable domains belonging to epitope bins 3 and 2.
WO 2013/128031 PCT/EP2013/054262 67 Preferred immunioglobulin single variable domains for use in these multiparatopic (preferably biparatopic or triparatopic) polypeptides of the invention are the monovalent polypeptides of the invention (as described above) belonging to the respective epitope bins, In a preferred aspect the first immrunoglobu in single variable domain belongs to epitope bin 3, and is preferably SEQ ID NO: 12. In 5 another preferred aspect the second mrnunoglobu in single variable domain belongs to epitope bin 2, and is preferab y SEQ ID NO: LIn yet another preferred aspect, the multiparatopic polypeptide of the invention is selected from any of SEQ ID NOs: 129, 134 and 137. in another aspect, the first immunoglobu in single variable domain and the second immunog obulin single variable domain present in the multiparatopic (preferably biparatopic or to triparatopic) polypeptides of the invention belong to the sarne epitope bin. Accordingly, the present invention relates to a rnultiparatopic (preferably biparatopic or triparatopic) polypeptide comprising two or rnore immunoglobulin single variable domains wherein both immunoglobulin single variable domains belong to the sarne epitope bin. While these immunoglobulin single vaniale dornains have different paratopes these immunoglobulin single variab e domains bind to cdosely related and/or overnapping 15epitopes and, as such, (cross)-cornpete with each other for binding to PcrV. Accordingly, the first immrunoglobutin single variable dornain of these multiparatopic (preferably biparatopic or triparatopic) polypeptides of the invention cross-blocks the binding to PcrV of the second irmmunoglobu in single variable dornain of these multiparatopic (preferably biparatopic or triparatopic) polypeptides of the invention and/or the first immunoglobulin single var ale is cross-b ocked from binding to PcrV by the In a preferred aspect the immunoglobulin single variable domains present in such rnultiparatopic (preferably biparatopic or triparatopic) po ypeptides of the invention belong to an epitope bin (1 to 3) as defined herein (see Tables B-4 and B-10). Accordingly, the present invention relates to a multiparatopic polypeptide com prising two or more immunoglobulin single variable dornains wherein both 25immunoglobu in single variable domains belong to the same epitope bin as defined herein, Pre erred immunoglobulin single v riable domai for use m these multparatopic (preferably biparatopic or triparatopic) po ypeptides of the invention are the monovalent polypeptides of the invention (s described earlier) belonging to the respective epitope bins. In another preferred aspect, the multipar poeptie of the invention is selected frorn any of SEQ ID NOs: 118-123. 3o Preferred combination for use in the multiparatopic (preferably biparatopic or triparatopic) polypeptides of the invention are - two immunoglobulin single variable dornains belonging to epitope bin 1; WO 2013/128031 PCT/EP2013/054262 68 -two immunoglobulin single variab e domains belonging to epitope biln 2. Preferred immunoglobu in single variable domains for use in these rnultiparatopic (preferably biparatopic or triparatopic) polypeptides of the invention are the monovalent polypeptides of the invention (as described earlier) belonging to the respective epitope bins. In preferred aspect, two s immunoglobulin single varnable domain present in the multiparatopic polypeptide of the invention belong to epitope bin 1, and one of the two or more immunoglobulin single variable dornains is preferably SEQ ID NO: 3. Preferred multiparatopic polypeptides of the invention include SEQ ID NOs 118, 120 and 121. In another preferred aspect, two immunoglobulin single variable domain present in the rnultiparatopic polypeptide of the invention belong to epitope bin 2 and one of the two or more io immunoglobulin single variable domains is preferably SEQ ID ND: 1. Preferred multiparatopic polypeptides of the invention include SEQ ID NOs: 122 and 123. The rnonovalent polypeptide of the invention and the rmultivalent (multiparatopic) polypeptide of 15the invention, may or may not further comprise one or more other groups, residues, moieties or binding units (these monovalent polypeptides as well as rnultivalent (multiparatopic) polypeptides (with or without additional groups, residues, rnoieties or bin ding units) are all referred to as "polyPpptide(s) of the invention"). If present, such further groups residues, moieties or binding units may or rnay not provide further functionality to the immrunoglobulin single variable domain (nd/or to the polypeptide in which it 20 is present) and rnay or rnay not modify the properties of the immunoglobu in single variable domain. For exarnpe, such further groups, residues, moieties or binding units rmay be one or more additional Irnino acid sequences, such that the polypeptide is a (usion) protein or (fusion) polypeptide. In a preferred but non- imiting aspect, said one or rnore other groups, residues, moieties or binding units are immunoglobulins, Even rnore preferably, said one or more other groups, residues, rmoieties or 25 binding units are immunoglobulin single variable domains chosen from the group consisting of Domain antibodies, arnino acids that are suitable for use as a domain antibody, single dornain antibodies, amino acids that are suitable for use as a single domain antibody, "dAb"'s, amino acids that are suitable for use as a dAb, or Na nobodies (such as e.g. VHH, hurnanized VHH). As described above, additional binding units, such asimmunogiobulin single variable domains so having different antigen specificity can be linked to form multispecific polypeptides. By combining immunoglobulin s ingle variable domains of two or rnore specificities, bispecific, trispecific etc. constructs can be formed. For example, a potypeptide according to the invention may comprise one, two or rnore WO 2013/128031 PCT/EP2013/054262 69 immunoglobulin single variable domains directed against PcrV and one irmmunoglobulin single variable domain against another target, Such constructs and modifications thereof, which the skilled person can readi y envisage, are all encompassed by the term "polypeptide of the invention" as used herein, In the polypeptides described above, the one, two or more immunoglobulin single variable 5 domains and the one or more groups, residues moieties or binding un'ts may be 'inked directly to each other and/or via one or more suitable linkers or spacers. For example, when the one or more groups, residues, moieties or binding units are am'no acid sequences, the linkers may also be amino acid sequences, so that the resulting polypeptide is a fusion (protein) or fusion (polypeptide). The one or more further groups, residues, rnoieties or binding units may be any sui'table and/or 10 desired arnino acid sequences. The further amino acid sequences may or rnay not change, a ter or otherwise influence the (biological) properties of the polypeptide of the invention, and may or may not add further functionality to the po ypeptide of the invention. Preferably, the further amino acid sequence is such that it confers one or rnore desired properties or functionalities to the polypeptide of to E~xarnple of such amino acid sequences will be clear to the skIled person, and may generally com prism e all arnino acid sequences that are used in peptide fusions based on conventional anti bodies and fragrments thereof (including but not limited to Sc~v's and single domain antibodies), Reference is for example rnade to the review by Holliger and Hudson (Nature Biotechno ogy 23 1126-1136, 2005) For examp e, such an amino acid sequence may be an amino aid sequence that increases the half 2 Iife, tie solubility, or the absorption, reduces the immunogenicity or the toxicity, eliminates or attenuates undesirable side effects, and/or confers other advantageous properties to and/or reduces the undesired properties of the polypeptide of the invention, compared to po ypeptide of the 'nvention per se. Some non-lirniting exarnp es of such amino acid sequences are serurn proteins, such as human serurn alburnin (see for exarnple WO 00/27435) or haptenic molecules (for exarnple haptens that are recognized in one specific aspect of the invention, a polypeptide is prepared that has an increased half-ife, compared to the corresponding polypeptide of the invention. Examples of polypeptides of the invention that comprise such half-life extending rnoieties for examp e irndude, without imitation, polypeptides in which the irmu noglobulin single variable dorna'ns are suitab e linked to one or rnore serurn proteins or 30 fragments thereof (such as (human) serum albumin or suitable fragrments thereof) or to one or more binding un'ts that can bind to serum proteins (such as, for exarnple, Domain antibodies, amino acids that are suitable for use as a domain antibody, single domain antibodies, amino acids that are suitable for use WO 2013/128031 PCT/EP2013/054262 70 as a single domain antibody, dA' s arnino aids that are suitable for use as a dAb or Nanobodies) that can bind to serum proteins such as serum albumin (such as human serum albumin )) serum immunoglobulin (such as Ig), tr ansferrin or one of the other serum proteins listed in WO 04/003019, polypeptides in which the immunoglobulin single variable domain is inked to an Fc portion (such as a 5 human c) or a uitable part or fragment thereof or polypeptides in which the one or more immunoglobulin single variable domains are suitable linked to one or more small proteins or peptides that can bind to serum proteins such as without limitation, the proteins and peptides described in WO 91/01743, WO 01/4574 or WO 02/076489). Reference is also made to the dAb's described in WO 03/002609 and WO 04/003019 and to Harmsen et al. (Vaccine 23: 4926-42 2005); to EP 0368684 as well o as to WO 08/028977, WO 08/043821 WO 08/043822 by Ablynx NV. and WO 08/068280. According to a specific, but nondimiting aspect of the invention, the polypeptides of the invention may contain, besides the two or more immunoglobuin single variable domains and/or monovalent polypeptdes o the invention against PcrV at east one Nanobody against human serum albumin. These Nanobodies against human serum albumin may be as generally described in the appliations by Ablynx 15 NV. cited above see for example WO 04/06251) Some particularly preferred Nanobodies that provide for increased half-life and that can be used In the poypeptides of the invention include the Nanobodies ALB-1 to ALB-10 disclosed in WO 06/122787 (see Tables A1 and Il) of which ALB-8 (SEQ 10 NO: 62 in WO 06/122787) is particularly preferred, as well as the Nanobodies disclose in WO 2012/175400 (SEQ ID NOs: 1-11 of WO 2012/175400. 20 The polypeptide of the invention may for example, be a trivalent, bispecific polypeptie, comprising two immunogobulin single variabe domains, preferably nonovalent polypeptides of the invention against PcrV and a third immunoglobulin single variable doain directed against (human) serun albumin in which sai first, second and third imunoglobulin single variable domain may optionally be lnked via one or more and in particular two, tinker sequences. 25 According to one specific aspect one or more polypeptides of the invention nay be linked (optionally via a suitable liner or hinge region) to one or more constant domains (for example, 2 or 3 constant donanis that can be used as part of/to orm an Fc portion) to an Fc portion and/or to one or mnre antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or may confer the ability to bind to one or more FC receptors. For s0 example, for this purpose and without being limited thereto, the one or more further amino acid sequences may comprise one or more C, 2 and/or CH 3 domains of an antibody such as from a heavy chain antibody (as described heren) and more preferably from a conventional human 4-chain ntibody; WO 2013/128031 PCT/EP2013/054262 71 and/or may form (part of) and Fc region, for example from !gG (e.g. from IgG1 gG2, gG3 or gG4), from IgE or fromn another human Ig such as IgA, IgD or 1gM. For example, WO 94/04678 describes heavy chain antibodies comprising a Camelid V . domain or a humanized d rivative thereof (i.e. a Nanobody in which the Camelidae 0 2 and/rC 3 domain have been replaced by human C 2 and CH 3 domains, so as 5 to provide an immunoglobulin that consists o 2 heavy chains each comprising a Nanobody and human
CH
2 and CH 3 domaIns but no CA domain k wch immunogIobuin has the efector function provided by the C.
2 and C.
3 domains and which immunoglobulin can function without the presence of any ight chains. Other amino acid sequences that c n be suitably linked to the polypeptides of the invention so as to provide an effector function wil be lear to the skilled person, and may be chosen on the basis of the 1 desired effector functions). Reference is for example made to WO 04/058820, WO 99/42077, WO 02/056910 an WO 05/0 7148, a well as the review by Holliger and Hudson supra; and to WO 09/068628. Coupling of a polypeptide of the invention to an Fc portion may aso lead to an increased half-hfe compared to the corresponding polypeptide of the invention. For some applications, the use of an Fc portion and/or of constant domains (.e. CH 2 and/or 0 H domains) that confer increased hale 15 wit out any biologically significant effector function may also be suitable or even preferred. Other suitable constructs comprising one or nore polypeptides of the invention and one or more constant domains with increased half-life in vivo wi be dear to the skilled person, and may for example comprise polypeptides linke to a C 3 3 domain, optionaly via a linker sequence. Generally any fusion protein or derivatives with increased half-ife wil preferably have a molecular weight of more than 50 kD, the cut 20 off vlue for rena borpon in another specific but non-rniting aspect, the polypeptides of the invention may be linked (optionally via a suitable linker or hinge region to naturally occurring, synthetic or semi-synthetic constant domans or analogs v giants, mutants parts or fra ments thereof) that have a reduced (or essential y no) tendency to self-associate into diners (i.e. compared to constant domains that natural 25 occur in convention 4-chain antibodies). Such monomeric (i.e not self-associating) Fc chain variants, or fragments thereof, wi be clear to the skilled person. For example, Helm et al. (J Biol. Chem, 271: 7494 1996), describe ronomeric F chain variants that can be used in the polypepti chains of the invention. Also, such monoreric F chain variants are preferably such that they are sting capable of binding to the complement or the relevant Fc receptors) dependingg on the Fc portion from which they are 30 derived), and/or such that the still have some or A of the eff actor functions of the Fc portion from which they are derived (or at a reduced level still suitable for the intended use). Atenatively, in uch a polypeptide chain o the invention, the monomeric F champ may be used to confer increased half-life WO 2013/128031 PCT/EP2013/054262 72 upon the polypeptde chain, in which case the monomeric c chain rnay also have no or essentially no effector functions Generally, the poypeptides of the invention with increased half-life preferably have a ha f-life that is at least 1 5 timne, preferably at least 2 times, such as at least 5 times, for example at least 10 times or 5 more than 20 times greater than the half-ife of the corresponding immunoglobulin single varable domain or polypepfde o the invention per se. Generally, the polypeptides of the invention with increased half-life preferably have a half-life that is increased with more than 1 hours preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 4 or 72 hours compared to the half-life of the 10 corresponding immunoglobulin single variable domain or polypeptide of the invention per se. In another preferred, but non-imiting aspect such polypeptides o the invention exhibit a serum halfi e in human of at least about 12 hours, preferably at least 24 hours more preferab y at least 48 hours even more preferably at least 72 hours or more. For example, poAypeptides of the invention may have a half-ife of at least 5 days (such as about 5 to 10 days), preferably at least 9 days such a about 9 15 to 14 days), nore preferably at least about 10 days (such as about 10 to 15 days), or at least about 11 day such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 12 days or more), or more than 14 days (such as about 14 to 19 days). The further amino acid residues may or may not change, alter or otherwise influence other (biological properties of the polypepitde of the invention and may or may not add further functionaLity 20 to the polypeptide of the invention. For example, such amino acid residues: a) can comprise an N-terminal Met residue for example as result of expression in a hetercog ous host celI or host organism b) may form a signal sequence or leader sequence that directs secretion of the polypeptide from a host cell upon synthesis (for example to provide a pre- pro- or prepro- form of the polypeptide of the 25 invention depending on the host cel used to express the polypeptide of the invention Suitable secretory leader peptides wi be clear to the skilled person nd may be as further described herein, Usually, such a leader se quence wi be linked to the N-termrnus of the polypeptide although the ivenon in its broadest sense i not limited thereto; c) may form a "t g" for examp e an amino acid sequence or residue that allows or facilitates the 30 purification of the polypeptide for example using affinity techniques directed against said sequence or residue. Thereafter said sequence or residue may be removed (e.g. by chemical or enzymatical cleavage) to provide the polypeptide (for this purpose the tag may optionally be lin ed to he WO 2013/128031 PCT/EP2013/054262 73 amino acid sequence or polypeptide sequence via a cleavable linker sequence or contain a cleavable notif). Some preferred, but non-limiting examples of such residues are multiple histidine residues, glutathione residues and a ny-tag such as AAAEQIUS EDLNGAA (Q KD NO: 206); d) nay be one or more amino acid residues that have been functionalized and/ r that can serve as 5 site or attachment of functional groups. Suitable amino acid residues and functional groups will be clear to the skiled person and include but are not limited to, the amino acid residues and functional groups mentioned herein for the derivatives of the polypeptides of the invention. The multivalent (such as biparatopic or triparatopi polypeptdes of the invention can generally be prepared by a method which omprises at least the step of suitably linking the immunoglobulin single 10 variable domain and/or monovalent polypeptide of the invention to one or more further immunog obulin single variable domains and/or monovalent polypeptides of the invention, optionaH via the one or mnore suitable liners so as to provide the multivalent polypeptide of the invention. Polypeptides o the invention can also be prepared by a method which general comprise t least the steps of providing a nucleic acid that encodes a polypeptide of te invention, expressing said nucleic cid 1s in a suitable manner, and recovering the expressed polypeptide of the invention. Such methods can be per ormed in a manner known per se, which will be clear to the skilled person, for example on the basis of the methods and techniques fur other descried herein. A method for preparing mrutipratopic polypeptides of the invention may comprise at least the steps of linking two or more immunoglobulin single variable domins and/or monovalent polypeptides of 20 the nventon and for example one or more lnkers together in a stable manner. The immunoglobulin single variable domains and/or monovalent polypeptides of the invention (and inkers) can be coupled by any m hod known in the art and as further descried herein. Preferred techniques include Lthe linking of the nucleic acid sequences that encode the immunoglobulin ingle variable domains and/or monovalent polypeptides of the invention (and Unkers) to prepare a genetic construct that expresse the 25 nultiparatopic polypeptide. Technique for linking amino acids or nucleic acids wil be clear to the skilled person, and reference is again made to the stan ard handbook, such as Sambroo et a and Ausubel et al., mentioned above, as well s the Examples below. Accordingly, the present invention also relates to the use of an immunoglobulin single variable domain and/or monovalent polypeptide of the invention in preparing a multivalent, preferably so multiparatopic polypeptide of the invention The method or the preparation of a multivalent polypeptide wil comprise the linking of an irmunoglobulin single variable domain and/or monovalent polypeptide of the invention to at least one further immunoglobulin single varnabe domain and/or WO 2013/128031 PCT/EP2013/054262 74 monovalent polypeptide of the invention, optionally via one or more linkers. The immunoglobulin single variable domain and/or rnonovalent polypeptide of the invenon isthen used as a bindingdoain or finding unit in providing and/or preparing the multivalent, preferably multiparatopic polypeptide comprising two (epg, n a bivalent polyp tidee, three (eg. in trivalent polypeptide or more (e, in a 5 multivalent polypeptide) binding unitsdn this respect, the immunoglobulin singe variable domain and/or the monovalent polypeptide of the invention may be used as a binding domain or binding unit in providing and/or preparing a multivalent (preferably multiparatopc), such as bivalent preferably biparatopic) or trivalent (preferably triparatopic polypeptide o the invention comprising two, three or more ending units. 10 Accordingly, the present invention also relates to the use of an immunoglobulin single variable domain and/or particularly, a monovalent polypeptide of the invention (as described herein in preparing a multivalent, preferably multiparatopic polypeptide. The method for the preparation of the multiva ent, preferably multparatopi polypeptide will comprise the inking of the immunoglobulin single variable domain and/or monovalent polypeptide o the invention to at least one further immunoglobulin single 15 variable dornain and/or monovalent polypeptide of the invention optionally via one or mre liners. Suitable spacers or linkers for use in multivalent, preferably multiparatopic polypeptides of the invention will be clear to the skilled person, and nay general be any liner or spacer used in the art to ink amino acd sequences. Pre erably id linker or spacer is suitable for use in constructing polypeptides that are intended for pharmaceutical use. 20 Some particularly preferred spacers include the spacers and linkers th at re used in the art to link antibody fragments or antibody domains. These include the lin ers mentioned in the general background art cited above as well as or example inkers that are used in the art to construc dabodies or ScFv fragments (in ths respect however, it should be noted that, whereas in diabodies and in ScFv fragment the inker sequence used should haven a lngtha degree of flexibility and other properties that allow the 25 pertinent V and V domain to corne together to forrn the complete antigen-binding site, there is no particular imitation on the length or the flexibility of the linker use in the polypeptid e of the invention, since each irmunoglobuin single variable domain by itself forms a complete ntigen-bining site For example, a liner may be a suitable amino acid sequence, and in particular amino acid sequences of between 1 and 50, preferably between j and 30 such as between 1 and 10 amino acid 0 residues. Some preferred examples of such amino acid sequences include gly-ser liners, for example of the type (glysery), such as (for example (gly 4 ser) 3 or (glyssers , as described in WO 99/42077, hinge-like WO 2013/128031 PCT/EP2013/054262 75 regions such as the hinge regions of naturally occurring heavy chain a antibodies or similar sequences (such as described in WO 94/04678 Some other particularly preferred liners are mentioned in Table A-8. of which GS40 (SEQ ID NO: 193) is particularly preferred. 5 Other suitable linkers generally comprise organic compound or polymers in particular those suit able for use in proteins for pharmaceutical use. For instance, polyfethylenegiycol moieties have been used io unk anibody domains see for example WO 04/01026. It as encornpased within the scope of the invention that the length, the degree of flexibility and/or other properties of the linker(s used (although not critical as it usually is for sinkers used in ScFv 1o fragments) may have some influence on the properties of the final polypeptide of the invention, including but not limited to the affinity, specificity or avidity for PcrV or for one or more of the other antigens. Based on the disclosure herein, the skilled person will e able to determine the optimal linker(s) for use in a specific polypeptide of the invention, optionally after some Imite rouine experiments. is it is also within the scope of the invention that the linker(s) used confer one or more other avourable properties or functionality to the polypeptides o the invention, and/or provide one or more site for the formation of derivative and/or for the attachment o funcional groups eg. as described herein for the derivatives of the polypeptides of the invention). For example liners ontaining one or more charged amino acd residues can provide improved hydrophilic properties, whereas liners that 20 form or contain small epitopes or tags can be used for the purposes of detection identification and/or purification. Again based on the disclosure herein, the skilled person will be able to determine the optimal liners for use in specific polypeptide of the invention optionally after some limited routine experiments. Finally, when two or more blinkers are used in the polypeptides of the invention these liners may s be the same or different. Again, based on the disclosure herein the skiled person wil be able to determine the optimal liners for use in a specific polypeptide of the invention, optionally after some limited routne experimnents Usually, or ease of expression and production, a polypeptide of he invention will be a linear polypeptide. However, the nvention in it broadest sense is not limited thereto. For example, when a ao polypeptide of the invention comprises three of more amino acid sequences or Nanobodies it is possible to linK them by use of a linker with three or more 'ars" which each "arm" being linked to n anino WO 2013/128031 PCT/EP2013/054262 76 acid sequence or Nanobody so as to provide a st r-shaped" construct. It is also possible, although usually less preferred, to use circular constructs. Also encompassed in the present invention are fused iromunoglobulin sequences, comprising tags or other functional moieties, e toxins, Iabels radiochemicals etc. 5 Alternatively the additional groups, residues, moieties or binding units mny for example be chemical groups, residues, moieties, which may or may not by themselves be biologically and/or pharmacologically active. For example, and without imitation, such groups may be linked to the two or more immunoglobulin single able domains or monovalent polypeptides so a to provide a "derivative" of the polypeptide of the invention. 10 Accordingly, the invention in its broadest sense also comprises derivatives of the polypeptides of the invention. Such derivatives can generally be obtained by modification, and in particular by chemical and/or biologicl e.g., enymatical modification of the polypeptides of the invention and/or of one or more of the amino acd resOues that form polypepide of the invention. Examples of such modifications, as well a examples of amino acid resiues within the polypeptide 15 sequences that can be modified in such manner (i.e. either on the protein backbone but preferably on a side chain), methods and techniques that can be used to introduce such modifications and the potential uses and advantages of such rmodifications will be clear to the skilled person. For example such a modification may involve the introduction (e.g, by covalent linking or in any other suitable manner) of one or more functional groups, residues or moieties into or onto the 20 p0ypeptide of the invention, and in particular of one or more functiona groups residues or moieties that confer one or nore desired properties or unctionalities to the polypeptide of the invention. Exarnple of such functional groups will be clear to the skilled person. For example, such modification may comprise e introduction (e.g, by covalent binding or in any other suitable manner of one or more functional groups that that increase the half-life, the solubility 25and/or the absorption of the polypeptide of the invention, thAt reduce the immunogenicity and/or the toxicity of the polypeptide of the invention that eliminate or attenuate any undesirable side fects of the polypeptide of the invention, and/or that confer other advantageous properties to and/or reduce the undesired properties o the polypeptide of the invention; or any combination of two or more of the foregoing. Examples of such unctional groups and of techniques for introducing them will be clear to the so skilled person, and can generally comprise al functional groups and techniques mentioned in the general bac ground art noted hereinabove as well as the functional groups and techniques known per se for the modification of pharmaceutical proteins, and in particular for the modification of antibodies or antibody WO 2013/128031 PCT/EP2013/054262 77 fragments (including ScFv s an sing e dom ain antibodies), for which reference is for example mad to Remington (Pharmaceutical Sciencs, 16th ed, Mack Publishing Co., Easton PA, 1980. Such function groups may for example be linked directly ( or example covalently) to a polypeptide o the invention or optionally via a suit ble linker or spa cer, as will again be clear to the skilled person. 5 One specific example is a derivative polypeptide of the invention wherein the polypeptide of the invention has been cheically modified to increase the half ife thereof (for example, by means of pegy aton) This i one of th most widely used techniques or increasing the half-ife and/or reducing the immunogenicity of pharmaceutical proteins and comrprises attachment of a suitable pharnacologically acceptable polymer, such as poly ethylenegycol PEG or derivatives thereof such as o methoxypoly(ethyleneglycol or mPEG). Generally any suitable orm of pegylation can be used, such s the pegylation used in the art for antibodies and ntibdy fragments (inclu ing but not limited to single domin ntibodies and Scv's); reference is made to for example Chapman Nat. Biotechnol. 54: 531-545, 2002), Veronese and Harris (Adv. Drug Deiv. Rev. 54: 453-456, 2003, Harris and Chess (Nat. Rev. Drug. Discov. 2: 214-221, 2003 and WO 04/060965 Variou reagents for pegylation of proteins are also 15 commercially available, for example from Nektar Therapeutics, USA. Preferably, site-directed pegylation is used, in particu ar via a cysteine-residue (see for example Yang et at (Protein Engineering 16: 761-770, 2003). For example, for this purpose, PEG may be att ched to a cysteine residue that naturally occurs in a polypeptide of the invention a polypeptide of the invention may be modified so as to sutably introduce one or more cysteine residues for attachment of 20 PEG, or an amino aid sequence comprising one or more cystein residues for attachrent of PEG may be fused to the N- and/or C-terminus of a polypeptide of the invention, all using techniques of protein engineering known per se to the sKiled person. Preferably, for the polypeptides of the invention a PEG is used with molecular weight of more than 5000, such as more than 10 000 and les than 200,000, such as less than 00,000; for example in 25 the range of 20,00080,000. Another, usually ess preferred modification omprises Nlinked or 0-linked gycosylation, usually as part of co-translational and/or post-translational modification, dependng on the host cell used for expressing the polypeptide of the invention. Yet another modifcation nay comprise the introduction of one or nore detectable labels or other 3o signal-generating' groups or moieties, depending on the in ended use of the Label ed polypeptide of the invention. Suitable labels and techniques or attaching, using and detecting them will be clear to the skilled person and for example include, but are not limited to, fluorescent labels (suc h as fluorescein, WO 2013/128031 PCT/EP2013/054262 78 isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine and fluorescent metals such as Eu or others metals from the lanthanide series), phosphorescent labels, cherniuminescent labels or bioluminescent labels (such as luninal, isoluminol, theromatic acridinium ester, inidazole, acridinium salts, oxalate ester, dioxetane or GFP and its analogs 5 ), radio-isotopes (such as US", m I, ' C, Cr, C1, "Co, "Co, PFe, and me), metals, metals chelates or metallic nations (for example metallic nations such as "Tc ALn,Ru, Cu, Ga, and "Ga or other metals or metallic cations that are particularly suited for use in in vivo, in vitro or in situ diagnosis and imaging, such as (Y'Gd, 'Mn, Dy, 2 Cr, and "Fe)), as well as chromophores and enzymes (such as malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol 10 dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, biotinavidin peroxidase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, g galactosidase, ribonuclease, urease, catalase, glucose-VQphosphate dehydrogenase, glucoamylase and acetylcholine esterase). Other suitable labels will be clear to the skilled person, and for example include moieties that can be detected using NMR or ESR spectroscopy. if Such labelled polypeptides of the invention may for example be used for in vitro, in vivo or in situ assays (induding immunoassays known per se such as EUSA, RIA, EIA and other "sandwich assays", etc.) as well as in vivo diagnostic and imaging purposes, depending on the choice of the specific label As will be clear to the skilled person, another modification may involve the introduction of a chelating group, for exarnple to chelate one of the metals or metallic nations referred to above. Suitable 20 chelating groups for example include, without limitation, diethyl-enetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Yet another modification may comprise the introduction of a functional group that is one part of a specific binding pair, such as the biotin-(strept)avidin binding pair. Such a functional group may be used to link the polypeptide of the invention to another protein, polypeptide or chemical compound that is 25 bound to the other half of the binding pair, Le. through formation of the binding pair. For example, a polypeptide of the invention may be conjugated to biotin, and linked to another protein, polypeptide, compound or carrier conjugated to avidin or streptavidin. For example, such a conjugated polypeptide of the invention may be used as a reporter, for example in a diagnostic system where a detectable signal producing agent is conjugated to avidin or streptavidin. Such binding pairs may for example also be used 30 to bind the polypeptide of the mvenion to a carrier, including carriers suitable for pharmaceutical purposes. One non-imiting example are the liposomal formulations described by Cao and Suresh WO 2013/128031 PCT/EP2013/054262 79 (Journal of Drug ITrgeting 8: 257, 2000) Such binding airs may also be used to Unk a thera poetically active agent to the polypeptide of the invention Other potential chemic a and enzymatical mo ifi nations will be lear to the s killed person. Such modifications may also be introdu ed for research purposes e.g. to study function-activty rel tionships 5 Re erence is for example made to Lundblad and Bradshaw (Biotechnol. A ppl. Biochem. 26: 143-151, 1997). Preferably, the derivatives are such that they bind to PcrV, with an affinity (suitably measured and/or expressed as a Kvalue actuall or apparent a KA-vaLue (actual or apparent a krate and/or a k -rate, or alternatively as an IC value, as further described herein) that is as defined herein (i.e. as to defined for the polypeptides of the invention Such denvatives will usually also have a PcrV neutralization efficacy and/or potency as defined herein. Such polypeptides o the invention and derivatives thereof may also be in essentially isolated form as defined herein). The invention further relates to methods for preparing the polypeptides, nucleic acids host cells, and compositions described here. The polypeptides and nucleic acids of the invention can be prepared in a manner known per se, will be cMear to the skilled person fror the further description herein, For example the polypeptides of the invention can be prepared in any manner known er se for the preparation of antibodies and in particular for the preparation of antibodyfragments including but not limited to ingle) domain antibodis and Scv fragments). Some preferred, but non-limiting methods for preparing the olypeptides and nucleic acids include the methods and technique described here. The method for producing a polypeptide of the invention may comprise the following steps: - the expression in suitab e host ce! or Iost organism (also referred to erein as ahost of th in vem on") or in another suitable expression system o a nucleic acid hat encode said 25 polypeptide of the invention (also referred to herein asa "nucc cdof th inventor"), optionally followed by: - isolating and/or purifying the polypeptide of the invention thus obtained. in particular, such a method may comprise the steps of: - cultivating and/or maintaining a host of the invention under conditions that are uch that said host 30 of the invention expresses and/or produces at least one polypeptide of the invention; optionally followed by: - isolating and/or purifying the polypeptide of the invention thus obtaed.
WO 2013/128031 PCT/EP2013/054262 80 Accordingly, the present invention also relates to a nucleic acid or nucleotide sequence that encodes a polypeptide of the invention (also referred to as "nucleic acid of the invention") A nucleic acid of the invention can be in the form of single or double stranded DNA or RNA, and is preferably in the form of double stranded DNA. For example, the nucleotide sequences of the invention may be genomic 5 DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cel or host organism). According to one embodiment of the invention, the nucleic acid of the invention is in essentially isolated from, as defined herein. The nucleic acid of the invention may also be in the form of, be present in and/or be part of a vector, such as for example a plasmid, cosmid or YAC, which again may be in 10 essentially isolated form. The nucleic acids of the invention can be prepared or obtained in a manner known per se, based on the information on the polypeptides of the invention given herein, and/or can be isolated from a suitable natural source. Also, as will be clear to the skilled person, to prepare a nucleic acid of the invention, also several nucleotide sequences, such as at least two nucleic acids encoding an 15 immunoglobulin single variable domain or a monovalent polypeptide of the invention and for example nucleic acids encoding one or more liners can be linked together in a suitable manner. Techniques for generating the nucleic acids of the invention wil be clear to the skilled person and may for instance include, but are not limited to, automated DNA synthesis; sitedirected mutagenesis; combining two or more naturally occurring and/or synthetic sequences (or two or more parts thereof), 20 introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and/or regions that may easily be digested and/or lighted using suitable restriction enzymes), and/or the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers. These and other techniques will be clear to the skilled person, and reference is again made to the standard handbooks, such as Sambrook et al and Ausubel et 25 al., mentioned above, as wel as the Examples below. The nucleic acid of the invention may also be in the form of, be present in and/or be part of a genetic construct, as will be clear to the person skilled in the art. Such genetic constructs generally comprise at least one nucleic acid of the invention that is optionally linked to one or more elements of genetic constructs known per se, such as for example one or more suitable regulatory elements (such as 30 a suitable promoterss, enhancer(s), terminator(s), etc.) and the further elements of genetic constructs referred to herein. Such genetic constructs comprising at least one nucleic acid of the invention will also be referred to herein as "genetic constructs of the nvention".
WO 2013/128031 PCT/EP2013/054262 81 The genetic constructs of the invention may be DNA or RNA, and are preferably double-stranded DNA. The genetic constructs of the invention may also be i a form suitable for transformation of the intended host cell or host organism, in a form suitable for integration into the genomic DNA of the intended host cell or in a form suitable for independent rep ication, maintenance and/or inheritance in 5 the intended host organism. For instance he genetic constructs of the invention may be in th for of a vector, such sforexampie a plasmid cosmid, YAC, a viral vector or transposon. in particular, the vector may be an expression sector i.e. a vector that can provide for expression in vtro and/or in vivo e.g. in a suitable host cell, host organism and/or expression system). In a preferred but non-limiting embodiment, a genetic construct of the invention comprises 10 a) at least one nucleic acid of the invention operably connected to b) one or more regulatory elements such a a promoter and optonall a suitabe Ierminator; and optionally also c one or more further elements of genetic constructs known per sr; in which the terms "regulatory element , "promoter, "terminator and operaby cone ted" have their 15 usual meaning in the art (as further described herein); and in which aid further e events present in the genetic constructs may for example be 3'- or 5'-UTR sequences, leader sequences, section markers expression markers/reporter genes and/or elements that may facilitate or increase (the efficiency of transformation or integration. These and other suitable elements for such genetic construct will be clear to the skilled person, and may for instance depend upon the typ of construct used the intended host 20 cell or host organism; the manner in which the nucleotide sequences of th invention of interest are to be expressed (e.g. via constitutive transient or nducible expression and/or the transform tion technique to be used. For example, regulatory sequences promoters and terminators known per se for the expression and production of antibodies and antibody fragments (including but no limited to (single) domain antibodies and ScFv fragments may be used in an essentially analogous manner. 25 Preferable, in the genetic constructs of the invention, said at least one nucleic acid of the invention and sad regulatory elements and option ly said one or more further elements, are 'operably knked" to each other, by which is generally meant that they are in a functional relationship with each other. For instance, a promoter i considered 'operably linked to a coding sequence if said promoter i able to initiate or otherwise control/regulate the transcription and/or the expression of a coding sequence (in o which said coding sequence should be understood as being "under the control of' sid promoter) Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and WO 2013/128031 PCT/EP2013/054262 82 usually also in the same reading frame They will usually also be essentially contiguous although this may aso not be required. Preferbly th regulatory and further elements of the genetic constructs of the invention are su h that they are capable of providing their intended biological function in the intended host cell or host 5 organism. or instance, a promoter, enhancer or terminator should be "operable" in the intended host cel or host organism by which is meant that (for example said promoter should be capable of initating or otherwise controling/regulating the transcription and/or the expression of a nucleotide sequence - eg., a coding sequence - to which it is operably linked (as de mfed herein), o Some p articul rly preferred promoters include, but nota t limited to, proro ers known per se for the expression in the host cels mentioned herein; and in particular promoters for the expression in th bacterial cells, such as those mentioned herein and/or those used in the Examples. A selection marker should be such that it allows - e., under appropriate selection conditions - host cells and/or host organism that have been successfuln) transformed with the nucleotide sequence o 15 the invention to be distinguished from host cells/organisms that have not been (successfully) transformed. Some preferred, but norIrmiting examples of such markers re genes that provide resistance against antibiotics (such as kanamycin or ampicilin), genes that provide or temperature resistance, or genes that aow the host cell or host organism to be maintained in the absence o certain fa actors, compounds and/or (food) components in the medium that are essential or survival of the non 20 transformed cell or org nsmrsa A leader sequence should be such that - in the intended host cel or host organism - it allows for the desired postranslational modifications n or such that i directs the transcbed mRNA to a desired part or organele of a cell A leader sequence may also allow for secretion of the expression product from said cell As such the leader sequence may be any pro- pre-, or prepro-sequence operable 25 in the host cell or hos organism. Leader sequence may not be required for expression in bacterial cell For example, leader sequences known per se for the expression and producion of antibodies and antibody fragments includingg but not limited to single main antibodies and ScFv fragments may be used in an essential analogous mnner, An expression marker or reporter gene should be such tha - in the host cell or host organism - it 30 allows for detection of the expression of a gene or nucleotide sequence present on) the genetic construct. An expression marker may optionally also aow for the localsation of the expressed product, e.g, in a specific part or organelle of a cell nd/or in (a) specific cel(s), tissuess, organs) or part(s) of a WO 2013/128031 PCT/EP2013/054262 83 multicellular organism. Such reporter genes may also be expressed s a protein fusion with the amino acid sequence or polypeptide of the invention, Some preferred, but non-limiting examples include forces nt proteins such as GP Some preferred, but non-limiting examples of suitable promoters, terminator and further elements 5 include those that can be used or the expression in the host cells mentioned herein; and in particular those that are suitable for expression in bacterial cells, such as those mentioned herein and/or those used in the Examples below. For some further non-limitng examples of the promoters selection markers, leader sequences, expression markers nd further elements that may be present/used in the genetic constructs of the invention such as terminators transcriptional and/or trans national enhances 10 and/or integration factors - reference is made to the general handbooks such as Sambroo et aL and Ausubel et al. mentioned above, as well s to the examples that are give n WO 9/07463 WO 9 /23810, WO 95/07463 WO 95/21191, WO 97/11094, W0 97/42320, W0 98/06737, W, 98/21355, US 7207,410, US 5,693,492 and EP 1085089. Other examples will be clear to the skil ed person Reference is also made to the genera background art cited above and the further references cited herein. 15 The genetic constructs of them mention may generally be provided by suitably linking the nucleotide sequence (s) of the invention to the one or nore further elements described above for example using the techniques described n the general handbooks such as Sambrook et aL and Ausubel et al mentioned above. aften, the genetic constructs of the invention will be obtained by inserting a nucleotide sequence 20 of the invention in a suitable (expression) vector known per se. Some preferred but non-limiting examples of suitable expression vectors are those used in the Exmples below, as well as those mentioned herein. The nucleic acids of the invention and/or the genetic constructs of the nvention may be used to transform a host cell or host organism i.e. for expression and/or production of the polypeptide of the 25 invention. Suitable hosts or host cells will be clear TO the killed person, and may for example be any suitable unga prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or (non human) eu aryotic organism, for example: - bacteria strain, including but not limited to gram-negative strains such as strains of Escherichio coli; of Proteus, for example of Proteus mn irabis; of Pseudomonas, for example of Pseudomonos 30 fluorescens; and gram-positive strains such as strains of Bn/us or example of Baoci/us sut o//s or of BL/us brevis; of Streptomynes, for exa m ple of Streptomyces //viduns; of Stapiyococcus, for exa mpe of Staphy ococcus carnosus; and of Lctococcus, for example of Lactucccus loctis; WO 2013/128031 PCT/EP2013/054262 84 - a fungal cell including but no limited to cells from species of Tchaderma, for exa mple from Trichoderma reeseI; of Neurospora, for exam pie from Neurospora crassa; of Sardari'a, for example from Sardaria macros para; of Asper g'Ilus, for exam pi f rom Aspergillus niger or fromn Aspergillus sajae; or from other filamentous fungi; 5 - a yeast cell including but not limited to cells from species of Saccharomyces, for example of Saccharomyces cerevisae; of Schizosaccharomyces for exam ple of Schizasaccharomyces pombe; of Pichia, for exm ple of Pichia pastors or of Pchia methanoica of hansenla, for example of H ansen ua pa ymarpha; a K uyveroryces, fo r exa m ple of K uv roamyc s actis; of Arxua, for example of Arxuia adeninivorans; of Yarrowia, for example of Yarrowia polyticac; 10 - an amphibian cell or cell line, such as Xenopus ocytes; - an insect-derived cell or cell ne, such as cels/cell lines derived from epdoptera induding but not limited to SpOdaptera SF9 and Sf21 cells or cells/cell lines derived from Drosophila such as Schneider and Kc cells; - a plant or plant cell for example in tobacco pants and/or 15 - a mammnalian cell or cel line, for example a cell or elI line derived from a human, a cell or a cel line from mammals including but not minted to CHO-ells BHK-cells (fore example BHK-21 ce s) and human cell or cell lines such as Heta, COS (for example COS-7) and PER.C6 cells; as wll as al other host cels or (non-human hosts known per se for the expression and production of antibodies and antibody fr gments (including but not limited to (single) domain antibodies and Scv 20 fragments), which will be clear to the skilled person. Reference is also made to the general background art cited hereinabove, as well as to for example WO 94/29457; WO 96/34103 WO 99/42077; Frenken et al Res Immunol 149: 89-99, 1998) Riechmann and Muylderman (1999), supra; van der Linden (J Biotechnot 80: 261-70, 2000); Joosten et at (Microb. Cell Fact. 2: 1, 2003) Joosten et a. (AppI Microbiol Biotechnol 66: 384-92, 2005); and the further reference cited herein. 25 The polypeptides of the invention may also be expressed as so-called "intrabodies", as for example described in WO 94/02610, WO 95/22618 and US 7,004,940; WO 03/014960; in Cattaneo and Biocca ('Intracellular Antibodies: Development and Applications Landes and Springer-Verlag, 1997) and in Kontermann (Methods 34 163-170, 2004). The po peptides of he invention can for exarnple also be produced in the milk of ransgenic o mammals for example in the milk of rabbits, cows, oats or sheep see for example US 741,957, US 6,304,489 and US 849,992 or general technique for introducing trans genes into mammals) in plants WO 2013/128031 PCT/EP2013/054262 85 or parts of plants including but not limited to their leaves, flowers, fruits, seed, roots or tubers (for exarnple in tobacco, mnaize, soybean or alfalfa) or in for exarnple pupae of the slkworm Bombix mor, Furthermore, the polypeptides of the invention can also be expressed and/or produced in cell-free expression systems, and suitable examples of such systems will be clear o tothe skiled person. Some 5 preferred, but non-limiting examples include expression in the wheat germ system; in rabbit reticulocyte lysates; or n te . Zubay system. Preferably, i i invention, an (in vivo or in vitro) expression system, such as a bacterial expression system, is used that provides the polypeptides of the invention n a form that is suitable for pharmaceutical use, and such expression systems wil again be clear to the skilled person, As also wih be 10 clear to the skilled person, polypeptides of the invention suitable for pharmaceutical use can be prepared using techniques for peptide synthesis. For production on industrial scale, preferred heterologous hosts for the (industrial) production of immunoglobulin single variable domains or immunoglobulin single variable domain-containing polypeptide therapeutics include strains of E coil, Pichi pastors, S. cerevisiae that are suitable for large is scale expression/production/fermentation, and in particular for large scale pharmaceutical expression/production/fermentation. Suitable examples of such strains wil be clear to the skilled person. Such strains and production/expression systems are also made available by companies such as Biovitrum (Uppsala, Sweden). Alternatively, mammalian cell lines, in particular Chinese hamster ovary (CHO) cells, can be used 20 for large scale expression/production/ferrnentation, and in particular for large scale pharmaceutical expression/production/fermentation. Again, such expression/production systems are also made available by some of the companies mentioned above. The choice of the specific expression system would depend in part on the requirement for certain post-translational modifications, rnore specifically glycosylation. The production of a immunoglobulin 25 single variable domain-containing recombinant protein for which glycosylation is desired or required would necessitate the use of mammalian expression hosts that have the ability to glycosylate the expressed protein, In this respect, it will be clear to the skilled person that the glycosylation pattern obtained (i.e., the kind, number and position of residues attached) will depend on the cell or ell line that is used for the expression. Preferably, either a human cell or cell line is used (i.e., leading to a protein 3o that essentially has a human glycosylation pattern) or another mammalian cell line is used that can provide a glycosylation pattern that is essentially and/or functionally the same as human glycosylation or at least mirnics human glycosylation. Generally, prokaryotic hosts such as E coil do not have the ability to WO 2013/128031 PCT/EP2013/054262 86 glycosylate proteins, and the use of lower eukaryotes such as yeast usually leads to a glycosyation pattern that differs from human glycosylation. Nevertheless, it should be understood that all the foregoing host cels and expression systerns can be used in the invention, depending on the desired polypeptide to be obtained, a Thus, according to one non-limiting embodiment of the invention, the polypeptide of the invention is glycosylated. According to another non-limiting embodiment of the invention, the polypeptide of the invention is non-glycosylated. According to one preferred, but non-limiting embodiment of the invention, the polypeptide of the invention is produced in a bacterial cell, in particular a bacterial cell suitable for large scale io pharmaceutical production, such as cells of the strains mentioned above. According to another preferred, but non-limiting embodiment of the invention, the polypeptide of the invention is produced in a yeast cell, in particular a yeast cell suitable for large scale pharmaceutical production, suc h as cells of the specie mentioned above According to yet another preferred, but non-limiting embodiment of the invention, the is polypeptide of the invention is produced in a mammalian cell, in particular in a human cell or in a cell of a human cell line, and more in particular in a human cell or in a cell of a human cell line that is suitable for large scale pharmaceutical production, such as the cell lines mentioned hereinabove. When expression in a host cell is used to produce the polypeptides of the invention, the polypeptides of the invention can be produced either intracellularly (e.g., in the cytosol, in the 20 periplasma or in inclusion bodies) and then isolated from the host cells and optionally further purified; or can be produced extracellularly (e.g., in the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified. When eukaryotic host cells are used, extracellular production is usually preferred since this considerably facilitates the further isolation and downstream processing of the polypeptides obtained. Bacterial cells such as the strains of E col 25 mentioned above normally do not secrete proteins extracellularly, except for a few classes of proteins such as toxins and hemolysin, and secretory production in E. coli refers to the translocation of proteins across the inner membrane to the periplasmic space, Periplasmic production provides several advantages over cytosolic production, For example, the N-terminal anino acid sequence of the secreted product can be identical to the natural gene product after cleavage of the secretion signal sequence by a 30 specific signal peptidase. Also, there appears to be much less protease activity in the periplasm than in the cytoplasm, In addition, protein purification is simpler due to fewer contaminating proteins in the periplasm. Another advantage is that correct disulfide bonds may form because the periplasr provides a WO 2013/128031 PCT/EP2013/054262 87 more oxidative environment than the cytoplasm. Proteins overex pressed in Eco are often found in insoluble aggregates so-called inclusion bodies These inclusion bodies may be located in the cytosol or in the peripiasm the recovery of biologically active proteins from these inclusion bodies requires a denaturation/refolding process. Many recombnant proteins, including therapeutic protein re 5 recovered frorn inclusion bodies. Alternatively as will be clear to the skilled person, recominant strains of bacteria that have been genetically modified so s to secrete a desired protein, and in particular a polypeptide of the invention, can be used, Thus according to one non-limiting embodiment of the invention the polypeptide of the invention is a polypeptide that has been produced intracellularly and tht has been isolated front the host ell, and 10 in particular from a batter i cell or from an inclusion body in a bacterial cell According to another non limiting embodient of the invention, the polypeptide of the invention is a polypeptide that has been produced extracellularly, and that has been isolated from the medium in which the host cell is cultivated. Some prefer ed, but nonriritng promoters for use with these host cels inc ude: - for expression n . col: lac promoter (and derivatives thereof u h as the lacUV promoter; 15 arabinose promoter; left PL) and rightwr (PR) promoter of phage lambda; promoter of the trp operon; hybrid lac/trp promoters (tac nd trc); T7promoter (more specifically that of T7-phage gene 10) and other 1-phage promoters; promoter of the Tn10 tetracycline resistance gene engineered variants of the above promoters that include one or more copies of an extraneous regulaory operator sequence; 20 - for expression in S cereviae: constitute: ADH1 (alcohol dehydrogenase 1), ENO (enolase), CYC1 cytochromee c iso-1), GAPDH (glyceradehydes-3-phosphate dehydrogenase), PGK1 (phosphog ycerate kinase), PYK1 (pyruvate kinase); regulated: GAL,10,7 (galactose metabolic enzymes ADH2 alcohol dehydrogenase 2, PHO (acid phosphatase) CUP copper metallothionein); heterologous: CaMV (cauliflower mosaic virus 35 promoter) 25 for expression in Pichia pastors the AOX1 promoter (alcohol oxidase I); - for expression in rmammaan ce Is: human cytomegalovirus (hCMV immediate early enhancer/promoter; hunan cytomegalovirus (hCMV immediate early promoter variant that contains Wo tetracycline operator sequences such that the promoter can be regulated by the Tet repressor; Herpes Simplex Virus thymidine kinase (TK) promoter Rous Sarcoma Virus long terminal 30 repeat (RSV LTR) enhancer/promoter; elongation factor la (hEF-la) promoter from human, chirpanzee, mouse or rat; the SV40 early promoter; HIV-1 long terminaI repeat promoter; -actin promoter; WO 2013/128031 PCT/EP2013/054262 88 Some preferred, but non-imiting vectors for use with these host celIs include: - vectors for expression in mammalian cells: pMAMneo (Clontech) pcDNA3 (Invitrogen), pMClneo (Stratagene , pG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-1 (8-2) (ATCC 371 10) pdBPV MMTneo (342-12) (ATCO 37224), pRSVgpt (ATCC37199) pRSVneo (ATCC37198), pSV2-dhfr (ATC 5 37 46 , pUCTag (ATCC 37460) and 1ZD35 (ATCC 37565, as well as viraKbased expression systems, such as those based on adenovirus vectors for expression in bacterial cells: pET vectors (Novagen) and pQE vectors (Qiagen - vectors for expression in yeast or other fungal cels: pYE2 (Invitrogen) and Pichia expression vectors (Invitrogen); 10 - vectors for expression insect cells: pBlueBacl (Invitrogen) and other baculovirus vectors - vectors for expression in plants or pWant cels: for example vectors based on cauliflower mosaic virus or tobacco mos ic virus, suitable strains of Agrobacterium, or Ti-plasmid based vectors, Some preferred, but non-limiting secretary sequences for use with these host cells include: - for use in bacterial cells such as E coli: PeB, Ba, OmpA, OmpC, OmpF, Omp Stil, PhoA, PhoE, 15 MalE, Lpp LamB and the like; TAT signal peptide, hemolysin C-terminal secretion signal; - or use in yeast: a-mating actor prepro-sequence, phosphatase (phol), invertase (Suc), etc.; for use in mammalian cells: indigenous signal in case the target protein is of eukaryotic origin; marine Ig K-chain V- 2-C signal peptide; etc. Suitable techniques for transforming a host or host cel of the invention wil be clear to th e skiled 20 person and may depend on the intended host cell/host organism and the genetic construct to be used. Reference is again made to the handbooks and patent apphcations mentioned above. After transformation, a step for detecting and selecting those host cells or host organisms that have been successful transformed Vt the nuceotde equence/genetic construct of the invention may be performed. This may for instance be a selection step based on a seectable marer present in the 25 genetic construct of the invention or a step involving the detection of the polypeptide of the invention e.g., using specific antibodies, The transformed host cell (which may be in the form or a stable cell line) or host organisms (which may be in the form of a stable mutant line or strain) form further aspects of the present invention. Preferably, these host cels or host organisms are such that they express, or are at least) capable so of expressing (e.g., under suitable conditions), a polypeptide of the invention (and in case of a host organism: in at least one cei, part tissue or organ thereof The invention also includes further WO 2013/128031 PCT/EP2013/054262 89 generations progeny and/or offspring of the host cel or host organism of the invention, that may for instance e obtainedbr yedivsin o by sexua or asexual reproduction. To produce/obtain express sion of the polypeptides of the invention, the transformed host cell or transformed host organisms may generally be kept, maintained and/or cultured under conditions such 5 that the desired) polypeptide of the inventim is expressed/produced. Suitable conditions wiH be lear to the skilled person and will usually depend upon the ost cell/host organism used, as well as on the regulatory elements that control the expression of the (relevant) nucleotide sequence of the invention. Again, reference is made o the handbook and patent applications mentioned above in the paragraphs on the genetic constructs of the invention. 10 Generally suitable conditions may include the use of suitable medium the presence of a suitable source of food and/or suitable nutrients the use of a suitable temperature, and optionaly he presence of a suitable inducing factor or compound (e.g, when the nucleotide sequences of the invention re under the control of an inducible promoter); al of which ray be selected by the skied person. Again under such conditions, the polypepties o the invention may be expressed in a consttutive manner in a 15 transient manner or only when suitably induced, It wil also be clear to he skiHed person that the polypeptide of the invention may (first be generated in an immature form as mentioned above), which may then be subjected to post translational edification, dependin on the host cell/host organism used. Also the polypeptide of the invention may be glycosylated, again depending on the host cel/host organism used. 20 The polypeptide of the invention nay then be isolated from the host cell/host organism and/or from the medium in which said host cel or host or nism was cultivated using protein isolation and/or purification techniques known per se, such as (preparative) chromatography and/or electrophoresis techniques, differential precipitation techniques, affinity techniques (e.g., using a specific, cleavable anino acd sequence fused with the polypeptide of the invention and/or preparative immunological techniques e. using antibodies against the poypeptide to be isolated) compositions of the invention The invention further relates to a product or opposition contain or comprising at least one polypeptide of the invention and/or at least one nucleic acid of the invention, and optionalyone or S mrnr further components of such compositions known per se, i.e. depending on the intended use of the composition. Such a product or composition may or example be a pharmaceutial composition (as described herein a veterinary composition or a product or composition for diagnostic use (as also WO 2013/128031 PCT/EP2013/054262 90 described herein). Some preferred but non-[riting examples of such products or compositions will become clear from the further description herein Generally for pharmaceutical use, the polypeptide of the invention may be formulated as a pharmaceutical preparation or composions comprising at least one polypeptide of the invention and at 5 east one pharmaceutically acceptable carrier, diluent or excipient anor adjuvant nd optionally one or more further pharmaceutically active polypeptides and/or compounds. Such suitable administration forms - which may be solid semi-solid or liquid depending on the manner of administration - as well as methods and carriers for use in the preparation thereof, will be clear to the skilled person, and are further described herein. 10 Thus, in a further aspect, the invention relates to a pharmaceutical composition that contains at least one polypeptide of the invention and at least one suitable carrier, diluent or excipient (i.e., suitable for pharmaceutical use), and optionally one or more further active substances. n a particular aspect, the invention relates to a pharmaceutical composition that contains a polypeptide o the invention selected from any of SEQ ID NOs: 11841 and at east one suitable carrier, diluent or excipient (i.e. suitable for 15 pharmaceutical us j and optionaly one or more further active substances. Generally the polypeptides of the invention can be formulated and administered in any suitable manner known per se, for which reference is for example made to the general background art cited above (and in particular to WO 04/041862, WO 04/041863, WO 04/041865, WO 04/041867 and WO 08/02007 as wel as to the stn har ndbok such as Remington s Pharmaceutical Sciences, 8 Ed., 20 Mack Publishing Company USA (1990) Remington the Science and Practice of Pharmacy, 21st Edition Lippincott Williams and Wilkins (2005); or the Handbook of Therapeutic Antibodies (S. Dubel, Ed.) Wiley, Weinheim 2007 (e for example pages 252-255) For example the polypeptides of the invention may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments (including ScFv's and 25 diabodies) and other pharmaceutically active proteins. Such formulations and methods for preparing the same will be clear to the skilled person, and for example include preparations suitable for parenteral administration (for example intravenous intraperitoneal subcutaneous, intramuscular intraluminal, intraarteral or intrathecal administration Preparations for parenteral administration may for example e sterile solutions, suspensions 30 dispersions or emulsions that are suitable for infusion or infection. Suitable carriers or diluents or such preparations for example include, without imitation, those mentioned on page 143 of WO 08/020079. Usually, aqueous solutions or suspensions will be preferred.
WO 2013/128031 PCT/EP2013/054262 91 The polypeptides of the invention may be administered intravenously or intraperitoneally by infusion or injection. Solutions of the polypeptides of the invention can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these 5 preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical dosage forms suiable for injectinsio i n include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form nust be sterile, fluid and stable under 1o the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, ycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The is prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. 20 Sterile injectable solutions are prepared by incorporating the polypeptides of the invention in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present 25 in the previousKy steri erec solutions, in a preferred aspect, the polypeptides of the invention and/or compositions comprising the same are administered to the pulmonary tissue. In the context of the present invention, "pulmonary tissue" is for the purposes of this invention equivalent with lung tissue or lung, The lung comprises 2 distinct zones: a conducting and a respiratory zone, within which the airway and vascular compartments lie (see 30 e.g, "Pulmonary Drug Delivery", Edited by Karonine Bechtold-Peters and Henrik Luessen, ISBN 978-3 87193-322-6 pp. 16-28, 2007).
WO 2013/128031 PCT/EP2013/054262 92 For pulmonar delivery, the polypeptides of the invention may be applied in pure form, ie., when they are liquids or a dry powder. However, it will be preferred to administer them to the pulmonary tissue as composition or formulation comprising a polypep ide of the invention and a carrier suitable for pulmonary delivery. Acordingly the pre ent invention al o relates to a pharmaceutic a i composition 5 comprising the polypeptide of the invention and a carrier suitable for pulnon ry delivery. Carriers suitable for pulmonary delivery are known in the art. The po peptides of the invention ay aso be administered as micro- or nanopartides of pure drugs with particle sizes and di tributons favorable or pulmonary delivery. Accordingly, the present invention also relatesto pharmaceutical device suitable for the 10 pulmonary delivery of the polypeptides of the invention nd suitable in the use of a composition comprising the same. This device may be an inhaler for liquids (e.g., a suspension of fine solid particles or droplets comprising the polypeptide of the invention. Preferaby this device is an arosol nebulizer or metered dose inhaler corprising the polypeptide of the invention. The device may aso be a dry powder inhaler comprising the polypeptide of the ivenion the form of a dry powder. 15 in a preferred method the administration to the pulmonary tissue is performed by inhaling the polypeptides of the invention and/or the opposition comprising the same in an aerosol cdoud According to the invention, inhaing of the aerosol loud can be performed by an inhaler device. The device should generate from a formulation comprising the polypeptides of the invention (and/or composition comprising the same) an aerosol cloud of the desired parties size (distribution) at the 2 appropriate moment of the mammal's inhalation cy de, conaining the right dose of the polypeptides of the invention "Pulmonary drug delivery" Bechtold-Peters and Luessen, eds., ISBN 978-3-87193-322-6, page 125, 2007) In the context of the present invention, "aerosol" denote a suspension of fine solid parties or liquid drop ets (or cornbination thereof) in g gs wherein, for the purposes of this invention, the particles 2s and/or droplets comprise the polypepudes of the invention The device should generate from the fornulation an aerosol coud of the desired partide size distribution) at the appropriate moment of the mammal inh action cyde, containing the right dose of polypeptides of the invention. V rious inha ation systems are e.g. described on pages 129 to 148 in the review ("Pulmonary Drug 30 Delivery BechtA Peters and Lue.sen, eds., supra) and include, ut are not limited to, nebulizers, metered dose inhalers, metered dose liquid inhaler and ry powder inhalers. Devices taking into account optinized and individualized breathing pattern for controlled inhalation mnnoeuvres may aso WO 2013/128031 PCT/EP2013/054262 93 be used (see AKITA* technology on page 157 of "Pulmonary Drug Delivery", Bechtold-Peters and Luessen, eds supra) However, not only the device is important to pulmonary delivery of the polypeptides of the invention but also the right formulation is critical to achieve an effective delivery. This can be in principle 5 achieved by using one of the fo lowing approaches: - Administration of a queou solutions or suspensions comprising the polypeptides of the invention (e.g, nasal drops into the naal cavities; - Nebulisation of aqueous solutions or suspensions comprising the polypeptides of the invention; -Atornization by means of liquefied propellants; and 1o .- Dispersion of dry powders. Hence formulations of the polypeptides of the ivention have to be adopted an adjusted to the chosen inhalation device. Appropriate formulations, i.e. the excipients in addition to the polypeptides of the invention re e.g., described in chapter IV of "Purnonary Drug Delivery Bechtold-Peters and Lessen, eds supra. In this respect, reference i also mde to WO 2010/0g18S6 15 The amount of the polypeptides of the invention required for use in treatment wilt vary not only with the particular polypeptide selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician, Also the dosage of the polypeptides of the invention will vary. The desired dose may conveniently be presented in single dose or as divided doses administered 20 at appropriate intervals, for examp e, as two, three, four or more sub-doses per day The sub-dose itself may be further divided e.g, into a number of discrete loosely spaced administrations such as multiple inhaatios from an insuffIator. An administration regimen could include long-term, daily treatment. By "long-term is meant at ea two weeks and preferably, several weeks months, or years of duration. Necessary modifications in 2 this dosage range may be determined by one of ordmary ski in the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutica Sciences (Martin .W, ed. 4) Mac Publishing Co., Easton, PA. The dosge can also be adjusted by the individual physician in the event of any compLiltation. 30 Uses of the DOpo endes of the inn The invention further relates to applications and use of the polypeptides, nucleic acids, hos cells, and compositions described herein as well as to method for the prevention and/or treatment P.
WO 2013/128031 PCT/EP2013/054262 94 aeruginosainfection. Some preferred but non-limitng applicatons and uses wil become clear from the further description herein. The polypeptides and compositions of tNe present invention can geeraly be used to neutralze P oruginosa such as to module, inhibit and/or prevent infectivity of P. oerugiosa to mo ute, inhibit 5 and/or proven colonization of the Iost by P. aeruginoso to modulate, inhibit and/or prevent TTSS virulence mechansms of P. oerugnoso, to modulate, inhbit an d/r prevent injection by P. oeruginoso into the host cell of various exotoxins, to modulate inhibit and/or prevent pore-mediated increases in host cell membrane permeability induced by P. aerugnos, to modulate, inhibit and/or prevent activation of broad cellular defence responses induced by P. aerugnos and/or to modulate, inhibit 10 and/or prevent triggering of tissue-damaging inflammation induced by P aerugnoso in one aspect, the polypeptides, and compositions of the present invention can neutralize P. aeruginos in ectivitya at least m. prrably t leat 5%, such as at least 10% or at least 25% preferably, at east 50%, at least 60%, at least 70%, at leat 80%, or at east 90% or more, suh as 100% compared to the P. aeruginosa infectivity under the same conditions but without the presence of th polypeptide of 1 the invention, measured in any suitable manner known per e, for example using one of the says described herein such as the TTSS- ependent cytotoxicity assay s described in the Example section). The invention also rea te to a method for the prevention and/or treatment of infection with P. oeruginoso, said method comprising administering to subject in need thereof a pharmaceutically active amount of a polypeptide of the invention, and/or of a pharmaceutical composition comprising the 20 same. As such the polypeptides and compositions of the present invention can be used for the prevention and/or treatment of P eruginosa infection. Exrnpes of patient groups susceptibe to P. aeruginsa infection will be clear the sied person based on the disclosure herein, and for example include (without being limiting the following patent groups ventilator- associated pneumonia (VAP), 2 hburn vctms mechanical ventiated patients Cystc Fbrosis (C patients, hem topoietic cell transplantation patients, bone marrow transplant patients, patients undergoing surgery, patients with chronic obstructive pulmonary disease (COPD), patients with bronchiectasis, patients with sepsis and patients with cancerassociated neutropena According y, the present invention alo relates to a method for the prevention and/or treatment of so P. aeruginoso infections in ventilator-associated pneumonia (VAP), burn victims, mechanical ventilated patients, Cystic Fibrosis (CF) patients, hematopoietic cell transp antation pa ients bone marrow transplant patients, sur ery, chronic obstruct tive pulmonary disease (COP) bronchiectasis, s psis, WO 2013/128031 PCT/EP2013/054262 95 cancer-associated neutropenia, said method comprising administerin to a subject in need thereof, a pharmaceutical active amount of at least one polypeptide of the invention or a compositon of the invention. The invention also relates to the use of a polypep ide of the invention in the preparation of a 5 pharmaceutical composition for prevention and/or treatment of P. aeruginosa infection including but not limited to P. aeruginose infections in ventilator-associated pneumonia (VAP), burn victirns, mechanical ventilated patients, Cystic Fibrosis (OF) patients, hernatopoietic cel transplantation patients, bone marrow transplant patients, surgery chronc obstructive pulmonary disease (COPD , bronchiectasis sepsis, cancer-assocated neutropenia; and/or for use in one or more of the methods to described herein. The invention also relates to a polypeptide of the invention or a composition comprising the same for prevention and/or treatment of P. aerugnosa in section such as P. aeruginosa infections inCuding (but not limited to P. aeruginosa infections in venti at or- associated pneumonia (VAP , burn victims mechanical ventilated patients, Cystic Fibrosis (CF patients, henm topoietic ceH transplantation patients, 15 bone m arrow transplant patients surgery, chronic obstructive pulmonary disease (COPD), bronchiectasis, sepss, cancer-associated neutropenia, In the context of the present invention, the term "prevention and/or treatment' not only comprises preventing and/or treating the infection but also generally comprises slowing or reversing the progress of the infection reducing the severity and/or the duration of the infection and/or preventing a 20 further increase in the severity of the infection, and generally any pharmacologica action that is beneficial to the patient being treated. In another aspect, the invention relates to a method for immunotherapyand in particular for passive immunotherapy, which method comprises a ministering to a subject suffering from or at risk of P. aeruginosa infe tion a pharmaceutical active amount of a polypeptide of the invent on and/or of a 25 pharmaceutical composition comprising the same, The subject to be treated may be any warm-blooded animal, but is in particular a marnmal, and more in particular a human being. As wiH be clear to the skilled person, the subject to be treated will in particular be a person suffering from, or at risk of P. aeruinosa infection, induding but not limited to the patient groups susceptible to P. aeruinose infect on mentioned herein. 30 Thus, in genera, the polypeptides according to the invention and/or the compose ions comprisin the same can be administered in any suitable manner. For example (but not limited thereto) the polypeptides according to the invention and compositions comprising the same can be administered WO 2013/128031 PCT/EP2013/054262 96 intranasally intratracheally, by inhalation and/or by any other suitable form of pulmonary delivery. Methods for pulmonary delivery, intranasal delivery, intrtracheally and/or delivery by inhalation of polypeptide of the invention will be known to the skilled person nd are e.g. described in the hand oo Drug Delivery: Principles and Applications" 2005 by Binghe Wang, Teruna Siahan and Richard Soltero 5 (Eds. Wiley lnterscience (John Wiley & Sons)); in "Pharmacology PreTest T Self-Assessment and Review" (11a [diton by Rosenfeld G. Loose-Mitchell DS. and in "Pharmcology" (3F Edition) by Lippincott Williams & Wi Ikins, New York; Shlafer M. McGraw-Hill Medical Publishing D'vision, New York; Yang K.Y. Graff LR, Caughey AB. Blueprints Pharmacology, Blackwell Publishing Accordingly, the present invention also relates to a method for administering an effective amount 10 o a polypepide of the invention and/or a composition comprising the same, wherein said method comprises the step of administering the polypeptide and/or composition comprising the same to the pulmonary issue In such method, the polypeptide and/or a composition comprising the same can be administered by any method known in the art for pulmonary delivery such as e.g., by use of an inhaler aerosol, metered dose inhaler, nebulier) or intranasal delivery device. 1 In a preferred aspect of the invention, the poIypeptide will bind and/or neutralize P ceruginos present in the pu monary tissue. Preferably in such method for pulmonary delivery at least 5%, preferably at east 10%, 20% 30% 40%, more preferably at least 50%, 0% 70%, and even more preferaby at least 80% or more of the polypeptide of the invention is stable in the pulmonary tissue for at least 12 hours, preferably at least 24 hours more preferably t least 48 hours. 20 Accordingly, the invention relates to a method for delivering a polypeptide of the invention to the pulmonary tissue of a subject without being inactivated, said method comprising the step of pulmonary administering said polypeptide of the invention to said subject. The invention also relates to a method for the prevention and/or treatment of P. oeruginosa infection, said method comprising administering to the pulmonary tissue of a subject in need thereof, a 25 pharmaceuticaly active amount of a polypeptide of the invention and/or of a pharmaceutical com position comprising the same. More in particular, the invention relates to method for the prevention and/or treatment of P. oeruginosa infections in ventilator-associated pneumon-a (VAP), burn victims, mechanical ventilated patients, Cystic Fibrosis (CF) patients, hematopoietic cell transplantation patients, bone marrow 30 transplant patients, surgery, ch ronic obstructive pulmonary disease (COPD), bronchiectasis, sepsis, cancer-associated neutropenia, said method comprising administering, to the pulmonary tissue ofa WO 2013/128031 PCT/EP2013/054262 97 subject in need thereof, a pharmaceutical active amount of a polypeptide of the invention and/or of a pharmaceutical composition omprising the same. The polypeptides of the in mention and/or the compositions comprising the same are adninstered according to reme of treatment that is suitable for preventing and/or treating the P. ieruginosa 5 infection. The clinician will generally be able to determine a suitable treatment regimen depending on factors such as the patient group to be treated, the seven of the infection nd/or the severity of th symptoms thereof, the specific polypeptide of the invention to be used, the specific route of administration and ph rmaceutica formulation or composition to be used, the age, gender, weight, diet, general condition of the pient and similar factors wel known to tm crin 10 Generally, the treatment regimen will comprise the administration of one or more polypeptides of the invention or of one or more compositions comprising the same in one or More pharmaceutical ly effective amount or doses. The specific amounts) or doses to administered can be determined by the clinician agan based on the actors cited above. Generally, for the prevention and/or treatment of the P. a ruginsa infection and depending on 15 the specific patient group to be treated, the potency of the polypeptide of the invention to be used, the specific route of administration and the specific pharmaceu ical formation or Composition used, the polypeptides of the invention wil generally be administered in an mount between 1 gram and 1 microgram per g body weight per day preferably between 0.1 gram and 10 microgram per kg body weight per day, most preferably between 0.01 gram and 100 microgram per kg body weight per day such 20 as about 0.1, 0.5 1, 2, 5 or 10 milligram per g body weight per day, either continuously (e. by infusion), as a sin e daily dose or as multiple divided oses durn the day. Polypeptides of the invention that contain a haf-life extending moiety may be administered in an amount between 1 milligram and 100 milligrar per kg body weight, pre ferably between 1milligram and 50 milligram per kg body weight such as about 10, 15, 20 or 30 milligram per kg body weight once or twice a month. The linician will 25 generally be able to determine a suitable dily dose depending on the f ctars mentioned herein. It wil also be clear that in specific cases the clinic n ma choose to deviate from these amounts, for example on the basis of te factors cited above and his expert judgment. When the polypeptide and/or a composition comprising the ae is adninis ered to the pulmonary tissue the treatment regime may be once or twice daily, preferab y once daily or once every 30 2, 3, 4, 5, 6, or 7 days. Usually in the above method, single polypeptide of the invention wil be use i.t is however within the scope of the invention to use two or more polypeptides of the invention in combination.
WO 2013/128031 PCT/EP2013/054262 98 The polypeptides of the invention may also be used in combination wth one or more further pharmaceutically active compounds or principles i.e. as a combined treatment regimen which may or may not lead to synergistic effect. Again, the ci nian wil be able to select such further compounds or principles, as well as a suitable combined treatment regimen based on the factors cited above and his 5 expert judgement, in particular the polypeptides of the invention may be used in combination with other pharmaceutical active compounds or principles that are or can be used for the prevention and/or treatment of P aeruginosa infection as a result of which a synergistic effect my or may not b obtained. Examples of such compounds nd principles, as well as routes, methods and pharmaceutical 10 formulations or composition for administerin hem will be clear to the clinician and include (without being uniting) Amnoglycosides tobramycin, gentamicin, sisomycin amikacin, netirnicin), luoroquinolones (sitafloxacin, ciprofloxacin levofoxacin, ofloxacin;, Polyryxins (polymyxin A, polymyxin B polymyxin C polymyxin D, polymyxin colistin; colimycin), UDP-N acetylglucosamne-3 enolpyruvyltransferase (NAM; MurA inhibitors (fosfomycin), Macrolides (azithrormycin) Oxazolidinones 15 (linezolid), Penicillins (methicillin; Carboxypenicillin: ticarcillin; Ureidopenicillins: piperacillin, alocillin), arbapenems (doripenem biapenem imipenem meropenem, topopenem), cephalosporins (ceftazidire, cefepime, aztreonam, ceftobiprole, CXA-101 (Calixa)), or other beta- actamase inhibitors such as clavu anate, and beta-lactamase inhibior combinations (combination of piperacillin and tazobactam, combination of ticarcillin and clavulanic acid, combination of imipenem and cilastatin); or 20 any combination thereof. When two or more substance or principles are to be used as part of a combined treatment regimen they can be administered via the same route of administration or via different routes of administration at essentially the same time or at different tmes ( essentially simultaneous consecutively, or according to an alerating regimeA When the substances or principles are to be 25 administered simultaneously vi the same route of administration, they mny be administered as different pharmaceutical formulations or compositions or part of a combined pharmaceutical formulation or composition s will be clear to the skilled person. Also, when two or more active substances or principles are to be used as part of a combined treatment regimen each of the substances or principles rnay be administered in the same amount and o according to the same regimen as used when the compound or principle is used on it own and s u h combined use may or may not lead to a synergistic effect. However, when the combined use of the two or more active substances or principles leads to a synergistic effect, it may also be possible to reduce the WO 2013/128031 PCT/EP2013/054262 99 amount of one more or all of the substances or principles to be administered while still achieving the desired therapeutic action This may for example be useful for avoiding, limiting or reducing any unwanted side-effects that are associated with the use of one or more of the substances or principles when they are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect. The effectiveness of the treatment regimen used according to the invention may be determined and/or followed in any manner known per se for the disease or disorder involved as will be clear to the clinician. The clinican will also be able, where appropriate and on a case-by-case basis, to change or modify a particular tre tent regimen, so as to achieve the desired therapeutic effect, to avoid, limit or 10 reduce unwanted side-effects, and/or to achieve an appropriate balance between achieving the desired therapeutic effect on the one hand and avoiding, limiting or reducing undesired side effects on the other hand. Generally, the treatment regimen will be followed until the desired therapeutic effect is ac eyved and/or for as long as the desired therapeutc ef ect is to be maintained. Again, this can be determined by 15 the clinician, Further uses of the polypeptides nucleic acids genetic constructs and hosts and host cells of the invention wil be clear to the skilled person based on the disclosure herein, For example, and without limitation, the polypeptides of the invention can be linked to a suitable carrier or solid support so as to provide a medium than can be used in a manner known per se to purinf PcrV protein fron P. eruginosa 20 rom compositions and preparations comprising the ame. Derivatives of the polypeptides of the invention that comprise a suitable detectable label can also be used as markers to determine (qualitatively or quantitatively the presence of PcrV protein of P. ueruginosa in a composition or preparation. The mventon wm now be furt her described by means of the following non-limiting preferred 25 aspects examples and figures. The entire contents of all of the references (including literature references issued patents published patent applications, and co-pending patent applications) cted throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove. 30 WO 2013/128031 PCT/EP2013/054262 100 EXAMPLES Example 1 Materials and Methods 1.1 Generation of recombinant PcrV protein (rPcrV) 5The gene encoding full length PcrV protein of reference strain PA01 amino acid residues 1-294, ee Table A-1) was cloned into an in house pUC119 dered expression ye tor. The vector contaied the La Z promoter, a resistance gene for kanamycin, amulticloning site and the pe 13 leader sequence. In frame with the rPvrV coding sequence, the vector co ed for a C-terminal His tag. Upon transformation in E coli (TG-1), expression cultures were grown and expression was induced by addition of 1 rnM IPTG 10 and allowed to continue fr4 hours at 37'C. Cells were harvested by center ugation and cell pellets were lysed by sonication. Cytcsolic fractions were isolated by centrifugation. The recombinant protein was purified from the crude extracts v immobilized net aaffinity chromatography (IMAC) on HisTrap FF crude 1 ml coluns and buffer exchange into D-PBS (HiPrep 26/10 column) followed by icn exchange chromatograph using a Soure15Q (2ml CV) column and finally a gel filtration with a Superdex75 10/300 15 GL column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The purity and homogeneity of the protein was confirmed by SDS-PAGE and analytical size exclusion. 1.2 Generation of GST-PcrV protein The PcrV coding sequence from strain PA1 (arino acid residues 1-294, see Table A-1) was cloned 20 into the expression vector pET42a(+ (EMRBiociences, Darmstadt Germany) to generate a GST-PcrV genetic fusion. The resulting vector was transformed into E ci BL21 DE3 cells (Invitrogen). GST-rPcrV fusion protein was expressed and puniied from E cir L21 DE3 as follows. Al liter culture batch of E col expressing GST-PcrV was grown and induced for expression by addition of 1rmM IPT Folowing further growth for 3 hrs at 37C, bacteria cells were pelleted by centrifugaton and lysed via sonication. 25 After the lysate was cleared by centrifugation it was passed over a gutathione sepharose column (STrap F) followed by desalting step (HiPrep26/10 column) and an additional ion exchange chromatography step using a Source15Q columrn (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). The purity (>90%) and homogeneity of the protein was confirmed by SDS-PACE. 30 13 Generation of PcrV sequence variant To determine whether an i-PcrV therapy is universe applcabe toa rety of P. a ruginoso clinical isolates, Lynch (Microbial pathogenesis 48: 197-204 2010) determined the genetic heterogeneity WO 2013/128031 PCT/EP2013/054262 101 of PcrV by sequencing PcrV from 90 clinica isolates collected from 3 distinct geographical areas. This way, they identi ied 14 dif erent PcrV variants (see Tabe A-1, PcrV variant 01- PcrV variant 15), Using a similar approach h, 207 clinical I solates - collected over geographically well spread locations (Prof. Vaneechoutte, UGent, Begum) - were used to provide additional information about the genetic 5 heterogeneity o PcrV, Eight novel PcrV sequence variants were identified this way (see Tabl A-, PcrV variant 16 - PrV vr-ant 23). For the generation of all PcrV sequence variants gene encoding the 23 different PcrV vanints (amino aid residues 1-294, see Table A-i were cloned to ether with the gene codng for the Hisr tag into the pET42a(+) ye tor (EMBBios ien es, Darmstadt, Germany) to generate GST-PcrV-H-is genetic 10 fusions. GST-PcrV-His fusion protein was expresed and purified from E coli (BL21 DE3) transformed with pET42a(+)-PcrV-Hi as fonows. A liter culture was grown and induced for expression y addition of 1mM IPTG, Following further growth for 3 hrs at 3TC, bacteria cells were pelleted by centrifugation and lysed via sonication. After he ysate was cleared by centrifugation, it was passed over GSTrap F column (GE Healthcare Bio-Sciences AB Uppsala, Sweden) and buffer exchanged to D-PBS after which 15 pure proteins (>90 %) were obtained. 1.4 Generation of anti-PcrV Fab molecules The gene segments encoding VL and VH frorn 3 Fab molecules, Fab 13.37 WO 2009/073631; SEQ ID NOs: 13 and 37), Fab 26.24 (WO 2009/073 31 SEQ ID NOs: 26 and 24) an Fab 3 .36 (WO 20 2009/073631; SEQ ID NOs: 35 and 36 were cloned into an in-house human Ig 1/K ab expression vector. This vector contained the LacZ promoter, a resist nce gene for kanamycine, two separate cloning sites precede by a pelB (light chan) or agene3 heavy chain leader sequence n frame wihthe heavy ch in coding sequence, the expression vector encoded a C-terminal HA tag and a Hi tag. The Fab fragments were expressed in E, col and purified under native, non-reducing conditions via immobilized 25 metal affinity chromatography (IMAC on HisTrap FF crude 1 rml (GE healthcare, Buckinghamshire, United Kingdom) folowed by affinity hrornatography for the human Fabt light chain (CaptureSelect iC-kappa (Hu), BAC) and size exclusion chromatography on a Superdex75 10/300 GL column GE Healthcare GE heathcare, Buckinghamshire, United Kingdom or dealtingvi Zeb spin columns (Pierce Rock ord, IL, USA) Arnino acid sequences of the va able heavy and varnible light chain of the 3 abs are shown in s3 Table A-2. The amino acid sequences of the constant heavy and light chain are shown in Table A-3.
WO 2013/128031 PCT/EP2013/054262 102 Example 2: Immunization of llamas with rPcrV protein, cloning of the heavy chain-only antibody fragment repertoires and preparation of phage 2J1 Immunizations After approval of the Ethical Committee of the faculty of Veterinary Medicine University Ghent, 5 Belgium), 4 Ilamas (designated No. 504 0, 506 and 507) were immunized with 4 intramuscular infection (25 or 10 ug/dose at two weekly intervals of rPcrV protein formuted in Stirmune (Prionics, 2.2 Cloning of heavy chain-only antibody fragment repertoires and preparation of phage 10 Following the final immunogen nation, two 150-mL b ood samples, collected 4 and 8 days After the last antigen injection were colected per animal Fror the blood samples periphera blood rnononuc:ear cell (PBMCs) were prepared using Ficoll-Hypaque according to the manufacturer's instructions (Amersharn Bioscience, Piscataway NJ, USA). From the PBMCs, total RNA was extracted and used as starting material or RT-PCR to amplify the VHH/Nanobody-encoding DNA segments. The 15 PCR-ampliied VHH repertoire was cloned via specific restriction items into vector designed to facial ate phage display of the VHH library. The vector was derived from pUC119 and contained the LacZ promoter, a M13 phage gll protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multiple cloning site and a hybrid g|l-peiB lea er sequence. In frame with the VHH/Nanobody coding sequence the vector encoded a C-terminal triple Flag tag and a Histag. Phage were prepared according to 20 standard protocols (see for example WO 04/041865, WO 04/041863 WO 04/062551, W 05/044858 and other prior art and applications filed by Ablynx NV. cited herein) and stored after filter sterilization at4*Cforfutheruse. Example 3: Selection of PcrV specific VHHs via phage display 25 VHH repertoires obtained rom all hamas and cloned in phage library were used in two selection strategies In afir t selection strategy, rPcrV protein in house produced, see Example 1, se tion 1,1) was immobilized at a concentration of 15 ug/ml on a Nunc Maxisorp plate next to negative control of 0 ug/mi ntigen. Following incubation with the phage librres and extensive washing, bound phages were eluted with trypsin ( 1mg/mL Eluted phage were amplified and applied in a second round of selection 30 on 10 ug/ml rPcrV and 0 ug/ml (control). In a second selection strategy, 50 nM and 5 nM of biotinylated rPcrV proemn (bio- PcrV; biotinylated according to the manuf Acturer in ructions using Sulfo-NHS-LC-Biotin (Pierce, Rockford, It, WO 2013/128031 PCT/EP2013/054262 103 USA)) were captured on a neutravidin coated (1 ug/mil) Nunc Maxisorp plate, next to a negative control of only neu ravidin (1 ug/m) Follown in cubation with the phage libraries and extensive washing, bound phages were eluted with trypsin 1 mg/mL). Fluted phages were used to infect L coli. Infected E col cells were either used to prepare phage for the next selection round on 5 nM, 0.5 nM, 0.05 nM, 5 0.005 nM bio-rPcrV and a control neutravidin 1 ug/mI) or plated on agar plates (LB+amp+glucose2%) or analysis o individual VHH clones. Outputs of all selection rounds were analyzed for enrichment factor (number of phages present in eluate relative to controls) and the best selection conditions were chosen for further analysis, In order to screen a selection output for specific binders, single colonies were picked from the agar plate and grown 10 in 1 mL 9-deep-well plates. LacZ-controlled VHH expression was induced by addition of IPTG (1mM final) in the absence of glucose. Periplasmic extracts (in a volume of C80 u) were prepared according to standard protocols see for example WO 03/035694, WO 04/041865 WO 04/041863, WO 04/062551 and other prior art and application filed by Ablynx N.V cited herein 15 Example 4: Screening of periplasmic extracts for functional blocking Nanobodies 4.1 Screening in EUSA In a first step, periplasmic extracts were ested for binding to bio-rPcrV by binding ELUSA. In brief 10 nM bio-rPrV protein was captured on neutravidin (2 ug/m ) coated 96-wel Maxiorp plates (Nunc, Wiesbaden, Germany. Wells were blocked with a Casen solution 1%). After addition of a 10-fo d 20 dilution of the periplasmic extracts Nanobody binding was detected using a mouse ant-F ag-HR P conjugate (Sigma) and a subsequent enzymatic reaction in he presence of the substrate esTMB (3,3' 5,'-tetramentylbenzidine) (SDT, Brussels, Belgium). Clones showing ELISA signals of >2-fold above background were considered to encode positive PcrV binding Nanobodies. 25 4.2 Sequence determination The DNA sequence of the positive clones was determined. The amino acid sequences o the ant PcrV Nanobodies are shown in Table A-4, 4.3 Off-rate determination 30 Off-rate nalysis of al unique PcrV-binding Nanobodies was done by means of surface plasrnon resonance on a ProteOn instrument BIoRad). To this end, recombinant PcrV protein was covalently bound to a GLC ProteOn Sensor chip vi amine coupling on one ligand channel after which remaining WO 2013/128031 PCT/EP2013/054262 104 reactive groups were inactivated. Periplasmic extracts prepared from . coli cells expressing anti-PcrV Nanobodies were diluted 10-fold n injected at a flow rate of 45 uL/min during 2 minutes for binding to the immobilized rPrV, Between sample injections, the surface was regenerated with ProteOn Pho phoric acid solution 0.85%. Off-rates were determined by fitting a :l inter action moel Langmuir 5 model) onto the individual dissociation curves. Determined off-rates ranged from below detection limit of 3 10 s< to 4 x 10 w 4 with the vast ma ority of the clones having off-rates in the range of 1 x 0 1 x 10 sQ 4.4 Analysis in TTSS-dependent cytotoxicity assay 10 To identify Nanobodies able to prevent Pseudomonas oerugnosTTSS-mediated infection representative cIones were tested in a TTSS-dependent cytotoxicity essay using P3-X63-Ag8 (P3X63) mouse myeloma cells (ECACC Cell line) as the target. Examples of Pseudomonas oerugnoso cytotoxicity assays are e.g provided in Frank et al, (The Journal of infectious diseases 18 : 64-73, 2002), Vance et al (Infection and Immunity 73: 1706-1 713, 2005 , E Solh et at (Am J Respir, Crit. Care Med. 178: 513-519 15 2008), A total of 2 x 10 P3X63 cells/well were seeded in 96 well plates. Periplasmic extracts (diluted %) containing PcrV binding Nanobodie were pre-incubated with P. oerugnosa strain PA103, which was grown under calcium-depleted conditions (LB medium - 5 mM EGTA; Kir, Microbiology 151: 3575-3587, 2005 to induce expression of the TTSS. Following pre-incubation the mixtures of periplasmic extracts and bacteria were added to the P3X63 cells and after an incubation step of 3h at 37C, P3X6 3 cells were 20 stained wth Propidium Iodide (Sigma-Aldrich, St Louis, MO and fixated with 2% formaldehyde (Sigma Aldrich, St Louis, MO). The ability to prevent Pseudomonas zeruginoso infection and mediated cell death was quantified by mnonitorng the uptake of the propidiun iodide dye by dead cells using a FACS Array (Becton Dick inson, USA) nd FC Express software (Denovo USA Infections with strain PA1O3 were done at an average multiplicity of infection (MOI of 8 bacteria to 1 myeoma cell (range 6:1- 10:1). The 25 data were analyzed using Prism5 software (Graphpad). Cytotoxicity was normalized to dead cells in untreated samples and normalized data were used to calculate the % nhibiton according to the following formula: 30 100 WO 2013/128031 PCT/EP2013/054262 105 Wherein: y = ra (% dead cells) of the PA103 treated wells incubated with irrelvant control Nanobody z o verage (% dead cells) of the untreated we! s, incubated with irrelevant control Naonobody x=%ofdeadcel ofevouat dot point 5 A summary of the peripiasmic extract screening data is given in Table B-1. Table B-: Screening of periplasmic extracts containing expressed antPcrV Nanobodie Nanobody binding EUSA off-rate (s) T cytotox assay (OD 45nm) (% inhibition) 1E11 2 563.5 x 10 3 26802 2____0___ 3.x 10 2.4isx i }3 2G09 2.37 1.0x10 45 0.527x 10 2 3 10 251 1.5x10 25 [4C03 0.89 6 8 x 10~ 22 ~4G10 ~ 260 1.3 x 10 81 .E2 2.43 5.7 x 10 71 5H012 4.9x10 76 ~~05 -, .551_8 x 032 7C10 2,55 82 x 10 r 7E09 2.59 4.0x10 16 10C05 { ND 6 0 x10~ 25 1B09 ND 4.8 x10 37 12B02 ND 4.4 x 10 14 13F07 ND 9 2x10~ 13 | 14B10 ND 9.2 x10 20 14E10 N~ D 6.0 >x0 22 10 WO 2013/128031 PCT/EP2013/054262 106 E example 5: Characterization of purified monovalent anti-PcrV Nanobodies 5.1 Preparation of selected Nanobodies Clones selected from the screening described in Example 4 were further characterized. Selected Nanobodies were subcloned into an in-house pUC119 derived expression vector. The vector contained 5 the LacZ promoter, a resistance gene for kanamycin, a muticloning site and the pel Bleader sequence,. In frame with the Nanobody coding sequence, the vector coded for a C-terminal triple Flag and Hise tag. Upon transformation in E. coli (TG-), expression cultures were grown and expression was induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37"C. After spinning the cell cultures, peripla mic extracts were prepared y freeze-thawing the pellet followed by centrifugation. These to extracts were then used as starting material for purification via IMAC on HisTrap FF crude 1 ml colurnns (GE healthcare, Buckinghamshire, United Kingdorn) followed by desalting via Zeba spin columns (Pier~e, Rockford lL, USA) resulting in 95% purity as assessed via SDS-PAGE. 5.2 Evaluation of PcrV blocking Nanobodies in cytotoxicity assay 15 The ability of the Nanobodies to prevent Pseudomonas oeruginosa TTSS-induced cytotoxicity was tested in a cytotoxicity assay with P3X63 cells as the target. In brief, serial dilutions of purified Nanobodies were pre-incubated with Pseudomonas aeruginosa PA1O3 which was cultured under TTSS inducing conditions (LB medium + 5 rnM EGTA; Kim, Microbiology 151: 3575-3587, 2005). The mixtures were added to the P3X63 cells and cell mediated death was analysed as in example 4.4. Infections were 2o done at an average MDI of 2.3 bacteria to 1 rnyelorna cell (range 4:1 - 1.5:1). Data are summarized in Figure 1 and Table B-2.
WO 2013/128031 PCT/EP2013/054262 107 Table B-2: Evaluation of monovalent anti-PcrV Nanobodies in P3X63 cytoxicity assay Nanobody MoI IC50 (nM) 95% Cl on Ic50 (n M) % inhibition _______ ___ _____ _____ _-i- --- - ___I______ 1111 4 23 1-5158 2B02 1 1732 577-5196 100 2E 10 4 647 428-976 100 2009 4 4 0.451 55 3B11 4 1145 640-2051 54 3E10 4 48 19-119 4C03 LS 8890 5500-14300 >20 4G10 1 105 39-284 100 5E02 4 38 16-91 100 5H01 1 7 4 1 100 6B05 3 435 162-1172 100 7C10 4 42 7-232 100 7E09 4 No blocking observed in tested concentration range 10CO5 3 >1000 0 100 11B09 3 100 29-336 100 12802 4 No blocking served in tested concentration range [13 F07 -1.5 ~ 35600 7100-178000 >20 14B10 31 3000 >70 14E10 3 >3000 0 100 4 8 4-15 100 Fab 13.37 1.5 1 0.7-1.5 100 3 3 0.4-1.5 100 4 48 24-96 100 Fab274.26 1.5 34I23-50 100 5.3 Affinity determination The kinetic binding parameters for a subset of Nanobodies was determined with Surface Plasmon Resonance (SPR) on a Biacore T100 instrument. To this end, recombinant GST-PcrV was immobilized onto a CM5 chip via amine coupling using EDC and NHS. Purified Nanobodies were injected for 2 minutes at different concentrations (between 1 and 1000 nM) and allowed to dissociate for 20 mi at a flow rate of WO 2013/128031 PCT/EP2013/054262 108 45 ul/min. Between sample injections the surfaces were regenerated with 10 mM glycine pH1.5 and 100 mM HC HBS-N (Hepes buffer pH7.4 was used as running buffer. The inetic constants were computed from the sensorgrams using the BlAEvaluation software (1:1 interaction) The affinities of the anti-PcrV Nanobodies ranged from 0.5 - 5 nM (Tabe B-3. 5 Table B-3: Affinity KD (nM) of purified Nanobodies for recombinant GST-PcrV fNanobody } ka (M) lkd (so) KD (nM) 2B05210n 1005 21 50 x 100m 310 270x 10 390 x10 1 4G10 5140 x060 x10 5 5EF02 1 0 x 10" 680 x 10 4 7C10 120x 10" 1 80 1 2 ___________L_ __ __ ___X__ _ __ __L_ __ __ _ 5A Epitope binning To sort Nanobodies in different epitope bins, competitive binding ELISA assays were performed. In 10 a first experiment 1 ugmL of GST-PcrV protein was coated in a 96-weH MaxiSorp plate (Nunc, Wiesbaden, Germany). Dlution series concernn ration range 100 nM - 6 4 pM) of the purified Nanobodies in PBS buffer containing 01% casein and 0,05% Tween 20 (Sigma) were incubated in the presence of1 nM Fab13.37 (SEQ ID NOs: 142 and 143). Residua binding of Fab13,37 (SEQ D NO: 142 and 143) to GST PcrV was detected using mouse anti-HA gG (Zymed Laboratories South San Francisco, California) 15 followed by horseradish peroxidase HRP) conjugated rabbit anti-mouse lgG (Dako, Glostrup, Denmark) and a subsequent enzymatic reaction in the presence of the subst rate esTMB (3,3'5,5' tetramentylbenzidine (SDT Brussels, Belgium in the second experiment, 2 nM biotinylated rPcrV protein was captured on a 96-well MaxiSorp plate (Nunc Wiesbaden Germany) coated with 2 ug/mn neutravidin, A mixture o periplasmic extracts 20 and Nanobody-phage particles from different Nanobodes (both prepared according to the standard protocol, see for example the prior art and applications filed by Ablynx N.y. cited herein) was incuated an residue binding of the phages to biotinylated rPcrV proteins was detected using an monoclonl ant M13-HRP conugate (GE healthcare, Buckinghamshire, United Kindom) and a subsequent enzymatic reaction in the presence of the substrate esTMB (3,3.45,5'-tetramentylbenzidine) (SDT Brussels, WO 2013/128031 PCT/EP2013/054262 109 Belgium) Based on the results from these two approaches Nanobodies were divded into different epitope bins (see Table B-4), Table B-4: Epitope bins of anti-PerV Nanobodies ~Nanobody -Nanobody epitope binning Fab epitope binning Epitope bin 1 1E11 bin 1 non- competing bin 1~ 2B02 bin 1 non competing bin 1 2810 bin 1 non competing bin I 2 09 bin 1 non- competing n r3B11 ND -~ 1 non~- com peting -___ ND/NC 3E10 ND competing ND/C 4C03 ND non- competing ND/NC 4G10 ND non- competing ND/NC 5E02 ND competing ND/C 5H01 bin 2 competing ___---bin 2 ~6B05 ______bin 1 non- competing bin 3 7C10 bm 2 competing bi2 7E09 bin 3non- competing bin 3 10C05 bm non- competing bmn1 11809 bm 1 non- c petog 1 12B302 ND non- competing ND/NC___ 13F07 __ I_ __ bin 3 non- competing bm I__ __ 14B10 ND non- competing ND/NC :1410 L bio 5 ND =not determined NC non-competing Example 6: Generation and screening of multivalent PerV blocking Na nobodies 61 Construction of a bivalent/biparatopic anti-PcrV Nanobody library 10 A bivalent/biparatopic anti-PcrV Nanobody library was constructed as follows. The coding sequences of 18 monovalent an-PcrV Nanobodies (Table A-4 were amplified by means of PCR in two separate reactions: N-terminal building block (5'-GAGGTGCAATTGGTGGAGTCTGGG-3'; SEQ ID NO: 150 and 5 -ACCGCCTCCGGAGGAGACCGTGACCAGGGT-3'; SEQ ID NO: 151) and C-terminal building block (5'- WO 2013/128031 PCT/EP2013/054262 110 TCTTGGATCCGAGGTGOCAGCTGGTGGAGTCTGGG-3; SEQ ID NO: 152 and 5'-TGAGGAGACGGTGACCAGGGT 3' SEQ ID NO: 153). For Nanobody 5H01 different primer sets were used for the N-terminal building block PCR (5 -GA GTGCAATTGGTGGAGTCTG-3 SEQ ID NO: 154 and 5' ACTTGAAGACCTCCGGAGGAGACCGTGACCAGGGT-3' SEQ ID NO: 155 and C-terminal building block PCR 5 (5-ACTTGAAGACTGGATCCGAGGTGCAGTTGGTGGAGTCTGGG-3 SEQ ID NO: 156 and 5 TGAGGAGACGGTGACCAGGGT-3' SEQ ID NO: 157 The N-terminal building block PCR pool of the 19 ant PcrV Nanobodies was cloned in an expression vector upstream and in frame with the coding information o a fe xible glycine-serine linker (40GS GGGSGGGGSGGGGSGGSGGGOSGGGGSGGGG5GGGGS; SEQ ID NO: 193) whereas the C-terminal pool oft e 19 anti-PcrV Nanobodies was cloned downstream and in 10 frame of the 40G'S linker coding sequence. The expression vector was derived from pUC119, containing the LacZ promoter to drive transgene expression, a resistance gene for kananycin, a rmultipe cloning site and the OnpA leader sequence. ln frame and downstream of the multivalent Nanobody coding sequence, the vector coded for a C-terminal triple Fag tag and a His. tag. The resulting plasmid pool harbouring the bivalent/biparatopic anti-PcrV 15 Nanobody brary (with a theoretical diversity of 19 x 19 = 361) was transformed into . co/l 'T1 cels and plated on agar plates LB Km + 2% glucose. 864 sing e colonies (representing an expected completeness of >90% of the 'br ry) were picked from the agar p ates. Periplasmic extracts were prepared as described above. 20 6.2 Screening of a bivalent/biparatopic anti-PcrV Nanobody library The periplasmic extracts of the bivalent/biparatopic library were used in a screening campaign to select for the most potent functional blocking anti-PcrV Nanobodies. To this end the periplasmic extracts (final dilution 1/150) were tested for their abi o preet TTSS nduced P. nergmoso infection in a cytotoxicity assay using P3X6cells as target (as described in Example 4 4). The screening results (% 25 inhibition) front the 48 most potent of 84 (13i library clones are summarized in Table B-S. Table B-5 captures all 361 potential bivalent/biparatopic combinations as a function of their N-terminal and C terminal building blocks, which are grouped by their relative epitope bins a- determined in Example 5. The corresponding sequences of the 48 most potent library clones were determined as shown in Table A-5. The sequence analysis revealed the following observations: within the 48 most potent 30 constructs ()no 0/48) rnospecific bivalent Nanobodies (consisting of two identical Nanobody building blocks) were present (i) the majority (42/48 clones consisted of building blocks from two different WO 2013/128031 PCT/EP2013/054262 111 epitope bins. After screening, 24 unique biparatopic Nanobodies (SQ ID NO: 118-141) were selected for further characterization. Table B-: Screening results (inhibition in P3X63 cyotoxicity assay) of 48 most potent bivalent anti 5 PcrV Nanobodies C-termial tuildmg block bmn bin bm3a NDNC NDIC LQ LfU Im IN 111 47 57 5249 67 52 44 2902 2910 S2G09 66 56 a 9B03 57 41 '54 10C05 1809 6C 82 52 49 14E10 62 5H05 4 42 85 8 47 57 7C10 61 43 G3 63 7E09 45 48 13F07 59-80 4 6 657 4 55 58 55 69 S4C03 0 4G1 81 12902 :" 10 2 85 A Z 5902 60 48 47 47 Example 7:Characterization of bivalent/biparatopic anti-PcrV Nanobodies 7.1 Preparation of selected Nanobodies Coding sequences from selected bivalent/biparatopic anti-PcrV Nanobodies were cloned into an in 10 house constructed plasmid allowing expression in Pichia pastors and secretion into the cultivation medium, The expression vector was derived frorn pPICZa (Invitrogen) and contained the AOX1 promoter for tightly regulated, methanol induced expression, a resistance gene for Zeocin'" a multicloning site and WO 2013/128031 PCT/EP2013/054262 112 the factor secretion signa In frame with the Nanobody coding sequence the vector coded for C terminal GlyAlaAla sequence followed by a triple Flag tag and a His6 tag. Upon transformation expression cultures were grown and Nanobody expression was induced by addition of methanol and allowed to continue for 48 hours at 30"C. The cleared supernatants were used as starting material for immobilized S meta lion affinity chromatography (IMAC) using a HisTrap column (GE Health are). Nanobodies were eluted fror the column using imidazole step gradient fror 20mM to 250mM. In a next step, Nanobodies were buffer changed to D-PBS (Invitrogen) using HiPrep T 26/10 desalting columns (GE Healthcare). 7.2 Evaluation of bivalent/biparatopic anti-PerV Nanobodies in cytotoxici assay 10 The bivalent/biparatopic antiPcrV Nanobodies were tested or their abili y to prevent TTS induced P. aeruginosa infection in the ytotox assay with P3X63 ceIs as target at an MCI of 12 (as described in section 5.2 The results, shown in Figure 2 demonstrate that al Nanobodies are able to prevent P. oeruginosa infection with IC50 values ranging from 3 x 10* to 37 x 10 M, as summarized in Table B-6. 15 Table P-6: Performance of bivalent/biparatopic anti-PcrV Nanobodies in c totoxicity assay with P3X63 cels as target Nanobody IC50 value (M) 95 Cl on 1C50 value (M) Efficacy % Fb 13.37 5___ y_10 - 3.4x1 to8 810 100 2565 4x>10 _____5 _3 to__4__1010 257 1.3x10 ~87 > 10' to 21 ' 100 258 9__0___10 "x 5.6 x10 ' tol1 4xy10 0 __00 259 3,9 10 22x10 o 8x10 100 261 4 8 10 ' 2 9 x 10X toI8 1 x __0 100 269 82x10 5.0x0~ oI14x10 3 100 271 5.9 x 10' ".5 x 10 " to 9 8x10 ~ 100 275 3.7x10 9 2,3>10 to 60x10_ 100 277 1.3x10 7.5y10to24 x10 100 237,6x10'" 4, xl0 to133x30 100 285 6,1> 10" 3.7 x 1~ to 9.9 x 10 100 308 x 8 > "2x 10'" to3.9>10 100 [--I C, 0 1 _ _ _ __ _ _ _ _ _ _ _ _ 319 __-_8110 5.3 x 10 to l2x10 100 335 6_____ .4x10' 3.9x10" to l.0x10' _100 WO 2013/128031 PCT/EP2013/054262 113 An additional cytotoxicity assay with human lung epithelial cells A549 cells as the target was also esta bished and use to character e the formatted Nanobodies. To this end, 6 x 10 A549 (ABL 161 ATCC: CCL- 8) cells per well were added to a96well -plate (Roche Cat No 05232368001 and placed in the xCELLigence RTCA workstation Roche Analyser Model W380 for -8 h at 37 *C. P. ceruginosa (strain 5 PA103) grown under inducing conditions for TTSS expression, was pre-ncubated during h at 37'C with 10 different concentrations of purified Nanobody and then added to the A549 cells and incubated during another 24h at 37 *C. Data were analyzed at the tirne points where drop in cell index of the treated wel s was between 75% - 90% of the cell index window. Data were normalized to the time point where the PA103 inoculum was added to the cells. Potencies of the tested bivalen /bipar topic anti-PcrV 10 Nanobodies from three independently performed experiments re presented as ratios relative to the potency of a reference molecule Fab 13.37( (ble B-7). Table B-7: Performance of bivalent/biparatopic anti-PcrV Nanobodies in cytotxicity assay with A549 cells as target Nanobody Ratio* (mean fold improvement Range (from three independent over Fab 13.37) experiments) 256 21 (7-50) 257 10 (1-54) 258 1 (1-1) 259 9 (1-180) 261 1(1-1) 2 69 1 (1-1) 271 -1 1 (1-1) 275 1 (1-1) 277 8 -(1-64) 283 1 ( 1) 285 11 (1-1) [308 -0.3 1(0.04-1) 319 2 (1-5.3) 15 Ratio IC50 value Fab 13,37/1C50 value Nanobody WO 2013/128031 PCT/EP2013/054262 114 7.3 Cross-reactivity to PcrV clinical variants Cross reactivity of the bivalent/biparatopic anti-PcrV Nanobodies to 23 PcrV sequence variants (see Table A-i) was evaluated by binding LISA. To this end, rnicrotiter plates were coated overnight with 1 ug/nI of each PrV van ant at 4C Nanobodies were applied as dilution nries concentrationn range: 5 nM 5 - 4 pM) in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma) Nanobody binding was detected using a mouse anti-Ha g-HRP conjugate (Sigma) and a subsequent enzymatic reaction in the presence of the substrate esTMB (3,3 5 M -tetramentiybenzidine) SD, Brussels Belgium All Nanobodies could bind to the al different PrV variants toa very similar extent as they bind to PcrV reference rom strain PA01. The range of C 50 values against the different PcrV variants and reference 1o strain PAO1 obtained for each Nanobody is presented in Table B-8. Table B-8: Binding of bivalent/biparatopic anti-PcrV Nanobodies to PcrV clinical variants Nanobody -EC50 Range (M) 256 7x10 -1x10 257 ~7 x 10-9 xi 1 259 3 x 30"2x 10 261 6 x 10- 7 x 10 271 6 x 10 1 x 10 7 6x10 -9x1 277 7 x 10 1 x 1 285 1x 10-1 x 10 398 x10 1 1 - 1x0 1 ____ 335 7 x 10 9 x 10 7.4 Stability of Nanobodies towards human neutrophil elastase and Pseudomonas aeruginosa 5 elastase Stability and maintenance of functionality in the presence of pathogen derived protease Pseudomonas aeruginoso last ase and host cell derived proteae humann neutrophil Elasase (HNE)) wa tested in vitro b m cuba ion of the Nanobodies with either HNE or Pseudomonas a ruginoso elastase nd subsequent ana ysi of the degradaton produce by b mindig ELS 20 To this end Nanobodies were incubated either with human neutrophil Elastase (1-2 U elastase/ug Nanobody) or Pseudomonos aeruginosa elastase 3 ug/ug Nanobody) in Tris pH 7.8, 150 mM NaCl; WO 2013/128031 PCT/EP2013/054262 115 10mM CaCl 2 buffer at 37'C for varying intervals of time (2 hr,4 hr ,24 hr and 48 hr). For sample ana ysis 1 ug/mL of recombinant PcrV protein was immobilized overnight at4 4C in a96-well MaxiSorp plate (Nunc, Wiesbaden, Germany). Wells were blocked with a casein solution (1%) and samples were applied as dilution series in PBS buffer containing 0.1% casein and 0.05% Tween 20 (Sigma). As reference untreated 5 Nanobody was taken along. Nanobody binding was detected using polyclonal rabbit anti-VHH antibody (Ablynx N.V,) followed by horseradish peroxidase (HRP) conjugated goat anti-rabbit lgG (Da ko, Glostrup, Denmark) and a subsequent enzymatic reaction in the presence of the substrate esTMB (3 3 5 5 tetrarnentylbenzidine) (SDT, Brussels, Belgium). Results are summarized in Table B-9. 10 Table 8-9: Proteolytic stability of selected bivalent/biparatopic anti-PcrV Nanobodies old decrease potency Nanobody [Pseudomonas elastase HNE 2hr -- 48hr }24 hr 48 hr 257 1 3 9 30 256 2 91 15 550 261 411227 350 IH H 271 38815 275 2 4 8 500 277 10 11 4 16 28562 3 319 1 2 1 2 33571 22 15 100 * Fold decrease in potency = EC50 value treated sample/EC50 value of untreated sample Example 8: Epitope mapping 15 A chimeric molecule-based strategy was used to map the conformational binding eptopes of selected ronovalent anti-PcrV Nanobodies. To this end, discrete solvent-exposed parts of PcrV were replaced by their counterparts of LcrV, a PcrV homolog from Yersnia spp. (Sato e al. Frontiers in WO 2013/128031 PCT/EP2013/054262 116 Microbiloog 2: 142, 2011). In total 7 chimeric molecules covering the most of the sequence range of PcrV protein were generated (see Figure 3; Table A-7 Chmeric molecules together with fu length PcrV protein (amino acids 1-294 see Table A-i) were cloned into an in house expression vector derived fror pUCi19 which contained the LacZ promoter, a resistance gene for kanamycin and a multicloning site. In frame with the chimera codin sequence, the vector coded for a C-terminal c-mye tag and a His tag. In addition, a small PcrV fragment (amino acids 144-257, see i ure 3) described as PcrV blocking epitope (Frank et at J. lnf. Ds. 18 : 64-73, 2002; US 6 827 935) was also cloned in the expression vector. Upon transformation in E cot (TOG-), expression cultures were grown and expression was induced by addition of 1mM PfTG and arowed to continue for 10 4 hours at 37C After spinning the cell cultures, periplasmic extracts were prepared by freeze-thawing the pellets fohowed by centrifugation The remaining cell pellets were resuspended and lysed by sonication. Cytosolic fractions were isolated by centrifugation and pooled with the periplasmic extracts. The recombinant proteins were purified vi immobilized metal affinity chromatography (IMAC) using Ni Sepharose Fast Flow beads (G Hea thcare, Uppsla, Sweden) followed by desalting using Zeba Desalt is Spin Columns (Thermo Scientific To evauate whether selected monovalent anti-PcrV Nanobodies could bind to the snaI PcrV fragment and the 7 chimeric molecules, a binding EUSA wa carried out. In brief, Nanobodies were coated overnight at 4"C t 3 ug/mL in a 96-well MaxiSorp plate (Nunc, Wiesbaden Germany). Wells were blocked with a casein solution (1% in PBS). PcrV proteins were applied (1/10) and binding was detected 20 using a biotinylated mouse anti-myc antibody (Serotec) followed by horseradish peroxidase (HRP) conjugated extravidi (Sigma St Louis, MO USA and a subsequent enzymatic reaction in the presence of the substrate esTMB (3,3',5,5'-tetramentylbenzidine) (SOT, Brussels, Belgium). As reference Fab i3.37 was taken long (9 ug/mI coating in 96 well plate). Since al tested Nanobodies bin to at least S out of 7 chimeric molecules with similar binding levels as on full-length PcrV and that each chimeric molecule is 25 bound by at least 3 out of 7 Nanobodies with similar binding levels as on ful-ength PcrV it could be concluded that only discrete parts of the PcrV protein were rep aced by their LcrV counterpart and that the overaH ternary structure of the chimeric molecules was not dramaticaHy affected n comparison with the full-length PcrV molecule. Comparison of the binding p patterns of the anti-PcrV Nanobodies to the different PcrV molecules revealed 3 major epitope groups see Table B-i0). These data are in line with 30 data retrieved from the epitope inning experiments as described in xarple 5.4. Nanobodies from bin 1 bind to PrcV region with amino acids i56-177 and amino acids 195-2i, Nanobodies from bin 3 recognize the PcrV region with amino acids 57-91 and Nanobodies from bin 2 bind to amino acids 209-249. More WO 2013/128031 PCT/EP2013/054262 117 so, this experiment further delineated the proposed binding epitope of Fab13,37 (Frank et al i lnf. Dis. 18 : 64-73 2002) and US ,827,935 from amino acids 144-257 to amino acids 209-249. A re presentatin of the 3 different PcrV blocking epitope regions s shown in Figure 3 5 Table B40: Epitope mapping of anti-PcrV Nanobodies Bi Bin2 Bin3 02B10 1410 01E11 06B05 05H01 13F07 07E09 Fab 137 Full enth PcrV B B B B E B B B (aa 1-294) i B' ~ crV fragment NB B B B NB NB B ( aa 144-257) Chimera 1 B BB {B B B B B IChimnera 2 B BB B RB R B Chimera 3 B B B B B B Chimera 4 NB NB NB RB B B B B ChimeraS 5B B B B B B B 'B Chimera NB NB RB RB B B B B Chimera 7 B B B RB B B RB B: Bindig: binding signal blinding signal on full length PcrV protein NB: No binding: binding signal background signal RB: Reduced binding: binding signa I= 30-90% reducton as compared to bnding signa on full length PcrV protein 10 Example 9: Nebulization of the polypeptides of the invention 9.1 Materials and methods Nanobodies 339 354, 360 and 37 (tagless versions of respectively Nanobodies 256 (SEQ ID NO: 129) 319 (SEQ ID NO: 138), 259 EQ ID NO: 137) and 258 SEQ ID NO: 134)) were produced using a is Picha pastors X33 standard fermentation set-up 2L or 10L ermentor sc le depending on expected expression level After fermentation all four Nanobodies were clarified using a generic tangential flow filtration step and further purfied via resin chromatography using a capture step, polish step and prep native Size Exclusion Chromatography. The Nanobodies were then concentrated to approximately 50 mg/ml both in D-PBS and D-PBS+ 0.01% Tween80, 20 A reference Nanobody SEQ ID NO: 207 DVQLVESGGGLVQAGGSLS1SCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVEGRFTISRDNAKNTG WO 2013/128031 PCT/EP2013/054262 118 YLQMNSLAPDDTAVYYCGAGTPLNPGAYIYDWSYDYWGRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQ AGGSLSISCAASGGSLSNYVLGWFRQAPGKEREFVAAINWRGDITIGPPNVELGRFT SRDNAKNTGYLQMNSLAPDDTA VYYCGAGTPLNPGAYIYOWSYDYWGRGTQVTVSSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGSLSISCAASGG SLSNYVLGWFRQAPGKEREFVAAINWR3GDITIGPPNVEGRFTSRDNAKNTGYLQMNSLAPDDTAVYYCGAGTPLNPG 5 AYIYDWSYDYWGRGTQVTV SS SE 0% D NO: 2 in WO 2011/098552) was also nebulised in parallel. This reference Nanobody was used at 55.2 mg/ml in 10mM NaH2PO4/Na2H PO4+0.13M NaCl, pH 7.0, The nebulisation sequence of the sarnples was based on OD 500 data obtained for the different Nanobodies from the stirring and freeze/thaw experiment: the most stable polypeptides were nebulised first, the polypeptfdes which showed more turbidity in stirring or F/T stress were nebulised later. The 10 reference Nanobody was nebulised before, in between and afterwards as control On day 1 all samples were nebulised according to the order (1 to 11) in Table B-11. The same experiment was repeated on day 2. The polypeptides were nebulzed by the AKiTA 2 APIXNEB nebulzer system (Activaero) according to the manufacturers instruction. The nebulisation experiment was performed in duplicate. 500 ul of 15 sample was nebulised continuously via a mesh nebulizer with a 4 pm mernbrane. The aerosol was collected in a 100 mL. glass bottle and then analysed. The sarnples before and after nebulisation were evaluated via turbidity measurement (OD 500) and SE-HPLC analysis. 20 9.2 Turbidity measurement 0D500 was measured as a turbidity parameter. Blank setting was done with D-PBS. The OD 500 values measured for the reference samples (before nebulissation) and the nebulised samples (day 1 and day 2) are shown Table B-li. 25 WO 2013/128031 PCT/EP2013/054262 119 Table B-11: Overview ODSO0 values before and after nebulisation Sample Reference Nebulized (dayl) Nebulized (day2. 1 Reference Nanobody 00202 0.0284 0.0391 2 Nanobody 339 Tween 80 0.0091 00149 0.019 3 Nanobody 360 + Tween 80 0 007 0.0167 0011 4 Nanobody 354 + Tween 80 0.012 0.0757 00699 5 Nanobody376 + Tween 80 0 0165 0 0702 0.0871 6 Reference Nanobody 00202 0.0201 04 7 Nanobody 339 0.0151 0.0107 0.024 8 aooy360 0.0065 0.0099 006 9 Nnbdy 354 0.0167 0.0171 0.071 10 Nanobody 376 OW16E.05 0 0162 11 Reference Nanobody 0.0202 0.0175 Q037 The OD500 values of the samples before and after nebulisation are further presented in Figure 7. 9.3 SE-HPLC analysis The SE-HPLC assay consisted of a BioSEC-5, 7.8 x 300 mm 5um, Agilent 5190-2521 (MEC85) column, a mobile phase consisting of 0.3M argininHCI + phosphate buffer pH6 and UV detection at A280 nm. The relative amount of specific protein impurity was expressed as relative area % and was calculated by dividing the peak area corresponding to the specific protein or protein impurity by the total 10 integrated area. 1ug sample was injected. The method is intended for quantification of higher molecular weight species (HMW's) which elute as pre-peaks in the chromatogram. An overview of the SE-HPLC integration data is given in Table B-12. As is known for the Reference Nanobody (see WO 2011/098552), an increase of pre-peaks (HMW's) was induced by the nebulisation. This phenomenon was not observed in all nebulised polypeptides of the invention. Nebulisation did not 15 affect the formation of high molecular weight molecules. The addition of Tween 80 to the samples did not seem necessary. ln the last column the total peak area on SE-HPLC is given which is relative to the concentration of the injected sample. No significant differences were seen for any of the samples between the group area before and after nebulization, indicating no loss in concentration.
WO 2013/128031 PCT/EP2013/054262 120 Table B-12: Overview SE-HPLC integration data of polypeptides and reference Nanobody before and after nebulisation Sample name Rei Area % R Area Rel Area Group area Pre-peak 2 Pre-peak 1 Main-peak 1 Post-peak 1 mAU* min Reference sarnples (before nebula nation) Ref. Nanobody 0.08 0.30 9 90.87 49.12 33 wen0 na 0.39.30.86302 354 +Tween80 na 0.40 98.89 0.71 33.89 360 +ween80 008 0.89 _98.36 0.67 33.43 376 -+Tween80 na , 0.17 199.19 10.63 36.73 339_ na 0.21 99.05 _ 1 0.733 na 1 0.36 99.02 0.62 34,04 [360 10.06 0.94 198.32 0.68 33.76 37 na 0.17 99.23 0.60 36.97 Nebulised samples (day 1 Re Nanobody 0.28 .20.38 47.88 33 + Twee n80 na 0.4 98.2 o.6 29.83 354 +Tween80 na 0.43 98.93 0.64 33.75 360+ITween80 10.08 11.03 98.27 T0.613.9 376 -Tween80 na 10.21 99.29 0.51 36.29 [339 jna 10.40 99.02 10.58 30.20 + T 354 na 0.42 98.95 0.63 34.33 - -------- ---- -_ _ _ __ _ _ _ _ _ _ 360 .08 .06 8.220.6534.25 0.21 99.28 0,5 37.15 Nebuhised samples (day 2} Ref Nanobody 027 1092 98.19 38 47.33 339 +Tween80 na 0.74 98.61 TO65 29.93 354 + Tween80 na 0.46 98.96 10.58 33.58____ 360 +Tween80 0.08 1.02 98.33 05 5 33.61 376 Tween80 na 0.23 i99.22 0.54 6.9 339 na 0.33 99.04 0.63 28.9 354 na 1041 98.99 o 33.87 360 0.0 1.05 I98.32 0 57 3376 376 na 0.19 1931 0.5 13744 WO 2013/128031 PCT/EP2013/054262 121 Example 10: In vivo efficacy of anti-PcrV Nanobodies An acute P aeruginosa infection mouse model was used to assess the n vvo efficacy of 3 Nanobodies, namely 339, 30 and 37, The model was conducted as described in Secher et al (Journal of Antimicrobial Chemotherapy 66: 1100-1109, 2011). In brief, P. aeruginosa strain PA2310.5 serotype 01 was grown at 37"C overnight with sha ing at 1 0rpm in BI medium (Brain Heart infusion) supplemented with 10nM nitriiotriacetic acid Sigma. Following the exponential growth phase, the inoculums were prepared by diluting the P. aerugnosa to 5x10 fu/m The Nanobodie or the Fab 13.37 were diluted in D-PBS to the appropriate concentrations and subsequently premixed with the Pseudomonas aenrugnosa culture at a 1 to I ratio, The premi (40 p) was immediately administered intrnasally to 8-10 week old 10 anaesthetized C57B/6 mice using an utra-fine pipette tip. The infected mice were monitored for 4 or 5 days and both survival and boy weights (7-8 animals per group) were recorded daidy throuho the study. AdditionaIy, 3-5 animaIs per group were sacrificed at 24 hours post-infection. In these animas, the right lung and th other lobe (except the big lobe of left lung were weighed and homogenized in 1.5 mL of isotonic sane solution using the sterile disposable 15 homogenization system Dispomix (Medic Tools AG. 300-Zug, Switzerland). Ten-fold serial diLutions of lung homogenates made in isotonic saline solution were plated on Blli agar pates to determine viable bacteria i1n the lung (cfu), After lung homogenization for CFU determination, lung homogenates were subsequently centrifuge the supernatant discarded and the pel et suspended in nL PBS containing 0.5% hexadecytrimethyl ammonium bromide (HTAB) and 5 nM ethylene-diarine tetra-acetic acid 20 (EDTA). After centrifugation, 100 pL of supernatants were placed in test tubes with 200pL PBS-HTAB EDTA, 1 mL Hanks balanced salt solution (HBSS), 100 pL of o-dianiside dihydrochloride (1.25 mg/mL), and 100 pL H 2 02 0.05%. The reaction was stopped with 100pL NaNs 1% a er I5 mn of incubation at 37 C in an agitator. The myeloperoxidase activity was determined as absorbance at 460 nm against medium. A low 0.028 pg/mouse) and a high dose 10 pg/mouse) of each Nanobody were tested in this 25 model The survival urve depicted in Figure 4 show that all the tested anti-PcrV Nanobodies completely protected the mice from P. aeruginoso infection-related lethlity at a dose of 10 pg/nouse. In contrast, the irrelevant Nanobody treated animals and buffer treated animals all died within the first 48 hours post-infection. At the lower dose of 0.028 pg/nouse still all three Nanobodes provided complete (A339 or near complete (A360 and A376) protection. o The prolonged survival of the infected Nanobody-treated or Fab1337 created mice versus the infected buffer-treated mice or the infected irrelevant Nanobody-treated mice was correlate.d with crease in lung nlanma tion parameters. More spec'ically, neutrophilic lung infil ration as measured WO 2013/128031 PCT/EP2013/054262 122 by the myeloperoxidase activity in the lung homogenates (Figure 5A and the relative ung weight (Figure SC) were clearly educed by a the nti-PcrV Nanobodies at a ose s low as 0.028 pg/mouse. in addition to these positive effects on lung inflammation bacterial burden at 24 hours was also reduced by rnore than 1.4og CFUs (Figure 5). Conversely, the irrelevant control Nanobody had no effect on these s parameters indicating th at the observed effects were anti-PcrV Nanobody related. Finally, also the percent weight loss was analyzed. A339 treated mice (Figure 6)had a maximal me<an percent weight loss of only 8.3% and reached this rninimum already at day 1 post-infection. Mice treated with an 0.028 pg/mouse dose of A376 and A360 or an 0.05 pg/mouse dose of Fab13.37 displayed mean percent weight loss of 11.9%, 14.1% and 13.5%, respectively which only reached a minimurnat 10 day 2 post-infection.
WO 2013/128031 PCT/EP2013/054262 123 TABLE ES Table A-1: Amino acid sequences (1-294) from PcrV variants PcrV SEQ ID sequence 0-- - -6 4 - - - - - L
I
T
AMELKVYSVTQSQ1NAALSAKQGIRIDAGGTDLVDPTLYGYAVGDPRWKDSP2EYALLSNLDTFSGKLSI K<DFLSGSPKQSGELKGLSDEYPFEKNNPVNATTVVSDRSRPLNDKVNEKTTLLNDTTSSFRYNSAVEAL ____ _______NRFTQKYDSVLRD1LSA1 __________ _____ 02 I 101 MEVRNLNAARELFLDELLAASAAPASAEQEELLALLRSEFRIVLAUAGQPLS£EAQVLKALIAWLLAANPSA - -- I 0 2 1 6 0 -L FT- - Q- 109 -- - KDFL SPQSGENLKLKQYPFEKDNNPGNTVDRSROOPLNDNETLLNDSSA 11~ 16 --- KDFLSGSPKQSGELKGLKDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNEKTTLLNDTSTSRYNSAVEAL 04126 MEVRNITRAELFLDELLDAAAPASAEQEELALL1RS ERIVLAHAGQPLSEAQVLKALAiLAANPSA WO 2013/128031 PCT/EP2013/054262 124 ~PPGQGEEVEEVLQARQ PAQWDlLRhFLVSAYF SLIGRL>DEDVIGVYKDVLQTQDGKRKALLTDELKAI TAELKVYS VIQSQ INAALSAKQGIR1DAGGIDLVDPT1LYGYAVGDPRWNKDS PEYALLTSNL 1 DT FSGKLS I KDFLISGS PKQSGEKGLhRDEYPFEKDNNPVGNFAT TVSDRSR PLNDKVNKTZ T LLNDT1SSRYNSAVTAL NRFMQQKYDSVLRZD1LSAI 3 M 1 lE5RNLNAGRELLFLDELLAAPAAPASAEQEELLALLR SERTIVLAHAGQT PSEAQVLKFALAWLLAAINA PPGQGL EVLREVLQARRZQPGAQWDLREFTLVSA~YFS LHGRLDEDTVI GVYKDVLQTQDGRKTALLDE LKTAL TAEFLKVYSVIQSQTINAALSAKQGIRITDAGIDLDPTmYGYAVG-DPRWKDS PEFYALLSNLDTFSGKLSU I KDFLSGUS PKQSGELKGLRDEYPFEKDNNPVGNFATTVNSDR SRPLNDKVNEKT"TLLNDTSESRYNSAVEAL 172 NRIQYDSVLRDILSAI 14 17 MEVRNFNAG.RELFL DELLAAPAAPASAEQKELLALLRSER11VfLAHAGQPLSEAQVLKALANLL AANP h SA PPGQGLE'VLR EVLQARRQ PGAQNDLREFLVSAYFS LEGRLDDVIGVJYKDVL QTQDGKRKALLDE LKAL TAE KVYSVQSQTINAALSAKQG1R IDAGGIDLVD PT LYGYAVGDPRWKDSPETYALLISNLDTFSGKL S 1 TUFTLSGSPKQSGEI LKG:LRDEY PFEKDNNPVGNFATTVSDR SRPLNDKVNEKTP'TLLNDT SSRYNSAVEAL _______I NRFIQKYDSVLRPDILSAI _ _________ 5 173 MEVRNFNASRELFLDELLAAPAAPASAEQELLALLRSERIVLAHAGQULSAQVL KANTLLAUANA PPGQLEVR ELQARQ GAQDLRELVSYFSHGRDEDIGVKDVQQGKRKALLDETLKAL TAET LKVS VTQSQ INAAL~SAKQG7I DAGGTU DDT LYGYAVGDPRWKDSPESYALLSNLDYTSGKLS I TKDFLSGSPKUQSGELKGLRDEY PbEKDNNPVGNF'ATTVSDRSRPLNDKVNE KT TLLNDT SSRYNSAVEAL NR FTQKYDSVL RDTLSAT ___________________ 16 17'4 MEVRNLNAARELFLDUELLIAASAAPASAEQEELLIATLLRSERl IAAGQPLSEAQVLTKAALLAANPSA PPGQGLEVLREVLQARRQ PGAQWDLREFPLVSAYFVSLH4GRLDEDVIGVYKDVL QTQDGKRKATLLDELKAL TAEVLKVYSVTQSQ INAAL SAKQGIR IDAGGDLVDPTLYTGYAVGDPRKDS PEYALL SNTTFSGKLSI TUFTLSGS KQSGFLKGLSDEYS FFKDNNPVGNFAT m TVSDR SRP LNDKVNEKTTLLNDT1 SRYNSAVEAL NRI QKYDSVLRDILSAT ____________ 17 t7 MEVRNFNAARELFLDEELAAPAAPS1SQldFWLALRSERI VLAHAGQPLSEAQVL KALJAWLLAANPSA -PPGQGLEVLTRENTLQARRQ PGAQWDLRQEFLVSAYF SLHGRLDED VIGVYKDVLQTIQDGKTRKALLDEFLKAL TAEL KVYSVT QSQ TNAATLSAKQGTR TDAGGIDLVDTLTYGYAVGDPRWKDS PEYALLSNLDT F SGITLS I KDFLSGSPKTQSGELKGLKDEY PFEKDNNPVGNFAT VSDSPLNDKVNE2TTTLLNDT SSRYNSAVEAL NR FTQKYDSVLRDTTLSAT PPGQGLEVLREVLQARRQPGAQWDLRFLVSAYFSL HGRLDENVIGVYKUVTLQTQDGKRKALLDE LKAL TAET LKVYSVTQSQ TNAALSAKQGIR IDAGGIDLVDPTLYG YAVGDPRWKDSPEYALL SNLDT FSGK V7 I - - KDFLSGSPKQSGETLKGT LKDEYPFEKDNN'PVGNFAT TVSDRSRIPLNDKVNEKT TLLNDT SSRYNSAVEAL - ______NRFbIQTYDSVLRDJILSA1I 2 72 TAE LKVYSVJIQSQ INAALSAKQGIR TDAGGTUTLVDPTLIYGYAVGD PRWKDSPFYAL LSNLDT FSGKTL S 1 KDFiLSGSPKQSGVLKGLRDEYI PbEKDUNNPVGNbFAll TVSDRSRPLNDKVNEKKTTLNDT SSRYNSAVEAL NFITQKYDSVLRDIL SATI _________________ 20 17/8 MEVRNLNAGRLFLDELLAAPAAPASAEQKELJ lARNERILAHAGQPLSEAQVLKAJLtGdliNUSA PPGQGLEVLRE VLQARRTQPGAQWDTL1REFLVSAYFbS LRGRL
T
DEDVTGVYKDVLQTQDGKRKALLDEFLKAL TAETLKVYSVTQSQTINAATSAKQGIR IDAGGTLVPTLYGYAVGDPRNKDS PEYALLSNLD TFSGKLSI FLSGS2 PKQSGEKGLRUDEYPFEKDNNPVGNFATTVSDRSPLNDKVNEIKTTLL NDTISSRY NSAVEAL NRFQKYBSVLRDILSAI 21 17 9 MEVRNFNAGRELjFLDELLAAPAAPASAEQEELMALLRSERI VLAHIAGQPLSFAQVLKAAWLLAANPSA PPGOQGLEVLREVLQARRQPG;AQNUDLREFLVSAVFSLTHGRLD EDVIGVYKDVLQTQDGKRKALLDELKAL~ TAE'LKVY SV1QSQI1NAALSAKQGIRIDAGGZIDLVDUTLYGYAVGDPRWKDSPE'YAL LSNLTFI SGKLI TUFTLSGSPKQSGELTGLRUEYPFESKUNKNPVGNF AT ~TDRRLNDKVNETTTLLUD T m S SRYNSAVEAL NRFIQKYDSVLRDILSAI ________________ 22 180 MEVRNILNAGRLFILDELLAAPAAUASAEQEELLTALLRSFRIVLAKAGQPL SEAQVLKAAWLLAAN2SA PPGQGLEVLREVLQARRQPGAQWDLREFLVINSAYFbSLHGRLDEDVIGVYKDVL QTDG'KKALLDELKAL TAT LKVYSVTQSQTNAATLSAKQG0IRIDAIDVDTPTLYGYAVCDPRWKDSPEIYALLSNLDTFSGKLS I5 KBIDFSSPKQSGELKGLSDEYPFEKDNNPVGNFATTVSDR SRPTLNDKVNEKTT LIT SERYNSAVEAL NRFTQKYDSVLRDILSAI 23 ~L61 MEVRNFNAAREFLDT 2 ELLIAASAAPASAEQEELLALLRSERTIVLAHAGQPULEAQVLKALJAWLLAANPSA PPGQGLEVLRELQAR RQPGAQWDLREIFLVSATFSLUIGRLDEDVTIGVYKDVL QTQDGKRKALLDELKAL TAELKV/YSVT QSQINAALSAKQGIRIDAGGIBLVD/PTILYGYAVGDPRNTDS PEYALLSNL DTFSGTLS I KDFLJSGS PKQSGELKGLTSDEYPFEKDNNPVGNFATTVSDRSRPLNDKVNKEKTLLNDTSSRYNSAVEAL NRFIQKYDSVLRDILSAI ___________ WO 2013/128031 PCT/EP2013/054262 125 Table A-2: Amino acid sequences of VH and VL regions present in 3 anti-PcrV Fab molecules (WO 2009/073631) [D Fab ' SEQ ID Sequence 7 - - - - - Fab 3s|42 E VQLVESGGVVQPGRSLRLSCAASGFTFSNYPHWVRQAPGKGLEWVAVISYDG 35 36 SEKYYADSVKGRFTI SRDNSKNTLYLEMNSLRPEDTAVYYCARNRGDIYYDFTYSA _________MDYWGQGTTVTVSS V 1~4 DIOMOQSSSIASUGDSVTTTCRASEGVRWAWYQQKPGRAPKLIDSL SGVPSFSGSSTEFSLTISSL<QPDDATYYCQHFWSTPYTFGQGTKLEIK 5 ~ab A-VSqene 144r consantVeaOCvyQnd SLtCa(OFT20YI09/073631)VA1 3C ASTKGPSTVVFPASSKTGTAGLVDFEVVWSGLSVTP VL 15 QGLQSPSVTPSSSLGTQTYIACVHKPSNTKVDKVEPK YAS1
V
1 7 -. IRTVAAPSVFIFPPSDQLKSGTASVLLNNFYAKQWPKNALQSGNSQV ___EQSSYS LSSTTLSKADYEKHKVACVTHQGLSSPKVKSNREC WO 2013/128031 PCT/EP2013/054262 126 Table A-4: SEQ ID NOs and amino acid sequences of monovalent anti-PcrV Nanobodies Nanobody SEQ 1 Seauence _____ ____ NO: 511023 1 FVQLVESGGGLVQPGGSLRL SCAASGS TDYYAIGWFRQAPTKEEVSCI TSSTYY225 SVKGRFTASRDNAKNTVYTLQMNSLR PETAV'7YYCVATIGC2ATLGGTLDJVQR3YYYRGSCQGTQV 7217 2120 -7 TVSS (VQLVEEGGLVQAGSLRLSCASGRLSSYMWRQAPEREFVAAMHT SGcPNTYA _______1__ DSVKGTCRPTISRDNAPNPYLQMNLPETAVYYWAT2APLKYPTYWGQGTQVTVSS 2510 3 !EQLVESEGGLVQPGGSLRILSCAASGR IFS INTMEWYRQAPKQRE31LVAGVINAITNYAD ISVKGR5FTIS RDNAKNTVYWLQMNSLJKPE;DTAVYYCHAWARSVSVAY SQN WGQETQVTVSS 2220 1 1EVQLVESGCLV G2523SLLSAASGRSSPYLLIMWRQAPARFVAIGUEST I SVKFTISDNAKVYLNSLKPDTAVYYLGVIREEHAYWGQGLVTVS 61302 7 f EVQLVESGGG-LVQAGGSLRLSCAASGNSTNPMYEWYRQAEGKQRELJVSISRGTVNYAD JSVRGR3FS ISRDNAKDTVYL QNSLKPQDT'AVYYCRLAASSSGTYYWGTLVrvs 10202 0 SVQLVESGGGLVQPGGSLJRLSCAASEGFTFDDYVIGWYRQAPGKEREGIVSTCISNQVPNYD I S VGRFTISDNAKNJTVYQMNAVPDTAVYCALVEASTGYTYPVHPEYDYWGQGTQ 1 109 51 EVQLVESGGGLVQAGGSLRLiTSCVIASTVNSDNMGWFROAPGKEREFNIDWSGGSTYP F ____ ~~SvKGscRPTSRDNAK3NTVYQMSLK13DTAVYYCAGYLTAPL2DWNYN2 WQG TV ~4K0 j 0 1EVVE2GGLVQPAGGS LLSCVISEDGVR' NYDMGWPRQAPG>KERFVACT2INAGGTNYA D11SVKGRFTVSRDNAKNTMY QMSLED 'TAVY YCVAAGFAYSLRANWNWGQGTV 713016 EVQLVESGGGLVQAGGSRLSCAASGFSLG'5FIGEWPRQAPGKEREGVACIDSSDGRTYA __________ D32VKGRFT72SRDNANVYLQMNSL KPT AVYY CATDLIESAGWGFQGTVTVS S7 310 12 / VQLVESGGGLVQAPGSLRLSCAASGRIFSINNMGWYHQAPGKQRELVATITMNGITNYAD ________ _____SVKGRETISRDNAKNTVYLQMNSLKPEDT'AVYYCSRSSGGDPYRDYWGQGT'Tvss -- _______jDSVKGRFTISRDNATSRDNTKITMALQMNSLKPEDVTALVYPYTLTcYPVANF WQTQT VSS 420 14 EVQLVESGGGLVQAGGSLRLSCAI ASTVLDIIWRQAPKEREGVASCSSGSTYYA D)SVKGERFTVSRVNAKNTVYLQMNSLKET91AVYYCAAAPS FYGYFPASGDNYWEKGTQV 4210 -______TVSS WO 2013/128031 PCT/EP2013/054262 127 Table A-5: Armino acid sequences of selected bivalent/biparatopic anti-PcrV Nanobodies Nanobdy SEQ ID Sequence K260 118 EVQLVFSGGGL0QPGGSL RLSCAASG0ST1RSVNPMAWFRQAPGRQREWVATISRSG61AT (1411-40G4 -2 10 YADSVKGRFT2I SGDNAKSSVYLQMNSLJKPEDTAVYY CVTGTYWG4QGTLVTVSSGGGGS GGGSGGGGGSGGGGS0-0-4CGGGCOS0(GGGSGGGGSEVQLVESGGLVQPGGSLRLSC ASGSIFSINNMGWYRQAPGKQRELVTVVTSNLTTYADSVKGRFTISRDNAKNTVYLQ MNSL0KPEDTAYYCNAWAR SVGSVPYSQ FWGQGTLVTVS7 2 72 19 EVQLVESG-GLTVQAGGSLRLS CAASRLTFNHYNMGWFRQNP0KERERVAEVTWSGDKI { 9GYVD'SVKGRFTISRDNTPNPR LVVS 7GGGGSGGGGSGGGGSGGGGSGGGGSGGGG0SGGGGSGGGGSERVQLVESGGGLV 200-400- 0009)Q PGGS LRLSCAASG50S I FS 1INTMGWYRQAPCKQRDLVAS ITMNNQVPNYADSVRFT S - ______RDNvRNITVHLQMNAVKPEDTAYFCNAWVRSSGAS00 PYTNYWCQGTLVTV 65 308 20 E1QVQU4ES0GLQ20GGSLRLSCAASGSIFSIINTMGWYRQAPGKQRELVSTIT1SNLOI) (05- 40 -1411, YADSVQGRFT ISRDNARNT VYLQMNSLKPQDTAHYY CNAW2ARS SCATPYTNYWGQGTL VTVSSGGGG0SCGGSGGSGGGSGGGGSGGGGSGGGG0-CCGSEVQL LESGGGLVQ PGG4LRLSCAASGSTRSVNPMA0WFRQAPGRQREWVATI SRSGYATYADSVRCRFT ISG ________________DNAKSSVYLQMNSLKPEDTAVYYCVTGTYWCQGT LVTVSS4 264 p121 EVJQVSGGLVQGGSLRLSCAASSSTRS VNPMAWFRQAPGRQREWVATIS0RSGYAT (212 1-40G4-2402) YADSVKGRFTISGDNKSSVYLQMNSLKPEDT7AVYYCVTGTYVWGQGTLVTVSSGGGGS SCGGGGSCGGGGSGGGGSGGGGSGGGGSGGGGSGGGCSEVQLVESCGGGLVQ PCDSLRLS CA 0A0GR0 LSTNNMVWYRQPGKQRE LVAHIITS SGSTGYADS VKGR0FT ISRDNAKINTEFYLQ 14TNLNPETAVYY CNCWVS SDSNPLKNOYWCQGTILVTVS S 302 122 E VQLVESGGG0VQOGGSLRLSAASSTLDYAWFFQAPGKEREGVISCTSONSOSTY 5401 -4004- 7010o YGGSVKGRFTA1'SRDNAKMTVTLQBNSI FR40EDTAVYCVAT IGCATLGGTLDVQRYYYR GIQCTLVTVSSGGGGSGGGGSGGGGSGGGG00SGGGGGGSGGGGSCGGGSEVQLvESG GGCLVQAGGSLRLSCTASGR TLSSYTMGWF RQAPGTEREFPVAAMTR SGFNTYYADSVKG4 R , FT1 I1SRDSTENTMALQM'1SSLKPEDTAVYYCTAGRGCL TSYRADYWGQGTLVS S 234 12 L'VQLVESGGLVQAGGSLRLSCT1ASCGRTLSSYTMGWFRQAPGTFNFEJAENTF' 7010- 4004- 901 YYADSVRFRTISRDSTENTMATLQMISSLKPEDTAVYYCTAGR0GLTSYRADYWGQ01TLY TVSSGGGGSGCSGGGGSGGGGSGGGGSGGGGSGGGGSGGGG0SEVQLVESGGLVQP 00G212 40CA30GSTLDYYAICWFRQAPGKEREGVSC CTSNSGTYYGGSVKGRE SF0R - - ---- _______ NA1KNTVYLQMNISLRPEDTAVYVATIGOCATLGCTLDVQRYYYRGQGTLVT VS 064 124 EVQLVESGGGLVQAGGLLSCAASGNTF0'STNPYWYRQAEGKQREL VASISS4CI ( 107-40G4-7010) YADSVRFCS ISRDNTKDT1VYLQMNSLKPEDITAVYYCRLASLSSGCTVYWGKGOTLTVS 40G00GSGGGGSGGGGSGGGG0-SGGSGGGGSGGGGGSEVQLVE SGGGL VQAGGS LRLSC041 CT RSSYTMGWF1(RQAPGT EREFVAAMT ROGFNTYYADSVKGR0 Fri 51RDST ENTMAL'QMSSLKPEDTAVYYCM TAGRGLTSYR9JYWG1QGT LVTV SR 275 125 EVQLVESGGGLVQPGGSLRILSCAOASGSFGDNYELYAMTWFRQAPGERRDFVASVTGDG (2G09-40C-5H410 STSYADS VKDRFISRDNAKKLM41( 1YLQMNSLKPEDT1AVYYCRKLLNYWGQGTLVTVSSCG GGSCGGSCGCGGSGGGG000GGGGSGGGSGGGCGGGSVQLVESGGGVQ PGGS L S 20A50GSTLDYYA IGWFRQAPGKEREGVSCTSNSGSTYYGGSVKGR FTASRD0 NAKNT I. YLQMNS0LR PEDTAV YY CVATTGCATLGCGTLDVQRYYYRGQGTLVITVS S 0 83 1226 EVQLVESGGGCLVQAGGSLRrUSCTASGR T LS1SY0 FRQAPGTIERE1FVAAMTRSCSGNI 7C10-40GS-11B 9' YYADSVKGR1FTI SRDSTENTMNALQM4SSLKPEDTAVYYCTAGRGLT1SYRADYWGQ0TLV TVSSGGGGSGGGGSGGGGSGG.GGSGGGGSGGGGSGGGGSCCGGSEVQL VESGGGLVQA CCSLRrLSCAARLFNYNMGWFQAPGKERFERVAEJVTNSGDKIYYVDIS VKGRF FISR DNTPNP71LYLQMNSLKPEDTA'VMYYCATAPRGL PYANCYWGCQCGTLVTV1SS 087 1 27 EVQLVESGGGLVQPGGCSLR LSCAASG4STRSVNPMAWFRQA PGRQREWVAISRSGYAT1 (1E11 -4004-2C00 YADSVKGR FT I SGDNKSVYLQMNSLK 1PEDTAVYY CVTGTYWGQGTLVTVS5GGGGS GGGGOSGGGGCSGGGGGGGSGGGGSGGGGSGGGG.SEVQ LVESGGGLVQAGGsLRLS 001 ASGRTLSSYTMGCWFRQAPCGTEREFVAAMTRISGFNYYASVKGRFTI'SRLSTENTMAL0 QMSS LKPEDTAVYY CTAGRGLTSYRADYWGQGTLVTVS 2 69 -128 EVQLVE 1SGGGLV~QPCSLRLSCAASGSIFSITNTMGWYRQAPCGKQRE'LVSTITSNLVPH 6B5 -4004-13F07 YADSVQGRFTISRDNRNTVYLQMNSLKPQTHYYCNAWARFSSCGATPYTINYWGQGTL VT VSOGGGGSGGGGSGGGGSGGGGSGGGGSGGGSGGGGSGGGGSEVQLVESGGGLVQ AGGSLRLSCAASNTSTNPMWYRQAEGKQRELVA 70ISSRGTTNYADSVRGR1(S ISR DNTKDTVYLQMNS405LKP'EDTAVYYCRLAOSLO OCT VYWGKGTLVITVS S 256 1 29 E'VQLVESGGGLVQAGG SLRLJSCAASTST 4 NPMYWYRQAEGKQRELVASISSRGITN0 (13 F07-4 0GS-5401) 100DSV RERSTISF04NTKDTVYLQMNSLK4PEDTAVYYCFLASLS4S0TVYWGKGTL1 VTVS SGGGGSGGGGSGGGGSCGGGSGGGGSGGGGSGGGGSCCGGS EVIQL VESCGGLVQPGGS WO 2013/128031 PCT/EP2013/054262 128 LRLS CAASOSl LDYYAIGWI'ROAPGKEREGVSOTSNSGSTYYGGSVKGRFTASRDNAK NTVYLQMNSLR PEDT A VYY CVA'TOCATLGGTLD VQRZYYYRGQTLVTVS S 2T'/ 130 EVQLVESGGGL.VQPGGSLRLSCAASGSTLDYY"AIGOWFRQAPGKEREGVSCTISNSCSTY ( 5H01- 40G- J-11B0 9YOGSVWGRFTASRDNAKNTVYLQMNSLRPEDTAVY{YCVATIGCATLGGTI LDVQRYYYR GQGTLVTIVSOGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLV SG25 00GLVQAGGSLRLSCAASRLFNYNMGOWFRQAPOKERERVAEVTIWSGDKlYYVDSVKGO RFT1ISRDNZTPNPVYLYLQMNISLKPED5TAVYYCATAPRGLPYANGYWGQGTLVTV75£ I25"i 131 2EVLVESGGGLVQAGGSLRLSCIAASGNTFSTNPYYRQ7AEGKQ2RELVAS ISSRGITN (13307 403GS-2310, YADSVRORFS ISRDNT1KDTVYLQMN4S LKPEDTIAVYYCOREASL0SSGT VYWGKGTLVTVS SGGGGSGGGGSGGGGSOGGGSGGGGSGGGGSGGOGSGGGGS EVQ2LVESGGGLVQPOGS LRLESCAASOS I1FS1INNMGWYRQAPOKQRZEVTVVTSNLITTYADSVKGR FT7ISRDNAK -NTVYLQ7MNSLKPEDITAVYYCNAWARSVOSVPYSQFEWGQGTLVTVSS0 235 32 EVQLVESGGGLVQ7AGGSLRILSCAASGNTFSTNPM4YWYRQ2AEGKQR2EVASI53RG1TN 13F07 -40GS2-2302' 3 Y<ADSVR1GRF1S ± SRDNT'KDTVYLQMNISLKPEDTAVYYCRLASLSSGTVYWOKOTLVTVS7 SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGDOS LRLS CAASORT J1LS INNM4VWYRQ7APGKQ7RELVAHIT1SSGSTGYADSVKGRFT11SRDNA1 ______________ ____ NTFYLQM1)TNLN3PEDTAVYYCNCWVSSDSNAPLKNYWGQGOTLVTVSS 1B009- 40GS- -30 I YYVDSVKGRFTISRDNTFPPVYLYLQ7MNSLKPEDTIAVYCAARGLYANYWGQGOT LVTV5SSGGGGSGGGGSGGGGSGGGGSGGGGOSGGGGSGGGGSOGGGS REVESGGGLV IQAGGOSLRLSCAASGNTF-ST£NPMYW/YRQ2AEGKQRZELVAS ISSRG01T'NYADSVRGRFS Is RDINTKDTVYIQMNSLKED2TAVYYCRLAS LSSGTVYWGKGTLVTVS55 253 134 EVQV2ESGGGL2VQAGGSLRLSCAASGNTFSTNPYWYR QAEGKQR7ELVASI1SS30iTN (13F07-4032-14310 YADSVRGRP'S I SDNTKDTYLQ7MNSLKPEDTAVYYCRLA71SSSGTV'WGKGTLVTVS SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEQESGGGLVQPGGS00 L RILSCAASGR IFSINMGW40YRQAPGKQ2REL VAGVTINAITNYADSVKGRFISRD1NAK32 ____________________ NTVWL2QMNSLKPEDTAVYYCHAWARSSGSAPYSQNWGQG7TLVTVS 2728 137EVOLESGGGVQAGOSIQLSCANSORTFRSSYING3WFRQ2APGKAREFVADIT7WGQRPY (7E09-40GS-6Bos2 YADSVKGRFVTISRDNAKNT'VYEMINSLKPEDTAVYYCAADLGVV1IEEAYWGQGTL7V TVSSGGGG'OSGGGGSGGSGGGGSGGGGGGGSGGGGSGGGGSEVQLVESGGGLVQPI OGSLRLESCAASGS IFS INTMGWYRQ112AGKQ7REVSTITSNLVPHYADS VQGRFTISRD$ ______NARNTVYLQMNSLK PQDT7ARYYCNAWAR SSGAT PYTNYWGQGTLVTV7S7 $ 271 136 E72QLVESGGGLVQAGGSLRLSCTASRTSSYTMGOWFRQAPGTE7SFREFVAAMTR'SGFNT' (731'-4035-14310 Y7AD$VKGRFT1ISRDSTENTM1ALQM3SSLKPEDTAVYYCTAGRGLTISYRADYWGQGTL V TVSSGGGGSGGGGSGGGGSGGGG5GG0G5 GGGGSGGGG000GGGGSE7LVESGGGLVQ GGSL2LSCAASGRIFSI NTWYWRQ7APGKQ7RELVAGVTINAITNYADSVKGRFTI'RD32 ______________________NAKNT'IVWLQMNISLKPEDTAVYYCHAWARSSG0SAPYSQNWGQGTL7VTV5SS 2 59 1 3 7 7EVQLV 0ESGGGLVQPGGSRLSCAASGSTR5V32NNWN2ONPG0OREN37n155A: (1E11-40GS-2H142 YADSVKGRFTISGDNAKSSVYLQMN3SLKPEDT37AYYCVTGTYWGQGTLVTVS73GGGG0 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE25VQLVE7SGGGLVQPGGSL RLSCA ASSTLDYYAIG WFRQ7APGKLREGVS CTSNSGST<YGGSVKGRFTASRDNAKNTVYL0Q MNSLRPEDTAVYY7CVATIGCA TLGGTLDVQ2RYYYRGQGTLVTVSS5 319 133 L'EVVSGGGLVQAGGSIRLSCAASG2NTFSTPYWYRQ7AEGKQRELVASISSRGITNI (13507-40G5-630)705 YDVRRFSI15RDNTKDTVYLQ2MNLKEDAYYCRALSSGTVYWGKGTLVTVS LE SS0 IFS 1NTM0WYRQAPGKQ2RELV7STITSNLV7PHYADSVQGRFTISRDNAR2 NT'Y LQMNSLKPQDT53 AHYYCNAWARSSGATPYTNYWGQGTLVTV7773 335 1 39 E7~VQLVSGGGLVQPGOSLRLSCAASGSTL DEYIOIWVR2ONPOKSE'075 07535057 (5201--4002-1E1 YGGSVKGRFTASRDNAKNOTVYLQMNSLRPEDTAVYYCVATIGCATLGGTLDVQR27YT 0270TLVTV7SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQL<VESG 261 1420 77 EQVESGGGLVQ7PGGSLRLSCAASGSTLDYAOWFRQ7APGKE2REGVSCTSN3SGST (51101 - 4 0G5 2-21 YGSVKG0RFTA'SRDNAKNTVYLQMNSL35RPEDTAVYYCVA'T IGACAGTLDVQ2RYYYR GGLVQ7PGGSLRLSCAASGS IFS'913iGWYRQ7APGKQRELVTVTSNLIT TYADSVKGR30 FT7ISRDNAKNTVYLQMNSLPEDT3AVYYCNAWARSVGSVPYSQF7WGQGTLVTVSS3 262 141 EVQLVESGGGLVQAGGSL01RILSCAASGRITFSSY1MGWFRQ7APGKAR'FVA2O1I3402<O (7305-4005-701) YAD225V1001FTISRDNAKNTVY7LEM(NSLKPEDTAVYCAADLGI07REEYWGQGTLVA TVSSGGGGSGGGGSGGGGSGGGG.SGGGGSGGGGSGGGGSGGGGEVQLESGGGLVQA72 0031RLSCT"ASGRTLSSYTMGWFRQ7APGTEREFVAMTRSGFNTYYADSVKGRFTISR15 DSTENTMALQMSSLKP3E0DTAVYYCTAGRGL T'SYRADYWGQGTLVTVSS ____ WO 2013/128031 PCT/EP2013/054262 129 F T- T - -i I o A o a9 0 A 0 A 0a a 0 o1o o I >4T (9 (9(994>I(994('4141Q( (94>1(94>5(9(9(9(94(9(9( (915944(19(i4(to 9(94' (9(5 (94> en m e emocoe 0le y 0.o 0> S 9 LO LCYLm o >0> e o 04 (9'> vs [ wVma;o km lop (9H o 5 o H (9. 4 p (;>o o' a o9 (9>e >>54>'94.. e >le9 e> se 9 e >e >o o> 1e s _ F 2 'jr i x 22 bx o >>(9>e'8 *> F r ±>' -J K' 44 44 44 i2 ShF 149( I9 9 (1 4 '(9' ( L. I6 C) it L. F 4 In mmm a O ~ I> 4> -' I 41 C9 C£4 vst o a 2C Ct to _ K 1 4ZA W94S94 Q (9(9; *d - - - -<3(9. - - -4 < F~c9Iu 9.-- - -K 4-94 - - -- (' (, - - --- 2 C)- + - - - - - - - - - i - - - - - - * U (9(>(-9>-94 > emer e 994>1.9>4'(94 C 4 82>1144>419)4>49494U44> (419> 2 414>I49(43>(8>49}4C949(99(4(44> 18> o ~ x 4o ozx 2a2a FZ(o93(oJJ<o( 949(1>941 s9"- -9"' 949(1494(4!994>4 9499.4,(C(9949409(Q(44(( C) ((9(1 (>4.ZI9494lp9~ 94 1g94<10(44(944($.4.-~(91Q94(9494(A, 4- (9n to[-" 9.>4AIA.- -. 49.a > 9'O I.4-<A. 4 o o o o a o v 9 >( o o o o o o o 4fl>4>41o o o o19 o o (4i(94)4o(e ( 194[(9( o o o e o9ose ' o ;0 0; ; 0 a&;o 4 ;(9I4e(994 e uou 9us u %uo94u ou 942 14. .2>294. 2942(19 <tt83 5IF3 <44K' 18 '4 94>o (9 4 {o a 1> s>~m em m u i '4- oc9a (9 4o Ioo o too o C et H((94(998 -49( H (91 8 17 4 o> Sr 4>'34 9491 a 4 49 > 9 >( z (9 '(1>4 i to (99.In>4>1991<( -9(5 4 > 4 9 >'4 (C s 9 9>42 o ~-9l~z~> ~~5 Z.-~~i~~~~ ~H ~~~~-l.>(H~ ~- T ~9-.--94~ ( (~ (9(9>~19>1> 1949>14>94' 4 4- N 9I 4t(9 H)4~4994(941 'j'>4f 40 WO 2013/128031 PCT/EP2013/054262 130 Table A-7: Amino acid sequences from PcrV fragment (arnino acids 14-257) and chimeric molecules used for epitope mapping Molecule SEQ ID Seqene (AA 144 -25 1-WAS- L -- T C2ra 99 --- - -_ T- 2 0 EVLAARSELFLELAAQLSAPAAQELLALLRSAPRIVQLVHAGPSE IQACAQWHDQQGKRKFESPTWLEFLVSAYFSLHGRLDEDIVKVQCDKKL VGVKLQT DGRKALDEKALVELVYSQQSINAALSAKQGIRIDG ILDPL2YV DAGWDDSPTLYGYADPRWSKDPYLSNLDTQSGKLSIDPLEGKD QSELGLDEPFKNPGNATTVSDRSRPLNDKVNEKTTLLNDTSSYNAELIEQYSR YNAELIKDVLRDILSAI Chier 3 200 MEVRNLNAAREL 4 FLDELLAASAAPASAEQEELLALLRSERIVLAGOACPLSE AQVLKALAWYDLNQPSAPPGLEVLRENQARRPGAWELVSAYRD VIGLYDICDDDLQVDHGKRKALLDELKALTAELKVY TT5 NASANQSQI5 NASGRDAGGIDLVDPTLYGYAVGDPRWKDSPEYALLSNLDTF ESGP KLSILKFCSKEKLSDEYPPEKDNNPVGNFARLDVTTRRLNDSS _________ ____V NSAVTLLNDTIQRYNSVESAIQYSLDLA Camera 3 202 NEVRNLNAARELFLDELLAASAAPASAEQELLALLRSRVLA-AGQPLS EAQVLKALAWLL AANPSAP PGQGLEVLREVLQARRQPGAQWDLREFLVSAY FSLHGIRDEDDVIVKDVLNQQKRKALLDELKALTAELKVYSVIQSQIN ALSASGIIDAKSILDLYGYAVGDPRWKDSPEYALLSNLDTSGK L JS IKDLSGSPKQSGELKGLSDSYPFEKDNNPVSNFATTVSDRSRPLDV I VNEKTTLLNDTJS SRYNSAVEALNRPIQKYSVLRDILSAT
~
1 ~ma 4 NESNLNARSFLDELAASAPAAEELLALRSR L HA7L T 2L '>LK LA5LAPC PQISLELAiQGQDR VA I ID HO L~hIDVOVYDVLQQDCPKALDELALCELKVSVITQ: WO 2013/128031 PCT/EP2013/054262 131 Table A-8: Linker sequences Linker SEQ ID NOl que - --- 182 GGGGS-- 76 1ikr83 sggsggs 8 ini1~84 ggggcggg 1 7 GGGSGGkSGGG 10 - j 8 GGGSGGeGGGG 189 GGGSGGGSGGGGSGGGGS 2 16 rik 190 GGGGSGGGGSGGGGS66GGGGGGGS 12 GGGGSGGGGSGGGGSGGGGSCGGGSGGGGS Jonq y cch n I S:194 S DKTHTCPCP _________________ - _______ jCPRCPEPKSCDTPPPCPRCP Equivalents e The foregoing written specification is considered to be sufficient to enable one killed in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as an illustration of certain aspects and embodiments of the invention. Other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention in addition to those shown and described herein will become. apparent to those skilled in the 10 art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encomnpassed by each embodiment of the invention. What is d aimed:

权利要求:
Claims (17)
[1] 1. Polypeptide comprising or consisting of two or more immunoglobulin single variable domains that are directed against PcrV wherein at least one "first" immunoglobulin single variable domain is S directed against airst eptope on PcrV and at lest one "second immunoglobulin single variable main is directed against a second epitope on PcrV different from the frst epitope on PcrV.
[2] 2. The polypeptide according to claim , which is capable of neutralizing PcrV with 100% efficacy and with an IC50 of 5.0x10 1M or lower in a cyotoxicity ssay with P3X3 cells as target at an MCI of 12. 10
[3] 3. The polypeptide according to any of claims 1 or 2, for which the decrease in potency afer 24 hours in the presence of P. euginosa elastase (3 ug/ug polypeptide) is maximal 5 old (e.g. 5 fold, 3 fold, 2 fold or lower) 15 4 Th e polypep ide cording to any of claims 1 to 3, for which the decrease in potency after 24 hours in the presence of human neutrophil elastase (1-2 ug/ug polypeptide) is maximal 15 fold (e.g. 10 oid 5 fod 3 ol old or lower).
[4] 5. The polypeptide according to any of claims 1 to 4 wherein the two or more immunoglobulin single 20 variable donains consist of a light chain variable domain sequence (e g. a Vsequence) or of a heavy chain variable domain sequence (e.g a V -sequencet
[5] 6. The polypeptide according to any of claims 1 to 5, wherein the two or more immunog obulin single variable domains consist of a heavy chain varinble domin sequence that is served from a 25 conventional four-chain antibody or consist of a heavy chain variable domain sequence that is derived from heavy chan antibody.
[6] 7. The polypeptide according to any of claims 1 to 6, wherein the two or more immunoglobulin sing e variable domains consist of a domain antibody (or an amino aid that suitable for use as a domain 30 antibody), of a single domain antibody (or an amino acid that is suitable for use as a singe domain antibody) of a "dAb" (or an amino acid that is suitable for use as a dAb) or of a Nanobody (including but not limited to a V"), WO 2013/128031 PCT/EP2013/054262 133
[7] 8. The pol peptide according to any of claims 1 to 7 wherein the two or more irnmunoglobun single variable domains consist o a partially or fully humanized Nanobody or a pa rtially or ully humanized VHH. 5
[8] 9. The polypeptide according to any of claims 1 to 8 wherein at least one of the immunoglobulin single variable domains insists of 4 framework regions FRi to FR4, respectively) and 3 complementarity determining regions (CDRi to CDR respective in which: 10 - CDRi is chosen rom the group consisting of: a) the amino cid sequences of SEQ iD NOs: 20-37; b) amino acid sequences that have at least 80% amino acid identity with t least one of the amino acid sequences of SEQ HD NOs: 20-37; c) amino acid sequences that have 3 2, or 1 amino acd difference with at least one of the amino 15 acid sequences of SEQ ID NOs: 20-37; and/or - CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NOs: 38-56; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amine 20 acid sequences of SEQ ID NOs: 38-56 f) amino acid sequences that have 3, or 1 minor acid difference with at least one of the amino acid sequences of SEQ ID NOs: 38-56; and/or - CDR3 is chosen frorn the group consisting of: 25 g) the amino acid sequences of SEQ ID NOs: 57-75 h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 57-75; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SQ ID NOs: 57-75. 30 WO 2013/128031 PCT/EP2013/054262 134
[9] 10. The polypeptide according to any of cairns 1 to 9, wherein at least one of the immunoglobulin single va ible domains consists of 4 framework regions (FRI to FR4, respectively and 3 complementarity determining regions (CDRI to CDR3, respectively) in which: 5 - CDRI is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NOs: 20-37 b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 20-37; c) amino acid sequences that have 3, 2, or I amino acid deference with at least one of the amino 10 acid sequences of SEQ ID NOs: 20-37; and - CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NOs: 38-56; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino 15 acid sequences of SEQ ID NOs: 38-56; f) amino acid sequences that have 3, 2 or amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 38-s and - CDR3 is chosen from the group consisting of: 20 g) the amino acid sequences of SEQ ID NOs: 57-7; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 57 75; i) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 57-75 25
[10] 11. The polypeptide according to any of claims 1 to1 wherein in at least one of the immunoglobulin single variable domains, the CDR sequences have at least 70% amino acd identity preferably at least 80% amino cid identity more preferably at least 90 amino and identity such as 950 amm and identity or more or even essentially 00% amino acid identity with the CDR sequences of at least one 30 of the immunoglobulin single variable domains with SEQ ID NOs: 1-19. WO 2013/128031 PCT/EP2013/054262 135
[11] 12. The polypeptide according to any of claims 1 to 11, where ein At least one of the immunoglobulin single variable domain cross-b ocs the bin Ing to PcrV of at le ast one of the immunoglobulin single var ible domain with SEQID NOs: 1-19 and/or is cros- blo ked from binding to PrV by at least one of the immunoglobulin ingle variable domain with SEQ ID NOs: 1-19, 5
[12] 13. The polypeptide according to any of claims 1 to 12, wherein at least one of the immunoglobulin single variable domains is selected from any of SEQ ID NOs: 1-19.
[13] 14. The polypeptide according to any of laims 1 to 13, wherein bot immunoglobulin single variab e 10 domains are immunoglobulin single variable domains as defied in any of claims 9to 13,
[14] 15. The polypeptide according to any of claims 1 to 14, wherein each of the two immunoglobulin single variable domains that are directed against PcrV belong to a different epitope bin. 5 16. The poly peptide according to any of claims 1 to 15, wherein the first immunoglobulin single variable domain does not cross-blocks the binding to PcrV of the second immunoglobulin single variable domain and/or wherein the first imunoglobulin single variable is not cross-blocked from binding to PcrV by the second immunoglobulin singe variable domain. 20 17. The po peptide according to cIaim 16, wherein: - the first imunoglobulin single variable domain cross-blocks the binding to PerV of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 3-10 epitope bin1] and/or is cross-blocke from binding to PcrV by at least one of the immunoglobulin single vanible domains with SEQ ID NOs: 3-10 [epitope bin1); and the second immunoglobuin single variable 25 domain ross-block the binding to PcrV of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 1-2 epitope bin2] and/or is ross-blocke front binding to PcrV by at least one of immunoglobulin single vanible domains with SEQ ID NOs: 1-2 [epitope bin2 - the first immunoglobulin single variable domain cross-blo ks the binding to PcrV of at least one of the immunoglobulin single friable domains with SEQ ID NOs:3-10 [epitope bin1 nd/or is so cross-blocked from binding to PcrV by at least one of the immunoglobulin single variable domains with SEQ ID NOs: 3-10 [epitope bin1] and the second immunoglobulin single variable domain cross-b rocks the binding to PcrV of at least one of he immunoglobulin single variable WO 2013/128031 PCT/EP2013/054262 136 domans with SEQ ID NOs: 11-12 [epitope bin3] and/or is cross-blocked from binding to PcrV by at least one o the immunoglobulin single variable domains with SEQ ID NO 11-12 epitope bin3] - the first immunoglo ulin single var able doain ross-bloc s the binding to PcrV of at least one 5 of the immunoglobulin single variable dornains with SEQ ID NOs: 1-2 epitope bin2] and/or is cross-blocked from binding to PrV by at leas one of the immunoglobulin single variable domains with SEQ ID NOs: 1-2 [epitope bin2]; and the second immunoglobulin single variab e domain cross-blocks the binding to PcrV of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 3-10 epitope bin1] and/or is cross-blocked from binding to PcrV by at 1 least one of the immunoglobulin single varable domains with SEQ ID NOs: 3-10 epitope bm - the first immunoglobulin single variable domain cross-blocks the binding to PcrV of at least one of the immunoglobulin single variable domains wih SEQ D NOs: 1-2 epitope bin2 and/or is cross-blocked from binding to PerV by at least one of the immunoglobulin single variable donains with SEQ ID NOs: -2 [epitope bin2]; and the second immunoglobulin single vaNble 15 domain cross-blocks the binding to PcrV of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 11-12 epitope in3 and/or is cross-blocked rom binding to PcrV by t least one of the immunoglobu in single variable domains with SEQ ID NOs: 11-12 epitope bin3]; - the first immunoglobulin single variable domain cross-blocks the binding to PcrV of at least one 20 of the immunoglobun single variable dorains with EQ ID NOs: 11-12 [epitope bin3} and/or is cross-blocked from binding to PcrV by at least one of immunoglobulin single variable domains with SEQ ID NOs: 11-12 [epitope bin3]; and the second immunoglobuin single variable domain cross-blocks the binding to PcrV of at least one of the immunoglobulin single vaable domains with SEQ ID NOs 3-10 epitope bini] and/or is cross-bloked rom binding to PcrV by at least 25 one of the immunoglobulin single variable domains with SEQ ID NOs: 3-10 [epitope bn] or - the first immunoglobulin single variable domain cross-b rocks the binding to PcrV of at leas one of the immunoglobulin single variable domains with SEQ D NOs: 11-12 [epitope bin3} and/or is cross-blocked rom binding to PcrV by t least one of the immunoglobulin single va liable domains with SEQ ID NOs: 11-12 [epitope in3 and the second irmunoglobulin single variable 30 domain cross-blocks the binding to PcrV of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 1-2 epitope bin2 and/or is cross-blocked from binding to PcrV by at least one of the irmunoglobulin single variable domains with SEQ ID NOs: 1-2 [epitope bin2 WO 2013/128031 PCT/EP2013/054262 137 18 The polypeptide cording to claiN 16, wherein: - the first immunoglobulin single varible domain is selected from the polypeptide o any of aims
[15] 47- 53 epitope bin1 and the second immunoglobulin single variable domain is selected from 5 the polypeptide of any of claims 40-46 epitope bin2; - the first immunoglobuWin single variable domain is selected from he polypeptide of any of claims 47-53 [epitope bin1]; and the second immunoglobulin single variable domain is selected from the polypeptide of any of claims 54- 0 [epitope bin3); - the first immunoglobulin single variable domain is selected from the polypeptide of any of claims 10 40-46 epitope bin2]; and the second immunoglobulin single variable domain is selected from the polypeptide of any of claims 47-53 epitope bin1 - the first immunoglobulin single variable domain is selected fron the polypeptide of any of claims 40-46 epitope bin2); and the second immunoglobulin single variab e omain i selected rom the polypeptide of any of cims 54-60 epitope bin3 15 - the first imrunoglobulin single variable domain is selected from the polypeptide of any of claims
[16] 54-60 epitope bin3 and the second immunoglobulin single variable domain i selected from the polypeptie of Any of claims 47-3 [epitope bin1; or - the first immunolobuiin single variable domam is selected from the polypeptide of any of claims 54-60 epitope bin3]; and the second immunoglobu in single variable domain is selected from 20 the polypeptide of any of claims 40-46 [epitope bin2], 19. The polypeptide according to any of clams I to 18 which is selected from any of SEQ ID NOs: 124
[17] 141. 25 20. The polypeptide according to any of claims 1 to 19 wherein the first immunoglobulin single variable domain is SEQ !D NO: 12. 21. The polypeptide according to claim 20 where the second immunoglobulin single variable omnin is selected from any of SEQ ID NOs: 1 and 10 30 22. The polypeptide according to claim 21, which is selected from any of SEQ ID NOs: 129 and 134, WO 2013/128031 PCT/EP2013/054262 138 23. The polypepti e according to any of claims I to 19, wherein the second immunoglobulin single variable domain is SEQ l NO: 1, 24. The polypeptide ac ording to claim 23, wherein the first immunoglobulin single varia le dom in is 5 selected from any of SEQ ID NOs: 3 and 12. 25. The polypeptide according to claim 24, which is selected from any of SEQ ID NOs: 129 and 137. 26. The polypeptide according to any of claims 1 to 14, wherein the two or more rnmunoglobulin single 10 vanable domains that are directed against PcrV belong to the same epitope bin. 27, The polypeptide according to laim 2 wherein the first immunoglobulin single variable domain cross-blocksthe binding to PcrVof theseond immunoglobulin single vriable domain and/or wherein the first immunoglobulin single variable domain is cross-blocked fron binding to PrV by the s second immunoglobulin single variable domain. 28. The polypeptide according to claim 27, wherein: - the first and the second rnmunoglobulin single variable domain cross-block the binding to PcrV of At s one of immunoglobulin ingle variable domains with SEQ ID NOs: 3-10 epitope bin1] 20 and/or the first and the second immunoglobulin single varable domain are cross-bocked from binding to PcrV by at least one of the immunoglobulin single variable domains with SEQ ID NOs: 3-10 [epitopoe bin1i - the first and the second immunoglobulin single variable domain cross-bock the binding to PcrV of at least one of the immunoglobulin single varible domain with SEQ ID NOs: 1-2 epitope 25 bin2 and/or the irst and the second immunoglobuln single variable domain are cross-b ocked from binding to PcrV by at least one of the immunogobulin single variable domains with SEQ ID NOs: 1-2 [epitope bin2 or -the first and the second immunoglobulin single variable domain cross-block the binding to PerV of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 11-12 epitope 30 bin3] and/or the first and the second immunoglobulin single variable domain are cross-blocked rom binding to PcrV by at least one of the immunoglobulin single variable domains with SEQ ID NOs: 11-12 [epitope bin3 WO 2013/128031 PCT/EP2013/054262 139 29. The polypeptide according to claim 27, wherein: the first and the second immunoglobulin single variable domain are selected from the polypeptide of any of claims 47-53 epitope bin1]; 5 - he first and the second immunoglobulin single variable domain are selected from the polypeptide of any of cims 40-46 [epitope bin2; or - the first and the second immunoglobu in single variable domain are selected rom the polypeptide of any of claims 54430 [epitope bin3). 10 30. The po ypeptide according to any of claims 26 to 29, which is selected from any of SEQ ID NOs: 118 123. 31. The polypeptide according to any of claims 26 to 30, wherein t least one immunoglobulin single variable domain is SEQ ID ND: 3. 32. The polypeptide according to claim 31, which is selected from any of SEQ ID NOs: 118, 120 and 121. 33. The polypeptide according to any of claims 2 to 30, wherein at least one immunoglobulin single variable domain is SEQ ID NO: 1. 20 34, The polypeptide according to claim 33, which is selected from any of SEQ ID NOs: 122 and 123. 35. Polypeptide that essential y consists of 4 framework regions (FR1 to FR4, respectively) and 3 complementarity determining regions (CDR! to CDR3, respectively) in which: 25 - DRi is chosen from the group consisting of. a) the amino acid sequences of SEQ ID NOs: 20-37; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 20-37; c) anno acid sequences that have 3 2, or 1 amno acd difference wth at least one of the amino 30 acid sequences of SEQ ID NOs: 20-37; and/or - CDR2 is chosen from the group consisting of: WO 2013/128031 PCT/EP2013/054262 140 d) the amino acid sequences of SEQ ID NOs: 38-56; e) amino acid sequences that have at least 80% minor acid identity with at least one of the amino acid sequences o SEQ ID NOs: 38-5 amino cid sequences that have 3, 2, or 1 amino acid difference with t least one of the amino 5 acid sequence of SEQ ID NOs: 38-56; and/or - CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NOs: 57-75; h) amino acid sequences th have at least 80% amino acid identity with at least one of the amino 10 acid sequences of SEQ PD NOs: 57-75; amino acid sequences that have 3, 2, or 1 anino acid difference with at least one of the amino acid sequences of SEQ1D NOs: 57-75. 36. The polypeptide according to clain 35, in which: 15 - CDR1 is chosen from the group consisting of: a) the amino acd sequences of SEQ ID NOs: 20-7; b) amino acid sequences that have at least 80% amino acd enity wi at east one of the amino acid sequences of QID NOs: 20-37; c) ino acid sequences that have 3, 2 or 1 amino acid difference with at least one of the arino 20 acid sequences of SEQL D NOs: 20-37; and - CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NOs: 38-56; e) arnio acid sequences that have at least 80% amino acid identity with at least one of the amino 25 acid sequences of SEQ ID NOs: 38-56; f) amino acid sequences that Iave 3, 2, or 1 arino acid difference with at least one of the amino acid sequences of SEQ D NOs: 38-S6; and - CDR3 is chosen from the group consisting of: so g) the arino acd sequences of SEQ ID NOs: 57-75; h) amino acid sequences that have at east 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 57-75; WO 2013/128031 PCT/EP2013/054262 141 ) amino add sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid equences of SEQ ID NOs: 57-75. 37. The polypeptide according to any o claim 35 or 36 in which the CDR sequences of said polypeptie 5 have at least 70% amino acid identity preferably at east 80% amino acid identity more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% amino acid identity with the CDoR sequences of at least one of the immunoglobulin single variable domains with SEQ 1D NOs: 1-19, 10 38. Polypeptide directed against PcrV, that cross-blocks the binding o PcrV of at le st one of the immunoglobulin single variable domains with SEQ ID NOs: -19 and/or that is cross-blocked front binding to PrV by at least one of the immunoglobulin single variable dom gains with SEQ ID NOs: 1-19, 39. The polypeptide according to any of claims 35 to 38, that is selected from any of SEQ ID NOs: 1-19, 15 40. The polypeptide according to any of claims 35 to 39, that belongs to epitope bin 2. 41. The polypeptide according to claim 40, that binds to full length PcrV (SEQ ID NU: 59) and that shows reduced binding 30-90% or no binding to chiner 7 NQ ID NO: 205). 20 42. The polypeptide accordingto any of caims 40 or 41 in which: - CDRi is chosen from the group consisting of: a) the amino acid sequences of SEQ ID NOs: 20-21 b) amino acid sequences that have at least 8Q% amino acid identity wit at least one of the amino 25 acid sequences of S Q ID NOs: 20-21; c amino acid sequences that have 3 2, or 1 amno acid difference wth at least one of the amino acid sequences of SEQID NOs: 20-21; and/or - CDR2 is chosen from the group consisting of: s0 d) the arino acid sequences of SEQ ID NOs: 38-39; e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ D NOs: 38-39; WO 2013/128031 PCT/EP2013/054262 142 f) amino acid sequences that have 3, 2, or 1 amino acd difference with at least one of the amino acid sequences of SEQ ID NOs: 38-39; and/or CDR3 is chosen from the group consiing of: g) the amino acid sequences of SEQ ID NOs: 57-58 h) amino acid sequences that have at least 80S amino aid identity with at least one of the amino acid sequences of SEQ ID NOs: 57-58; i) arino acid sequences that have 3 2, or 1 amino acd difference with at least one of th amino acid sequences of SEQ ID NOs: 57-58 10 43. The polypeptide according to clam 42, in which - CDR is chosen rom the group consisting of: a) the amino acid sequences of SEQ ID NOs: 20-21; b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino 15 acid sequences of SEQ ID NOs: 20-21; c) amino acid sequences that have 3 2, or I amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 20-21 and - CDR2 is chosen rom the group consisting of: 20 d) the amino acid sequences of SEQ ID NOs: 38-39 e) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 38-39 f) amino aci sequences that have 3, 2 or 1 amino acid difference with at least one of the amino acd sequences of SEQ ID NOs: 38-39; 25 and - CDR3 Is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NOs: 57-58; h amino acid sequences that have at le st 80% anino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 57-58; so i amino acid sequences that have 3, 2 or 1 amrnio acid difference with at least one of the amino acid sequences of SEQ ID NOs: 57-58, WO 2013/128031 PCT/EP2013/054262 143 44. The polypeptide according to any of claims 40 to 43 in which the CDR seq uences of said polypeptide have at least 70% amno acid identity, preferably at least 80% amno acid identity, more preferably at least 90% amino acid identity such as 95% amino acid identity or more or even essentially 100% mino acd identity with the CDR sequences of at least one of the immunoglobulin single vanible 5 domains with SEQ ID NOs: 1 nd 2. 45, The polypeptide directed against P rV, that cross-blocks the binding to PcrV of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 1 and 2 and/or that is cross-blocked from binding to PcrV by at least one of the immunog obulin single variable domains with SEQ ID NOs: 1 10 and 2 46. The polypeptide according to any of claims 40 to 45, that is selected from SEQ ID NOs: 1 and 2. 47. The polypeptide according to any of claims 35 to 39 that belongs to epitope bin 1. 15 48. The polypeptide according to claim 47, that binds full length PcrV (SEQ ID NO: 159) an that shows reduced binding (30-9 ) or no binding to chimera 4 (SEQ ID NO: 202) and camera 6 (SEQ ID NO: 204). 20 49. The polypeptide according to any of claims 47 or 48, in which: - CDI is chosen rom the group consisting of a) the amino a id sequences of SEQ ID NOs: 22-28; b) amino acid sequences that have at east 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 22-28; 25 c) amino acid sequences that have 3, 2, or 1 amno acid difference with at least one of the amino acid sequences of SEQ ID NOs: 22-28; and/or - CDR2 is chosen rom the group consisting of: d) the amino acid sequences of SEQ ID NOs: 40-47; 30 e) amino acid sequences that have at least 80 amno add identity with at least one of the amino acid sequences of SEQ ID NOs: 40-47; WO 2013/128031 PCT/EP2013/054262 144 f) arino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 40-47; a nd/ or - CDR3 is chosen front the group consisting of: 5 g) the amino acid sequeces of SEQ ID NOs: 59-66 h) amino acid sequences that have at least 80% amino acid identity with at least one of the arino acid sequences of SEQ ID NOs: 59- 6; i) amino acid sequences thA t have 3, 2 or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOS: 59-6. 10 50. The polypeptide according to claim 49 in which: - CDRis chosen from the group consist ing of: the amino acid sequences of SEQ ID NOs: 22-28 b) amino acid sequences that have at least 80% amino acid identity with at least one of the amino 15 acid sequences of SEQ D NOs 22-28; c) amino acid sequences that have 3 2, or 1 aino acid difference with at least one of the anino acid sequences of SEQ ID NOs: 22-28; and - CDR2 is chosen from the group consisting of: 20 d) the minor add sequences of SEQ ID NOs: 40-47; e) amno acid sequences that have at least 80% amino acid entity with at least one of the amino acid sequences of SEQ ID NOs 40-4; f amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 40-47; 25 and - CDR3 is chosen from the group consisting of: g) the amino acid sequences of QID NOs: 59-66; h) amino acid sequences that have at least 80% arno acid identity with at least one of the anno acid sequences of SEQ ID NOs: 59-66; 30 i) amino acid sequences that have 3 2, or 1 amino acid difference with at least one of the amino acid sequences o SEQ ID NOs: 59-66. WO 2013/128031 PCT/EP2013/054262 145 51 The polypeptide according o any of aims 47 to 50, in which the CDR sequences of said polypeptide have at least 70' amino acid identity, preferably at least 80% amino aci identity, more pre erably at least 90% amino acid identity such as 95% amino acid identity or more or even essential y 00% amino aci identity with the CDR sequences of at least one of the immunoglobulin single variable 5 domains with SEQ ID NOs: 3-10. 52 Polypeptide directed against PcrV that cross-blocks the biding to PcrV of at t one of the immunoglobulin singe varable domains with SEQ ID NOs: 3-10 and/or that is cross-blocked from binding to PcrV by at east one of the immunoglobulin single variable domains with SEQ ID NOs: 3-10, 10 53. The polypeptide according to any of claims 47 to 52 that is elected from any of SEQ ID NOs: 3-0, 54. The polypeptide according to any of claims 35 tc 39, that belongs to epitope bin 3. 15 55. The polypeptide according to claim 54 that binds full ength PcrV (SEQ ID NO: 159) and that shows reduced binding (30-90%) or no binding to chimera 2 SEQ ID NO: 200), 56. The polypeptide according to any of claims 54 or 55 in which: - CDR1 is chosen from the group consisting of: 20 a) the amino acid sequences of SEQ ID NOs: 29-30; b) amino acid sequences that have at east 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 29-30, c) amino acd sequences that have 3, 2 or 1 amino acid difference with at least one of the amino acid sequel ces of SEQ ID NOs: 29-30 26 and/or - CDR2 is chosen from the group consisting of: d) the amino acid sequences of SEQ ID NOs: 48-49 e amino acid sequences that have at least 80% amino ac ientty wIth at least one of the anmno acid sequences of SEQ ID NOs: 48-49 30 f) amino acid sequences that have 3, 2, or 1 amino acid difference with at leas one of the amino acid sequences of SEQ ID NOs: 48-49; and/or WO 2013/128031 PCT/EP2013/054262 146 - CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NOs: 67-68; h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 67-8; 5 ) amino acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ D NOs: 67-8. 57, The polypeptide according to claims 56 in which: - CDR1 is chosen from the group consisting of: wo a) the anmno acid sequences of SEQ ID NOs: 29-30; b) amino acd sequences that have at east 80% aino acid identity with at least one of the amino acid sequences o SEQ ID NOs: 29-30; c) amino acid sequences that have 3, 2 or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 29-30; 15 and - CDR2 is chosen from the group consisting of: d the amino acid sequences of SEQ ID NOs: 48-49; e) amino acid sequences that have at least 80% amino aid identity with at least one of the amino acid sequen es of SEQ ID NOs: 48-49; 20 f) minor acid sequences that have 3, 2, or 1 amino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 48-49; and - CDR3 is chosen from the group consisting of: g) the amino acid sequences of SEQ ID NOs: 67-68 25 h) amino acid sequences that have at least 80% amino acid identity with at least one of the amino acid sequences of SEQ ID NOs: 67-68; i) amino acid sequences that have 3, 2, or I anino acid difference with at least one of the amino acid sequences of SEQ ID NOs: 67-68. 30 58. The polypeptide according to any of claims 54 to 57, in which the CDR sequences of said polypeptide have at least 70% amino acid identity preferably at least 80 amino acid identity, more preferably at least 90% amino acid identity, such as 95% amino acid identity or more or even essentially 100% WO 2013/128031 PCT/EP2013/054262 147 amino acid identity with the CDR sequences of at least one of the immunoglobulin single variable domains with SEQ ID NOs: 11 and 12. 59. Polypeptide dire ted against PcrV, that cross-blocks the binding to PcrV of at least one of the S immunoglobulin single variable domains with SEQ ID NOs: 11 and 12 and/or that is cross-.blocked from binding to PcrV by at least one o the immunoglobulin single variable domains with SEQ ID NOs: 11 and 12. 60. The polypeptide according to any of claims 54 to 59, that is selected from any of SEQ ID NOs: 11 and 0 12, 61. Tne polypeptide according to any of claims 35 to 60, that essentially consists of n immunoglobulin single variable domain selected from a light chain variable domain sequence (e.g. a V -sequence and of a heavy chain variable domain sequence (e.g. a Vrsequence) 15 62. The polypeptide according to any of claims 35 to 61, that essentially consists of an inmunoglobulin single variable domain selected from a heavy chain variable domain sequence that s derived from a conventional four-chain antibody and a heavy chain variable domain sequence that is derived from heavy chain ntibody. 20 63. The polypeptide acordingto any of cl im 35to 2 that essentially consists of an inmunoglobulin single variable domain selected from a domain an body or an amino acid that is suitable for use as a domain antibody), a single domain antibody (or an amino acid that is suitable for use as a single domain antibody a dAb" or an amino acid that is suitable for use as a dAb) or a Nanobody 25 (including but not limited to a V. or a humanized VH 64. The polypeptide according to any of claims 35 to 63, that essentially consists of partial or fully humanzed Nanobody such as a partially or fully humanized VHH. 30 65. Use of a polypeptide according to any of claims 35 64 in preparing a polypeptide according to any of caim 1 to 34. WO 2013/128031 PCT/EP2013/054262 148 6. Use o a polypeptide according to any of claims 35 to 64, as a binding domain or oin dng unit in preparing a polypeptide according to any of claims 1 to 34. 67. The polypeptide accordin to any of claims 1 to 64, further comprising one or more other groups, 5 residues moieties or binding units, optionally linked via one or more peptidic linkers. 68, The polypeptide according to claim 6 in which said one or more other groups, residues moietie or binding units are chosen from the group consisting of domain antibodies arino acids tht are suitable for use as a domain antibody, single domain antibodies amino acids that re suitable or use 10 as a single domain antibody dAb's , amino acids that are suitable for use as a dAb, or Nanobodies. 69. The polypeptide according to any of claims 67 or 8, which has an increased half-fe, compared to the corresponding poypeptide according to any of claims 1 to 64, per se. 15 70. The polypeptide according to claim 9, n whic -h said one or more other groups residues moieties or binding units provide the polypeptide with increased half-life, compared to he corresponding polypeptide according to any of claims to 64, per se. 71. The polypeptide according to claim 70, in which said one or more other groups, residue, moeties or 20 binding units that provide the polypeptide with increased half-li e is chosen from the group consisting of serum proteins or fragments thereof, binding unts that can bind to serum proteins, an Fc portion, and small proteins or peptides that can bind to serum proteins. 72. The polypeptide according to any of claims 70 or 71, in which sai one or more other groups, 25 residues, moieties or ending units that provide the polypeptide with increased half-life is chosen from the group consisting of human serum albumin or fragments thereof. 73. The polypeptide according to any of laims 70 or 71, n which said one or more other binding units that provides the polypeptide with increased half-life are chosen from the group consisting of 30 binding units that can bind to serum albumin (such as human serum albumin) or serum immunoglobulin such as gG WO 2013/128031 PCT/EP2013/054262 149 74. The polypeptide according to claims 73 in which said one or more other binding units that provides the polypeptide with increased half-life are chosen rom the group consisting of domain antibodies, amino acids that are suitable for use as a domain antibody, single domain antibodies amino acids that are suitable for use as a single domain antibody, dAb"'s amino acids that are suitable for use as 5 a dAb, or Nanobodies that can bind to serum albumin such as human serum albumin or a serum immunoglobulin (such as IgG). 75, The polypeptide according to any of claims 70 to 74, that has serum half-life that is at least L5 times, preferably at least 2 times such as at least 5 times, for example at least 10 times or more than 1o 20 times greater than the half-life of the corresponding polypeptide according to any of claims ito 64 per se 76. The polypeptide according to any of claims 70 to 75 that has a serum half-life that is increased with more than 1 hours, preferabi more than 2 hours more preferably more than 6 hours such as more 1s than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding polypeptide according to any of claims 1 to 64, per se 77. The polypeptide according to any of claims 70 to 76, that has a serum half-lfe n h human of at least about 12 hours, preferably t least 24 hours, more preferably at least 48 hours, even more 20 preferably at least 72 hours or more for example, of at least 5 days (such as about 5 to 10 days), preferably at least 9 days (such as about 9 to 14 days morere eferably at least about 10 days (such as about 10 to 15 days), or at least about 11 days such as about 11 to 16 days), more preferably at least about 12 days (such as about 12 to 18 days or more), or more than 14 days (such as about 14 to 19 days). 25 78. Nuclec acid or nucleotide sequence that encodes a polypeptide according to any of claims to 6 and 67 to 77. 79. Nucleic acid or nucleotide sequence according to claim 78, that is in the form of a genetic construct. 30 WO 2013/128031 PCT/EP2013/054262 150 80. Use of a nuceic acid or nucleotide sequence according to daim 78, that encodes polypeptide according to any of claims 35 to 64, for the preparation of a genetic constru t that encodes a polypeptide according to any of claims 1 to 34 5 8L Host or host cell that expresses, or that under suitable circumstances is capable of expressing a polypeptide according to any of daims 1 to 64 and 67 to 77; and/or that comprises a nudeic acid or nucleotide sequence according to claim 78 or a genetic construct according to daim 79. 82. Composition comprising at least one polypeptde according to any of claims 1 to 64 nd 7 to 77, or 10 nudeic acid or nucleotide sequence according o any o claims 78 and 79. 83. Composition according o claim 2 which is a pharmaceuticaI composition. 84, Composition according to daim 83, which is a pharmaceutic composition that further comprises at 15 least one pharmaceutically acceptable carrier, diluent or recipient and/or adjuvant, and tha optionally comprises one or more further pharmaceutcall active polypeptides and/or compounds. 85. Composition according to claim 84, comprising at least one polypeptide according to any of caims 1 to 64 and 67 to 77, or nucle acid or nucleotide sequence according to any of daims 78 and 79 and 20 a carrier suitable for pulonary delivery. 86. Pharmaceutical device suitable for the pulmonary delivery of at least one polypeptide according to any of aims to 64 and 67 to 77, or nudeic acid or nuceotide sequence according to any of caims 78 and 79, comprising at least one polypeptide according to any of claims 1 to 64 and 7 to 77 or 25 nucec acid or nucleotide sequence according to any of laims 78 and 79. 87. Pharmaceutical device according to daim 86 that is selected from an inhaler for liquids (e.g. a suspension of fine solid parties or droplets), a nebu izer, metered dose inhaler aerosol and a dry powder inhaler. 30 88. Method for producing a polypeptide according to any of daims 1 to 4 and 67 to 77, said method at least comprising the steps of: WO 2013/128031 PCT/EP2013/054262 151 a) expressing, in a suitable host cel or host organism or in another suitable expression system, a nucleic acid or nucleotide sequence according to claim 78, or a genetic construct according to claim 79; optionally followed by: 5 b) isolating and/or purifying the polypeptide according to any of claims 1 to 64 and 67 to 77, thus obtained. 89. Method for producing a polypeotide according to any of claims 1 to 64 and 67 to 77, said method at least comprising the steps of: 10 a) cultivating and/or maintaining a host or host cell according to claim 80 under conditions that are such that said host or host cell expresses and/or produces at least one polypeptide according to any of claims 1 to 64 and 67 to 77; optionally followed by: b) isolating and/or purifying the polypeptide according to any of claims 1 to 64 and 67 to 77, thus 15 obtained. 90. Method for preparing a polypeptide according to any of claims 1 to 34, said method comprising at least the steps of linking two or more polypeptides according to any of claims 35 to 64, and optionally one or more linkers. 20 9L Method for the prevention and/or treatment of infection with P. aeruginossaid method comprising administering, to a subject in need thereof, a pharmaceutically active amount of at least one polyneptide according to any of claims 1 to 64 and 67 to 77, and/o r com position according to any of claims 83 and 84, 25 92. Method according to claim 90, for the prevention and/or treatment of infection with P. oeruginosa in at least one of ventilator-associated pneumonia (VAP), burn victirns, mechanical ventilated patients, Cystic Fibrosis (CF) patients, hematopoletic cell transplantation patients, bone marrow transplant patients, surgery, chronic obstructive pulmonary disease (COPD), bronchiectasis, sepsis, cancer 30 associated neutropenia, said method comprising administering, to a subject in need thereof, a pharmaceuticay active amount of at least one polypeptide according to any of cims 1 to 64 and 67 to 77 and/or composition according to any of claims 83 and 84. WO 2013/128031 PCT/EP2013/054262 152 93. Method according to clain 91 for the prevention and/or treatment of infection with P. aergnosa, said method comprising administering to the pulmonary tissue of a subject in need thereof, a pharnaceutica active amount of at least one polypeptide according to any of claims 1 to 4 and 7 5 to 77 and/or composition according to any of caims 83 to 85. 94. Method for the prevention and/or treatment of infection with P. aeruginosa in at least one of ventila or-associated pneumonia (VAP) burn victims mechanical ventilated patients Cystic Fibrosis C patients, hematopoietic cell transp antation patients, bone marrow transplant patients, surgery, 10 chronic obstructive pulmonary disease (COPD) bronchiectasis, sepsis, cancer-associated neutropenia, said method comprising administering to the pulmonary tissue of a subject in need thereof, a pharmaceutically active amount o at least one polypeptide according to any of claims 1 to 4 and 67 to 77, and/or composition according to any of claims 83 to85, M 95. Use of a polypeptide according to any of claims 1 to 64 and 67 to 77and/or composition according to any of claims 83 to 8 in the preparation of a pharmaceutical composi ion for prevention and/or treatment of infection with P. eruginosa; and/or or use in one or nore of the methods according to cairns 91 to 94 20 96 Use of a polypeptide according to any of claims 1 to 64 and 67 to 77 and/or composition according to any of claims 8 and 5 in the preparation of pharmaceutical composition for prevention and/or treatment of infection with P. aeruginosa in at least one of ventilator-associated pneumonia (VAP), burn victims mechanical ventilated patients, Cystic Fibrosis CF), hematopoietic cell transplantation patients bone narrow transplant patients surgery chronic obstructive pulmonary disease (CO PD), 2 bonchiectasis sepsis. cancerassociated neutropenl 97. Polypeptide according to any of aims 1 to 64 and 67 to 77 and/or composition according to any of claims 83 to 85, for prevention and/or treatment of infection with P. Ueruginosa. 30 98. Polypeptide according to any of claims 1to 67 and 67 to 77 and/or composition according to any of claims 83 to 85, for prevention and/or treaty nt of infection with P. aeruginosa in at least one of ventilator-associated pneumonia (VAP), burn victims, mechanic cal venti ated patients Cystic Fibrosis WO 2013/128031 PCT/EP2013/054262 153 (CF, hematopoetic cell transpantation patients, bone marrow transplant patients, surgery chronic obstructive pulmonary disease (COPD bronchiectasis, sepsi , cancer-associated neutropenia.

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同族专利:

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公开号 | 公开日

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EP2820043A1|2015-01-07|

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JP6415987B2|2018-10-31|

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KR20140132748A|2014-11-18|

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CA2863468A1|2013-09-06|

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US20150044215A1|2015-02-12|

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RU2014139911A|2016-04-20|

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US20200190217A9|2020-06-18|

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AU2013224851B2|2018-03-01|

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JP2019013239A|2019-01-31|

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SG11201404532XA|2014-08-28|

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US20190055322A1|2019-02-21|

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MX2014010495A|2014-11-14|

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WO2013128031A1|2013-09-06|

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PH12014501818A1|2014-11-17|

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JP2015509723A|2015-04-02|

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CN104144945A|2014-11-12|

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US10072098B2|2018-09-11|

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EP2820043B1|2020-01-15|

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法律状态:
2018-06-28| FGA| Letters patent sealed or granted (standard patent)|

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2020-10-01| MK14| Patent ceased section 143(a) (annual fees not paid) or expired|

优先权:

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申请号 | 申请日 | 专利标题

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US201261606094P| true| 2012-03-02|2012-03-02||

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US61/606,094||2012-03-02||

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PCT/EP2013/054262|WO2013128031A1|2012-03-02|2013-03-04|Pseudomonas aeruginosa pcrv binding single variable domain antibodies|

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