monoclonal antibody that binds to human b cells (bcma), pharmaceutical composition and its use

专利摘要:
The invention relates to new antibodies against BCMA, their manufacture and use.
公开号:BR112018001955B1
申请号:R112018001955-0
申请日:2016-08-03
公开日:2021-05-11
发明作者:Minh Diem Vu；Klaus Strein；Oliver Ast；Marina Bacac；Camille Delon；Lydia Jasmin Duerner；Anne Freimoser-Grundschober；Christian Klein；Ekkehard Moessner；Samuel Moser；Pablo Umana；Tina Weinzierl
申请人:Engmab Sárl；
IPC主号:

专利说明:

[0001] The present invention relates to antibodies against BCMA, their manufacture and use. BACKGROUND OF THE INVENTION
[0002] Human B cell maturation antigen, also known as BCMA; TR17_HUMAN, TNFRSF17 (UniProt Q02223), is a member of the tumor necrosis receptor superfamily that is preferentially expressed in differentiated plasma cells (Laabi et al. 1992; Madry et al. 1998). BCMA is a non-glycosylated type III transmembrane protein that is involved in the maturation, growth and survival of B cells. BCMA is a receptor for two ligands of the TNF superfamily: APRIL (a proliferation-inducing ligand), the ligand for high affinity BCMA and B cell activating factor BAFF, the low affinity ligand for BCMA (THANK, BlyS, stimulator B lymphocytes, TALL-1 and zTNF4). APRIL and BAFF show structural similarity and overlap, however, distinct receptor binding specificity. The TACI negative regulator also binds to both BAFF and APRIL. Coordinated binding of APRIL and BAFF to BCMA and/or TACI activates the transcription factor NF-KB and increases the expression of pro-survival Bcl-2 family members (eg, Bcl-2, Bcl-xL, Bcl-w , Mcl-1, A1) and the down-regulation of pro-apoptotic factors (eg, Bid, Bad, Bik, Bim, etc.), inhibiting apoptosis and promoting survival. This combined action promotes B cell differentiation, proliferation, survival, and antibody production (as reviewed in Rickert RC et al., Immunol Rev (2011) 244 (1): 115-133).
[0003] Antibodies against BCMA are described, for example, in MP Gras. et al. Int Immunol. 7 (1995) 1093-1106, WO200124811, WO200124812, WO2010104949 and WO2012163805. Antibodies against BCMA and their use for the treatment of lymphomas and multiple myeloma are mentioned, for example, in WO2002066516 and WO2010104949. WO2013154760 and WO2015052538 refer to chimeric antigen receptors (CAR) comprising a BCMA recognition unit and a T cell activation unit. Ryan, MC et al., Mol. Cancer Ther. 6 (2007) 3009-3018 refer to anti-BCMA antibodies with ligand blocking activity that could promote the cytotoxicity of multiple myeloma (MM) cell lines as naked antibodies or as antibody-drug conjugates. Ryan showed that SG1, a BCMA inhibitory antibody, blocks APRIL-dependent nuclear factor-KB activation in a dose-dependent manner in vitro. Ryan also mentioned the SG2 antibody which inhibited APRIL binding to BCMA non-significantly.
[0004] A wide variety of recombinant bispecific antibody formats have been developed in the recent past, for example by fusion of, for example, an IgG antibody format and single chain domains (see, for example, Kontermann RE, mAbs 4 : 2, (2012) 1-16). Bispecific antibodies in which the variable domains VL and VH or the constant domains CL and CH1 are substituted for one another are described in WO2009080251 and WO2009080252.
[0005] An approach to circumvent the problem of mismatched by-products, known as "knobs-in-holes", aims to force the pairing of two different antibody heavy chains, introducing mutations in the CH3 domains to modify the contact interface. In one chain, bulky amino acids were replaced with amino acids with short side chains to create a "hole". On the other hand, amino acids with large side chains were introduced into the other CH3 domain, to create a "button". By co-expressing these two heavy chains (and two identical light chains, which must be appropriate for both heavy chains), high yields of heterodimer formation ("button-hole") versus homodimer formation ("hole-hole" or "button-button"') has been observed (Ridgway JB, Presta LG, Carter P. Protein Eng. 9, 617-621 (1996); and WO1996027011). The percentage of heterodimer could be further increased by remodeling the interacting surfaces of the two CH3 domains using a phage display approach and introducing a disulfide bridge to stabilize the heterodimers (Merchant AM, et al. Nature Biotech 16 (1998) 677-681; ATwell S, Ridgway JB, Wells JA, Carter P., J Mol. Biol 270 (1997) 26-35). New approaches to button-in-hole technology are described, for example, in EP 1870459A1. While this format looks very attractive, no data describing progression towards the clinic is currently available. An important constraint of this strategy is that the light chains of the two parent antibodies have to be identical to avoid misadjustment and the formation of inactive molecules. Thus, this technique is not suitable for the development of bispecific recombinant antibodies against two targets from two antibodies against the first and the second target, as the heavy chains of these antibodies and/or the identical light chains must be optimized. Xie, Z., et al, J Immunol. Methods 286 (2005) 95-101 refers to a bispecific antibody format using scFvs in combination with button-in-hole technology for the FC part.
The T-lymphocyte TCR/CD3 complex consists of an alpha (α)/beta (β) or gamma (y)/delta (δ) TCR heterodimer co-expressed on the cell surface with the CD3-labeled gamma invariant subunits (y), delta (δ), epsilon (ε), zeta (Ç), and eta (^). Human CD3ε is described under UniProt P07766 (CD3E_HUMAN).
[0007] An anti-CD3ε antibody described in the prior art is SP34 (Yang SJ, The Journal of Immunology (1986) 137; 1097-1100). SP34 reacts with both primates and human CD3. SP34 is available from Pharmingen. Another anti-CD3 antibody described in the prior art is UCHT-1 (see WO2000041474). Another anti-CD3 antibody described in the prior art is BC-3 (Fred Hutchinson Cancer Research Institute, used in phase I/II trials of GvHD, Anasetti et al., Transplantation 54:844 (1992)). SP34 differs from UCHT-1 and BC-3 in that SP-34 recognizes an epitope present only on the ε chain of CD3 (see Salmeron et al., (1991) J. Immunol. 147:3047) whereas UCHT-1 and BC -3 recognize an epitope contributed by both the ε and Y chains. Other additional anti-CD3 antibodies are described in WO2008119565, WO2008119566, WO2008119567, WO2010037836, WO2010037837, WO2010037838 and US8236308 (WO2007042261). The CDR, VH and VL sequences of another anti-CD3 antibody are shown in SEQ ID NO: 7 and 8.
[0008] Bispecific antibodies against CD3 and BCMA are mentioned in WO2007117600, WO2009132058, WO2012066058 and WO2012143498. BCMA antibody CAR compounds are mentioned in WO2013154760, WO2013154760 and WO2014140248.
[0009] Cell-mediated effector functions of monoclonal antibodies (such as antibody-dependent cellular cytotoxicity (ADCC)) can be enhanced by manipulating their oligosaccharide composition into Asn297 as described in Umana, P., et al., Nature Biotechnol. 17 (1999) 176-180; and US6602684. WO1999054342, WO2004065540, WO2007031875 and WO2007039818, Hristodorov D, Fischer R, Linden L., Mol Biotechnol. 2012 Oct 25. (Epub) also relate to the manipulation of antibody glycosylation to enhance Fc-mediated cellular cytotoxicity.
[00010] Also several amino acid residues in the hinge region and the CH2 domain influence the cell-mediated effector functions of monoclonal antibodies (Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319 (1995) , Chemical Immunology, 65, 88 (1997)] Chemical Immunology, 65, 88 (1997)] Therefore, modification of such amino acids can improve cell-mediated effector functions. are mentioned in EP1931709, WO200042072 and comprise in the Fc part substitutions at the position(s) of amino acids 234, 235, 236, 239, 267, 268, 293, 295, 324, 327, 328, 330 and 332 Other antibody modifications to enhance cell-mediated effector functions are mentioned in EP1697415 and comprise amino acid replacement of EU amino acid positions 277, 289, 306, 344 or 378 with a charged amino acid, a polar amino acid or a non-polar amino acid.
[00011] Antibody formats and multispecific and bispecific antibody formats are also pepbodies (WO200244215), antigen ("NAR") (WO2003014161), diabody-diabody "TandAbs" dimers (WO2003048209), oxide-modified polyalkalene scFv (US7150872), humanized to rabbit antibodies (WO2005016950) synthetic immunoglobulin domains (WO2006072620), covalent diabodies (WO2006113665), flexibodies (WO2003025018), domain antibodies, dAb (WO2004058822), vacibody (WO2004076489), antibodies with frame of New World primate (WO2007019620), drug-antibody conjugated with cleavable linkers (WO2009117531), IgG4 antibodies with removed hinge region (WO2010063785), bispecific antibodies with IgG4 as CH3 domains (WO2008119353), camelid antibodies (US6838254) , nanobodies (US7655759), CAT diabodies (US5837242), bispecific (scFv)2 directed against the target antigen and CD3 (US7235641),), sIgA plAntibodies (US6303341), minibodies (US5837821), IgNAR (US2009148438) , antibodies with modified hinge and Fc regions (US2008227958, US20080181890), trifunctional antibodies (US5273743), triomabs (US6551592), troybodies (US6294654).
[00012] WO2014122143 describes anti-human BCMA antibodies characterized in that the binding of said antibody is not reduced in 100 ng/ml of APRIL for more than 20%, measured in an ELISA assay as OD at 405 nm compared to the binding of said antibody to human BCMA without APRIL, said antibody does not change the activation of APRIL-dependent NF-kB by more than 20% compared to APRIL alone, and said antibody does not change the activation of NF-kB without APRIL by more than 20% compared to without said antibody. WO2014122144 describes bispecific antibodies that specifically bind to the two human targets CD3ε and human BCMA, comprising anti-human BCMA antibodies from WO2014122143. An anti-human BCMA antibody with unique properties, especially with regard to its therapeutic use as a bispecific T cell binder, is the 83A10 antibody, characterized in that it comprises as regions CDR1H of SEQ ID NO:15, CDR2H of SEQ ID NO16:, CDR3H of SEQ ID NO: 17, CDR1L of SEQ ID NO: 18, CDR3L of SEQ ID NO: 19, and CDR3L of SEQ ID NO: 20, described also in WO2014122143 and WO2014122144. SUMMARY OF THE INVENTION
[00013] The invention comprises monoclonal antibodies that specifically bind to the human B cell maturation antigen (BCMA). Antibodies according to the invention comprise as CDR3H and CDR3L regions the same CDR regions as the 83A10 antibody.
[00014] The antibodies according to the invention comprise in an embodiment as regions of CDR3H and CDR3L the same CDR regions as the 83A10 antibody, but show especially potent and efficient advantages compared to the 83A10 antibody for killing MM cells in aspirates of the patient's bone marrow.
[00015] The invention comprises a monoclonal antibody that specifically binds to BCMA, characterized in that it comprises a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO: 20 and a combination of CDR1H, CDR2H, CDR1L region and CDR2L selected from the group of a) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22, CDR1L region of SEQ ID NO: 23 and CDR2L region of SEQ ID NO: 24, b) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22, CDR1L region of SEQ ID NO: 25 and CDR2L region of SEQ ID NO: 26, c) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22, CDR1L region of SEQ ID NO: 27 and CDR2L region of SEQ ID NO: 28, d) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30, CDR1L region of SEQ ID NO: 31 and region CDR2L of SEQ ID NO: 32, e) CDR1H region of SEQ ID NO: 34 and CDR2H region of SEQ ID NO: 35, CDR1L region of SEQ ID NO: 31 and CDR2L region of SEQ ID NO: 32, and f) CDR1H region from SEQ ID NO: 36 and CDR2H region from SEQ ID NO: 37, C region DR1L of SEQ ID NO: 31 and CDR2L region of SEQ ID NO: 32,
[00016] The invention comprises a monoclonal antibody that specifically binds to BCMA, characterized in that it comprises a VH region comprising a CDR1H region of SEQ ID NO: 21, a CDR2H region of SEQ ID NO: 22 and a CDR3H region of SEQ ID NO: : 17 and a VL region comprising a CDR3L region of SEQ ID NO: 20 and a combination of CDR1L and CDR2L region selected from the group of a) CDR1L region of SEQ ID NO: 23 and CDR2L region of SEQ ID NO: 24, b) CDR1L region of SEQ ID NO: 25 and CDR2L region of SEQ ID NO: 26, or c) CDR1L region of SEQ ID NO: 27 and CDR2L region of SEQ ID NO: 28.
[00017] The invention provides an antibody according to the invention, characterized in that it comprises a VL region selected from the group consisting of VL regions of SEQ ID NO: 12, 13 and 14 wherein amino acid 49 is selected from the group of amino acids tyrosine (Y), glutamic acid (E), serine (S) and histidine (H). In one embodiment, amino acid 49 is E within SEQ ID NO: 12, S within SEQ ID NO: 13 or H within SEQ ID NO: 14.
[00018] The invention provides an antibody according to the invention, characterized in that it comprises a VL region selected from the group consisting of VL regions of SEQ ID NO: 12, 13 and 14 wherein amino acid 74 is threonine (T) or alanine (THE). In one embodiment, amino acid 74 is A within SEQ ID NO:14.
[00019] The antibodies according to the invention comprise in an embodiment as CDR3H, CDR1L, CDR2L and CDR3L regions the same CDR regions as the 83A10 antibody. The invention comprises a monoclonal antibody that specifically binds to BCMA, characterized in that it comprises a VH region comprising a CDR3H region of SEQ ID NO: 17 and a VL region comprising a CDR1L region of SEQ ID NO: 31, a CDR2L region of SEQ ID NO: 32 and a CDR3L region of SEQ ID NO: 20 and a combination of CDR1L and CDR2L region selected from the group of a) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30, b) CDR1H region of SEQ ID NO: 34 and CDR2H region of SEQ ID NO: 35, or c) CDR1H region of SEQ ID NO: 36 and CDR2H region of SEQ ID NO: 37.
[00020] The invention provides in one embodiment an antibody according to the invention, characterized in that it comprises a VL region of SEQ ID NO: 12 and a VH region selected from the group comprising the VH regions of SEQ ID NO: 38, 39 and 40. The invention provides an antibody according to the invention, characterized in that it comprises a VL region SEQ ID NO: 12, wherein amino acid 49 is selected from the group of amino acids tyrosine (Y), glutamic acid (E), serine (S) and histidine (H). In one embodiment, amino acid 49 is E.
[00021] The invention provides in one embodiment an antibody according to the invention, characterized in that it comprises as VH region a VH region of SEQ ID NO: 10. The invention provides in one embodiment an antibody according to the invention, characterized in that it comprises as VL region a VL region selected from the group consisting of VL regions of SEQ ID NO: 12, 13 and 14. The invention provides in one embodiment an antibody according to the invention, characterized in that it comprises as VH region a VH region of SEQ ID NO: 10 and as VL region a VL region of SEQ ID NO: 12. The invention provides in one embodiment an antibody according to the invention, characterized in that it comprises as VH region a VH region of SEQ ID NO: 10 and as region VL a VL region of SEQ ID NO: 13. The invention provides in an embodiment an antibody according to the invention, characterized in that it comprises as VH region a VH region of SEQ ID NO: 10 and as VL region a VL region of SEQ ID NO: 14.
[00022] The invention provides in one embodiment an antibody according to the invention, characterized in that it comprises as VH region a VH region selected from the group consisting of SEQ ID NO: 38, 39 and 40. The invention provides in one embodiment an antibody according to the invention, characterized in that it comprises as VH region a VH region of SEQ ID NO: 38 and as VL region a VL region of SEQ ID NO: 12. The invention provides in one embodiment an antibody according to the invention, characterized in that it comprises as VH region a VH region of SEQ ID NO: 39 and as VL region a VL region of SEQ ID NO: 12. The invention provides in one embodiment an antibody according to the invention, characterized in that it comprises as VH region a region VH of SEQ ID NO: 40 and as VL region a VL region of SEQ ID NO: 12.
[00023] In one embodiment, the antibody according to the invention is further characterized in that it also specifically binds to BCMA of cynomolgus. In one embodiment, an antibody of the invention shows relative to BCMA binding a cyno/human affinity gap between 1.5 and 5 or 1.5 and 10 or 1.5 and 16 (Table 5).
[00024] The bispecific antibody according to the invention is, therefore, in a modality characterized by specifically binding to CD3 cynomolgus. In one embodiment, the bispecific anti-BCMA/anti-CD3 antibody of the invention shows a cino/human CD3 Mab range between 1.25 and 5 or between 0.8 and 1.0.
[00025] In another embodiment of the invention, the antibody according to the invention is an antibody with an Fc part or without an Fc part including a multispecific antibody, a bispecific antibody, a single chain variable fragment (scFv) such as as a bispecific T cell engager, a diabody, or tandem scFv, an antibody mimetic such as DARPin, a naked monospecific antibody, or an antibody drug conjugate. In one embodiment, a multispecific antibody, a bispecific antibody, a bispecific T cell engager, a diabody, or a tandem scFv is specifically bound to BCMA and CD3.
[00026] Based on an antibody according to the invention, it is possible to generate antibody-drug conjugates against BCMA and multispecific or bispecific antibodies against BCMA and one or more additional targets in different formats with or without an Fc portion known in the state of the art (see for example, above under "background of the invention"), single chain variable fragments (scFv) such as bispecific T cell splices, diabodies, tandem scFvs and antibody mimetics such as DARPins, all of these are also embodiments of the invention. Bispecific antibody formats are well known in the art and, for example, also described in Kontermann RE, mAbs 4: 2 1-16 (2012); Holliger P., Hudson PJ, Nature Biotech.23 (2005) 1126-1136 and Chan AC, Carter PJ Nature Reviews, Immunology 10, 301-316 (2010) and Cuesta AM et al., Trends Biotech 28 (2011) 355- 362.
[00027] Another embodiment of the invention is a bispecific antibody against the two human CD3ε targets (also called "CD3") and the extracellular domain of human BCMA (also called "BCMA"), characterized in that it comprises as a binding portion of BCMA and anti-BCMA antibody according to the invention.
[00028] The invention relates in an embodiment to a bispecific antibody against BCMA and CD3, characterized in that it comprises within the BCMA binding portion a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO: 20 and a CDR1H, CDR2H, CDR1L, and combination of CDR2L region selected from the group of a) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22, CDR1L region of SEQ ID NO: 23 and CDR2L region of SEQ ID NO: 24, b) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22, CDR1L region of SEQ ID NO: 25 and CDR2L region of SEQ ID NO: 26, c) CDR1H region of SEQ ID NO :21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28, d) CDR1H region of SEQ ID NO:29 and CDR2H region of SEQ ID NO:30, CDR1L region of SEQ ID NO: 31 and CDR2L region of SEQ ID NO: 32, e) CDR1H region of SEQ ID NO: 34 and CDR2H region of SEQ ID NO: 35, CDR1L region of SEQ ID NO: 31 and CDR2L region of SEQ ID NO: 32, and f) CDR1H region of SEQ ID NO: 36 and C region DR2H of SEQ ID NO: 37, CDR1L region of SEQ ID NO: 31 and CDR2L region of SEQ ID NO: 32,
[00029] The invention relates in an embodiment to a bispecific antibody against BCMA and CD3, characterized in that it comprises a VH region of an antibody according to the invention (hereinafter referred to as "BCMA VH") comprising a CDR1H region of SEQ ID NO: 21, a CDR2H region of SEQ ID NO: 22 and a CDR3H region of SEQ ID NO: 17, and a VL region (also called "BCMA VL") comprising a CDR3L region of SEQ ID NO: 20 and a combination of region CDR1L and CDR2L selected from the group of a) CDR1L region of SEQ ID NO: 23 and CDR2L region of SEQ ID NO: 24, b) CDR1L region of SEQ ID NO: 25 and CDR2L region of SEQ ID NO: 26, or c) CDR1L region of SEQ ID NO: 27 and CDR2L region of SEQ ID NO: 28.
[00030] The invention provides in one embodiment a bispecific antibody according to the invention, characterized in that it comprises as VH BCMA region a VH region of SEQ ID NO: 10.
[00031] The invention relates in an embodiment to a bispecific antibody against BCMA and CD3, characterized in that the BCMA VL is selected from the group consisting of VL regions of SEQ ID NO: 12, 13 and 14. The invention provides in a An antibody according to the invention, characterized in that it comprises as VH BCMA region a VH region of SEQ ID NO: 10 and as VL region a VL region of SEQ ID NO: 12. The invention provides in one embodiment an antibody according to the invention, characterized in that it comprises as VH BCMA region a VH region of SEQ ID NO: 10 and as VL region a VL region of SEQ ID NO: 13. The invention provides in one embodiment an antibody according to the invention, characterized in that it comprises as VH region a VH region of SEQ ID NO: 10 and as VL region a VL region of SEQ ID NO: 14.
[00032] The invention provides a bispecific antibody according to the invention, characterized in that it comprises a VL region selected from the group consisting of VL regions of SEQ ID NO: 12, 13 and 14 wherein amino acid 49 is selected from from the amino acid group tyrosine (Y), glutamic acid (E), serine (S) and histidine (H). In one embodiment, amino acid 49 is E (SEQ ID NO: 12), S (SEQ ID NO: 13) or H (SEQ ID NO: 14). The invention provides a bispecific antibody according to the invention, characterized in that it comprises a VL region selected from the group consisting of VL regions of SEQ ID NO: 12, 13 and 14 wherein amino acid 74 is threonine (T) or alanine (THE). In one embodiment, amino acid 74 is A within SEQ ID NO:14.
[00033] The invention relates to a bispecific antibody against BCMA and CD3, characterized in that it comprises a VH BCMA region comprising a CDR3H region of SEQ ID NO: 17 and a VL BCMA region comprising a CDR1L region of SEQ ID NO: 31, a CDR2L region of SEQ ID NO: 32 and a CDR3L region of SEQ ID NO: 20 and a combination of CDR1L and CDR2L region selected from the group of a) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30, b) CDR1H region of SEQ ID NO: 34 and CDR2H region of SEQ ID NO: 35, or c) CDR1H region of SEQ ID NO: 36 and CDR2H region of SEQ ID NO: 37.
[00034] The bispecific antibody against BCMA and CD3 is characterized in an embodiment comprising an anti BCMA antibody according to the invention and an anti CD3 antibody, wherein
[00035] the light chain and heavy chain of an antibody that specifically binds to one of said CD3 and BCMA targets; and
[00036] the light chain and the heavy chain of an antibody that specifically binds to the other of said targets, wherein the variable domains VL and VH or the constant domains CL and CH1 are substituted for each other.
[00037] In one embodiment, a VH domain of said anti-CD3 antibody portion is linked to a CH1 or CL domain of said anti-BCMA antibody portion. In one embodiment, a VL domain of said anti-CD3 antibody portion is linked to a CH1 or CL domain of said anti-BCMA antibody portion.
[00038] In one embodiment, the bispecific antibody comprises no more than one Fab fragment of an anti-CD3 antibody portion, no more than two Fab fragments of an anti-BCMA antibody portion, and no more than one Fc portion , in a modality a human Fc part. In one embodiment, no more than one Fab fragment of the anti-CD3 antibody portion and no more than one Fab fragment of the anti-BCMA antibody portion are linked to the Fc part and binding is accomplished via C-terminal binding of the fragment. Fab(s) for the hinge region. In one embodiment, the second Fab fragment of the anti-BCMA antibody portion is linked via its C-terminus to the N-terminus of the Fab fragment of the anti-CD3 antibody portion or to the hinge region of the Fc portion, and thus between the Fc part and the anti-CD3 antibody portion. Preferred bispecific antibodies are shown in Figures 1 to 3.
Especially preferred are bispecific antibodies comprising only the Fab fragments and the Fc part as specified, with or without "aa substitution":
[00040] Fab BCMA-Fc-Fab CD3 (bispecific format, fig. 1A or 1B),
[00041] Fab BCMA-Fc-Fab CD3-Fab BCMA (bispecific format fig.2A or 2B),
[00042] Fab BCMA-Fc-Fab BCMA-Fab CD3 (fig.2C or 2D bispecific format),
[00043] Fab BCMA-Fc-Fab CD3 (bispecific format, fig. 3A or 3B),
[00044] Fc-Fab BCMA-Fab CD3 (bispecific format fig. 3C or 3D).
[00045] As shown in Figures 1 to 3 "Fab BCMA-Fc," Fab BCMA-Fc-Fab CD3 "and" Fab BCMA-Fc-Fab CD3 "means that the Fab fragment(s) is (are) linked via its(their) C-terminus to the N-terminus of the Fc fragment. "Fab CD3-Fab BCMA" means that the Fab fragment CD3 is linked with its N-terminus to the C-terminus of the Fab fragment BCMA "Fab BCMA - Fab CD3" means that the Fab BCMA fragment is linked from its N-terminus to the C-terminus of the Fab fragment CD3.
In one embodiment, the bispecific antibody comprises a second Fab fragment of said anti-BCMA antibody linked with its C-terminus to the N-terminus of the CD3 antibody portion of said bispecific antibody. In one embodiment, a VL domain of said first anti-CD3 antibody portion is linked to a CH1 or CL domain of said second anti-BCMA antibody.
[00047] In one embodiment, the bispecific antibody comprises a second Fab fragment of said anti-BCMA antibody linked with its C-terminus to the Fc part (like the first Fab fragment of said anti-BCMA antibody) and linked with its N-terminus to the C - terminus of the CD3 antibody portion. In one embodiment, a CH1 domain of said anti-CD3 antibody portion is linked to a VH domain of said second anti-BCMA antibody portion.
In one embodiment, the bispecific antibody comprises an Fc part linked with its N-terminus to the C-terminus of said Fab fragment of the CD3 antibody. In one embodiment the bi-specific antibody comprises an Fc part linked with its first N-terminus to the C-terminus of said Fab fragment of CD3 antibody and a second Fab fragment of said anti-BCMA antibody linked with its C-terminus to the second N- terminal of the Fc part. In one embodiment, the CL domain of the Fab fragment of the CD3 antibody is linked to the hinge region of the Fc portion. In one embodiment, the CH1 domain of the BCMA antibody Fab fragment is linked to the hinge region of the Fc portion.
[00049] Fab fragments are chemically linked together by using an appropriate linker according to the state of the art. In one embodiment a linker (Gly4-Ser1) 3 is used (Desplancq DK et al., Protein Eng. 1994 Aug; 7(8): 1027-33 and Mack M. et al., PNAS July 18, 1995 vol. 92 no. 15 7021-7025). "Chemically linked" (or "linked") means according to the invention that the fragments are linked by covalent bonding. As the linker is a peptide linker, such covalent linkage is usually performed by recombinant biochemical means, using a nucleic acid encoding the VL and/or VH domains of the respective Fab fragments, the linker and, if appropriate, the partial Fc chain .
[00050] The invention relates in an embodiment to a bispecific antibody against BCMA and CD3 according to the invention, characterized in that the VH variable domain of the anti-CD3 antibody portion (termed as "CD3 VH") comprises the chain CDRs heavy chain of SEQ ID NO: 1, 2 and 3 as respectively heavy chain CDR1H, CDR2H and CDR3H and the variable domain VL of the anti-CD3 antibody portion (designated as "CD3 VL") comprises the light chain CDRs of SEQ ID NO: 4, 5 and 6 as light chain CDR1L, CDR2L and CDR3L respectively.
[00051] In one embodiment, such a bispecific antibody according to the invention is characterized in that the variable domains of the anti-CD3ε antibody portion are from SEQ ID NO: 7 and 8.
[00052] The invention relates to a bispecific antibody according to the invention, characterized in that the anti-CD3 antibody portion is linked at its N-terminus to the C-terminus of an anti-BCMA antibody portion and the variable domains VL and VH of the anti-CD3 antibody portion or the CL and CH1 constant domains are substituted for each other.
[00053] In one embodiment, a VH domain of said anti-CD3 antibody portion is linked to a CH1 or CL domain of said anti-BCMA antibody portion. In one embodiment, a VL domain of said anti-CD3 antibody portion is linked to a CH1 or CL domain of said anti-BCMA antibody portion.
[00054] An antibody portion according to the invention is in one embodiment a Fab fragment of the respective antibody.
[00055] In another embodiment of the invention, the bispecific antibody in which the VL and VH variable domains in the light chain and the respective heavy chain of the anti-CD3 antibody portion or the anti-BCMA antibody portion are substituted in turn, is characterized by comprising a constant domain CL of the anti-CD3 antibody portion or the anti-BCMA antibody portion wherein the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and in the respective constant domain CH1 the amino acid at position 147 and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D). In one embodiment the antibody is monovalent for CD3 binding. In one embodiment, in addition to the amino acid substitution at position 124 in the CL constant domain, the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine (H) (still called "charge variant swap "). In one embodiment, the antibody is monovalent for CD3 binding and amino acid 124 is K, amino acid 147 is E, amino acid 213 is E, and amino acid 123 is R. In one embodiment, the bispecific antibody further comprises the same anti - BCMA binding moiety again (in one modality a Fab fragment). It also means that if the first anti-BCMA binding moiety comprises the charge variant exchange, then the second anti-BCMA binding moiety comprises the same charge variant exchange. (The amino acid numbering is according to Kabat).
[00056] The invention relates to a bispecific antibody according to the invention, characterized in that it comprises a) the first light chain and the first heavy chain of a first antibody that specifically binds to BCMA; and b) the second light chain and the second heavy chain of a second antibody which specifically binds to CD3, and wherein the VL and VH variable domains in the second light chain and second heavy chain of the second antibody are substituted for each other; and c) in which in the constant domain CL of the first light chain under which a) the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and wherein, in the CH1 constant domain of the first heavy chain under which a) the amino acid at position 147 and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering accordingly with Kabat) (see for example Figures 1A, 2A, 2C, 3A, 3C).
[00057] In one embodiment, the bispecific antibody described in the last preceding paragraph is further characterized in that said bispecific antibody further comprises a Fab fragment of said first antibody (also called "BCMA-Fab") and in the CL constant domain of said BCMA-Fab, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein, in the constant domain CH1 of said BCMA-Fab , the amino acid at positions 147 and the amino acid at position 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat) (see, for example, Figures 2A and 2C).
[00058] The invention further relates to a bispecific antibody according to the invention, characterized in that it comprises a) the first light chain and the first heavy chain of a first antibody that specifically binds to BCMA; and b) the second light chain and the second heavy chain of a second antibody which specifically binds to CD3, and wherein the VL and VH variable domains in the second light chain and second heavy chain of the second antibody are substituted for each other; and wherein c) in the constant domain CL of the second light chain under which b) the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and in that, in the constant domain CH1 of the second heavy chain under which b) the amino acid at position 147 and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat) .
[00059] In one embodiment, in addition to the substitution of amino acids at position 124 in the CL constant domain of the first or second light chain, the amino acid at position 123 is independently substituted by lysine (K), arginine (R) or histidine ( H).
[00060] In an embodiment in the constant domain CL, the amino acid at position 124 is replaced by lysine (K), in the constant domain CH1, the amino acid at position 147 and the amino acid at position 213 are replaced by glutamic acid (E) . In one embodiment, additionally, in the constant domain CL the amino acid at position 123 is replaced by arginine (R).
[00061] In a preferred embodiment of the invention, the bispecific antibody according to the invention consists of a Fab fragment of an antibody that specifically binds to CD3 (also called "CD3-Fab") and a Fab fragment of an antibody anti-BCMA according to the invention (also called "BCMA-Fab(s)") and an Fc part, wherein the CD3-Fab and BCMA-Fab are linked via their C-terminus to the hinge region of said part Fc. CD3-Fab or BCMA-Fab comprises a substitution and CD3-Fab comprises crossover (Figures 1A and 1B).
[00062] In a preferred embodiment of the invention, the bispecific antibody according to the invention consists of a single CD3 Fab and a BCMA-Fab Fab and an Fc part, wherein the CD3 Fab and the Fab of BCMA are connected via its C-terminus to the hinge region of said Fc part and a second BCMA-Fab, which is connected with its C-terminus to the N-terminus of the CD3-Fab. CD3-Fab comprises crossover and CD3-Fab or both BCMA-Fab include an aa substitution (Figures 2A and 2B). Especially preferred is a bispecific antibody comprising BCMA-Fab-Fc-CD3-Fab-BCMA-Fab, wherein both BCMA-Fab include an aa substitution and the CD3-Fab comprises a VL/VH crossover (Figures 2A). Especially preferred is a bispecific antibody consisting of BCMA-Fab-Fc-CD3-Fab-BCMA-Fab, where both BCMA-Fab include an aa substitution Q124K, E123R, K147E and K213E and the CD3-Fab comprises VL/VH crossover . Especially preferred is that both BCMA-Fab include as CDRs the CDRs of antibody 21, 22 or 42, or as VH/VL the VH/VL of antibody 21, 22 or 42.
[00063] In a preferred embodiment of the invention, the bispecific antibody according to the invention consists of two BCMA Fabs and an Fc part, wherein the BCMA Fab and the CD3 Fab are linked via their C-terminus to the hinge region of said Fc part and a second BCMA Fab, which is linked with its C-terminus to the N-terminus of the CD3 Fab. CD3-Fab comprises crossover and CD3-Fab or both BCMA-Fab include an aa substitution (Figures 2A and 2B).
[00064] In a preferred embodiment of the invention, the bispecific antibody according to the invention consists of two BCMA Fabs and a part of Fab, wherein the BCMA Fabs are linked via their C-terminus to the hinge region of the said Fc part is a CD3 Fab, which is linked from its C-terminus to the N-terminus of a BCMA Fab. The CD3-Fab comprises crossover and the CD3-Fab or both BCMA Fabs include an aa substitution (Figures 2C and 2D).
[00065] In a preferred embodiment of the invention, the antibody according to the invention consists of a single CD3-Fab, which is linked via its C-terminus to the hinge region of said Fc part, and a Fab BCMA, which is linked to its C-terminal to N-terminal of CD3-Fab. The CD3-Fab comprises crossover and the CD3-Fab or both BCMA Fabs include an aa substitution (Figures 1A and 1B).
[00066] In a preferred embodiment of the invention, the antibody according to the invention consists of a single CD3-Fab, which is linked via its C-terminus to the hinge region of said Fc part, and a Fab BCMA, which is linked to its C-terminal to N-terminal of CD3-Fab. The CD3-Fab comprises crossover and the CD3-Fab or both BCMA Fabs include an aa substitution (Figures 3A and 3B).
[00067] In a preferred embodiment of the invention, the antibody according to the invention consists of a single BCMA-Fab, which is linked via its C-terminus to the hinge region of said Fc part, and a CD3 Fab, which is linked to the its C-terminus to the N-terminus of BCMA-Fab. The CD3-Fab comprises crossover and the CD3-Fab or both BCMA Fabs include an aa substitution (Figures 3C and 3D).
[00068] Fab fragments are chemically linked together by using an appropriate linker according to the state of the art. In one embodiment a linker (Gly4-Ser1) 3 is used (Desplancq DK et al., Protein Eng. 1994 Aug; 7(8): 1027-33 and Mack M. et al., PNAS July 18, 1995 vol. 92 no. 15 7021-7025). The linkage between two Fab fragments is carried out between the heavy chains. Therefore, the CH1 C-terminus of a first Fab fragment is linked to the VH N-terminus of the second Fab fragment (no cross) or to the VL (crossover). The linkage between a Fab fragment and the Fc part is carried out according to the invention as linking between CH1 and CH2.
[00069] The first and second Fab fragments of an antibody that specifically binds BCMA are in a modality derived from the same antibody and in an identical modality in the CDR sequences, VH and VL variable domain sequences and/or the constant domain sequences CH1 and CL. In one embodiment, the amino acid sequences of the first and second Fab fragment of an antibody that specifically binds BCMA are identical. In one embodiment the BCMA antibody is an antibody comprising the CDR sequences of antibody 21, 22 or 42, an antibody comprising the VH and VL sequences of antibody 21, 22 or 42, or an antibody comprising the sequences VH, VL, CH1 and CL of antibody 21, 22 or 42.
[00070] In one embodiment, the bispecific antibody comprises Fab fragments and part of Fc, no more than one Fab fragment from an anti-CD3 antibody, not more than two Fab fragments from an anti-BCMA antibody portion, and not more than an Fc part, in a human Fc part modality. In one embodiment, the second Fab fragment of an anti-BCMA antibody is linked via its C-terminus to the N-terminus of the Fab fragment of an anti-CD3 antibody or to the hinge region of the Fc portion. In one embodiment, binding is performed between CH1 of BCMA-Fab and VL of CD3-Fab (VL/VH crossover).
[00071] In one embodiment, the antibody portion that specifically binds to human CD3, in one embodiment the Fab fragment, is characterized by comprising a variable domain VH comprising the heavy chain CDRs of SEQ ID NO: 1, 2 and 3 as respectively heavy chain CDR1, CDR2 and CDR3 and a variable domain VL comprising the light chain CDRs of SEQ ID NO: 4, 5 and 6 as respectively light chain CDR1, CDR2 and CDR3 of the anti-CD3ε antibody (CDR MAB CD3). In one embodiment, the antibody portion specifically linked to human CD3 is characterized in that the variable domains are of SEQ ID NO: 7 and 8 (VHVL MAB CD3).
[00072] The invention relates to a bispecific antibody that specifically binds to the extracellular domain of human BCMA and human CD3, characterized in that it comprises a set of heavy and light chains selected from the group consisting of polypeptides
[00073] SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51 (2x); (set 1 TCB of antibody 21),
[00074] SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54 (2x) (set 2 TCB antibody 22), and
[00075] SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 (2x) (set 3 TCB of antibody 42).
[00076] In one embodiment, the bispecific antibody according to the invention is characterized in that the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain meet at an interface comprising a unique interface between the CH3 domains of the antibody; wherein said interface is altered to promote the formation of the bispecific antibody, wherein the alteration is characterized by:
[00077] the CH3 domain of a heavy chain is altered, so that within the original interface the CH3 domain of a heavy chain that meets the original interface of the CH3 domain of another heavy chain within the bispecific antibody, an amino acid residue is substituted by an amino acid residue having a larger side chain volume, thus generating a bulge within the interface of the CH3 domain of one heavy chain that is positionable in a cavity within the interface of the CH3 domain of the other heavy chain and
[00078] the CH3 domain of the other heavy chain is altered, so that within the original interface of the second CH3 domain that meets the original interface of the first CH3 domain within the bispecific antibody, an amino acid residue is replaced by a residue of amino acid that has a smaller side chain volume, thus generating a cavity within the interface of the second CH3 domain into which a bulge within the interface of the first CH3 domain is positionable.
[00079] In one embodiment, such a bispecific antibody is characterized by said amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).
[00080] In one embodiment, such a bispecific antibody is characterized in that said amino acid residue has a smaller volume of the side chain being selected from the group consisting of alanine (A), serine (S), threonine (T), valine ( V).
[00081] In one embodiment, such a bispecific antibody is characterized in that both CH3 domains are further altered by introducing cysteine (C) as amino acid at the corresponding positions of each CH3 domain.
[00082] In one embodiment, such a bispecific antibody is characterized by the constant heavy chain CH3 domains of both heavy chains being replaced by a constant heavy chain CH1 domain; and the other constant heavy chain domain CH3 is replaced by a constant light chain domain CL.
[00083] The invention further relates to an antibody according to the invention, comprising a modified Fc part inducing cell death of 20% or more cells of a BCMA-expressing cell preparation after 24 hours at a concentration of said antibody of 100 nM by ADCC versus a control under identical conditions using the same antibody with the main Fc part as a control. Such an antibody is in one embodiment a naked antibody.
[00084] In one embodiment, the antibody according to the invention is an antibody with an amount of fucose of 60% or less of the total amount of oligosaccharides (sugars) in Asn297 (see, for example, US20120315268).
[00085] In one embodiment, the Fc part comprises the amino acid substitutions that are introduced into a human Fc part and disclosed in SEQ ID NOs: 55 and 56.
[00086] Another embodiment of the invention is a chimeric antigen receptor (CAR) of an anti-BCMA antibody according to the invention. In such an embodiment, the anti-BCMA antibody consists of a single-chain VH and VL domain of an antibody according to the invention and a CD3-zeta transmembrane and endodomain. Preferably, the CD3 zeta domain is connected via a spacer to the C-terminus of said VL domain and the N-terminus of the VL domain is connected via a spacer to the C-terminus of said VH domain. Useful BCMA antibody chimeric antigen receptors, transmembrane domains and endodomains and methods for production are described, for example, in Ramadoss NS. et al., J. Am. Chem. Soc. J., DOI: 10.1021/jacs.5b01876 (2015), Carpenter RO et al., Clin. Cancer. Res. DOI: 10,1158/1078-0432.CCR-12-2422 (2013), WO2015052538 and WO2013154760.
[00087] Other embodiments of the invention are the Mab21, Mab22, Mab42, Mab27, Mab33 and Mab39 antibodies as described in this document by their CDR sequences and/or VH/VL sequences together with the CL and CH1 sequences described, as antigen-binding fragments, especially Fab Fragments, such as bispecific antibodies that bind BCMA and CD3, with and without Fc part, as bispecific antibodies in the formats described, especially the 2+1 format and bispecific antibodies with heavy and light chains as described in this document, especially as described in table 1A.
[00088] Another embodiment of the invention is a method of generating an anti-BCMA antibody which depletes, in the bispecific format according to the invention, human malignant plasma cells in bone marrow Multiple Myeloma aspirates at least 80% after a treatment 48 hours at a concentration of between 10 nM and 1 fM inclusive, characterized in panning of a variable heavy chain (VH) and variable light chain (VL) phage display library of the 83A10 antibody (VH library, VL library) with cyno BCMA 1 -50 nM in 1-3 rounds and selecting a variable light chain and a variable heavy chain that have properties as such a bispecific T cell binder. Preferably, the panning is performed in 3 rounds, using cynoBCMA 50nM for cyBCMA 1st round, 25nM for cyBCMA round 2 and 10nM for round 3. Libraries are probably randomized to either the CDR1 or CDR2 light chain or the chain heavy CDR1 and CDR2. Preferably, a light and heavy chain is identified which each binds as a Fab fragment, further comprising the corresponding VH or VL of the 83A10 antibody, huBCMA with a Kd of 50pM to 5nM and Cyno BCMA with a Kd from 0.1 nM to 20 nM. Preferably, the bispecific format is the format in fig. 2A, comprising the respective VL and VH constant domains of the replacement of CD3 Fab for each other and within BCMA Fabs K213E and K147E amino acid exchanges in the CH1 domain and amino acid exchanges E123R and Q124K in the CL domain.
[00089] Another embodiment of the invention is a method for preparing an antibody according to the invention comprising the steps of
[00090] transforming a host cell with
[00091] vectors comprising nucleic acid molecules encoding the light chain and the heavy chain of an antibody according to the invention,
[00092] culturing the host cell under conditions that allow the synthesis of said antibody molecule; and
[00093] recovering said antibody molecule from said culture.
[00094] Another embodiment of the invention is a method for preparing a bispecific antibody according to the invention comprising the steps of
[00095] transforming a host cell with
[00096] vectors comprising nucleic acid molecules encoding the light chain and the heavy chain of an antibody that specifically binds to the first target
[00097] vectors comprising nucleic acid molecules encoding the light chain and the heavy chain of an antibody that specifically binds to the second target, wherein the variable domains VL and VH or the constant domains CL and CH1 are substituted for each other ;
[00098] culturing the host cell under conditions that allow the synthesis of said antibody molecule; and
[00099] recovering said antibody molecule from said culture.
[000100] Another embodiment of the invention is a host cell comprising vectors comprising nucleic acid molecules encoding an antibody according to the invention. Another embodiment of the invention is a host cell comprising vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody that specifically binds to the first target and vectors comprising nucleic acid molecules encoding the light chain and the chain weight of an antibody specifically binding to the second target, wherein the variable domains VL and VH or the constant domains CL and CH1 are substituted for one another.
[000101] Another embodiment is a pharmaceutical composition comprising an antibody according to the invention and a pharmaceutically acceptable excipient.
[000102] Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention for use as a medicine.
[000103] Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention for use as a medicament in the treatment of cellular plasma disorders.
[000104] Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention for use as a medicament in the treatment of Multiple Myeloma.
[000105] Another embodiment of the invention is pharmaceutical composition comprising an antibody according to the invention for use as a medicine in the treatment of systemic lupus erythematosus.
[000106] Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention, including a monospecific antibody, an ADCC improved naked antibody, an antibody-drug conjugate, a multispecific antibody or a bispecific antibody for use as a medicine in the treatment of antibody-mediated rejection.
[000107] In one embodiment, an antibody according to the invention can be used for the treatment of plasma cell diseases like Multiple Myeloma MM or other BCMA expressing plasma cell disorders as described below. MM is a malignant disease of plasma cells characterized by monoclonal expansion and accumulation of abnormal plasma cells in the bone marrow compartment. MM also involves circulating cells from clonal plasma cells with the same IgG gene rearrangement and somatic hypermutation. MM arises from an asymptomatic premalignant condition called monoclonal gammopathy of unknown significance (MGUS), characterized by low levels of bone marrow plasma cells and a monoclonal protein. MM cells proliferate at a low rate. MM results from a progressive occurrence of multiple structural chromosomal alterations (eg, unbalanced translocations). MM involves the mutual interaction of malignant plasma cells and bone marrow microenvironment (eg, normal bone marrow stromal cells). Clinical signs of active MM include monoclonal antibody spike, plasma cells overcrowding the bone marrow, lytic bone lesions, and bone destruction as a result of osteoclast overstimulation (Dimopulos & Terpos, Ann Oncol 2010; 21 suppl 7: vii143- 150). Another plasma cell disorder involving plasma cells that is, expressing BCMA is systemic lupus erythomatous (SLE), also known as lupus. SLE is a systemic and autoimmune disease that can affect many parts of the body and is represented by the immune system attacking the body's own cells and tissues, resulting in chronic inflammation and tissue damage. It is a Type III hypersensitivity reaction in which antibody-immune complexes precipitate and cause a further immune response (Inaki & Lee, Nat Rev Rheumatol 2010; 6: 326-337). Other plasma cell disorders are plasma cell leukemia and AL-Amyloidosis (see also Examples 19 and 20). In all these plasma cell disorders the depletion of plasma cells/malignant plasma cells by antibodies according to this invention is expected to be beneficial to patients suffering from such disease.
[000108] Another embodiment of this invention is an antibody according to the invention for the treatment of antibody-mediated allograft rejection involving plasma cells and alloantibodies including acute and chronic antibody-mediated rejection (AMR). Acute AMR is characterized by graft dysfunction that occurs over days and is the result of de novo specific antibodies from preformed or developed post-transplant donors. It occurs in about 5-7% of all kidney transplants and causes 20-48% of acute rejection episodes among patients with positive presensitized disorders (Colvin and Smith, Nature Rev Immunol 2005; 5 (10): 807- 817). Histopathology in patients with acute AMR often reveals endothelial cell swelling, neutrophil infiltration of peritubular glomeruli and capillaries, fibrin thrombi, interstitial edema, and hemorrhage (Trpkov et al. Transplantation 1996; 61 (11): 1586-1592). AMR can be identified with C4d staining or other improved methods of detecting antibodies in allograft biopsies. Another form of AMR is also known as chronic allograft lesion, which also involves donor-specific antibodies, but manifests itself months and even years after transplantation. It is seen as transplant glomerulopathy (also known as chronic allograft glomerulopathy) in kidney biopsies and is characterized by glomerular mesangial expansion and capillary basilar membrane doubling (Regele et al. J Am Soc Nephrol 2002; 13(9): 23712380) . Clinical manifestations range from patients who are asymptomatic in the early stages to have nephrotic gamma proteinuria, hypertension, and allograft dysfunction in the advanced stages. Disease progression can be quite rapid, especially with ongoing acute AMR, resulting in graft failure within months (Fotheringham et al. Nephron - Clin Pract 2009; 113 (1): c1-c7). The prevalence of transplant glomerulopathy in patient biopsies ranges from 5% at 1 year to 20% at 5 years (Cosio et al. Am J Transplant 2008; 8:292-296).
[000109] Another embodiment of the invention is an antibody according to the invention for use as a medicine.
[000110] Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention for use as a medicine.
[000111] Another embodiment of the invention is a pharmaceutical composition comprising a naked antibody or a bispecific antibody according to the invention for use as a medicine.
[000112] Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention with improved effector function for use as a medicine.
[000113] Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention with decreased effector function for use as a medicine.
[000114] Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention as bispecific antibody for use as a medicine.
[000115] Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention as multispecific antibody for use as a medicine.
[000116] Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention as conjugated with a therapeutic agent (drug conjugate), for example, with a cytotoxic agent or radiolabel for use as a medicine.
[000117] Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention as a diabody for use as a medicine.
[000118] In one embodiment, the antibody according to the invention, especially when it is a bispecific antibody against CD3 and BCMA, is administered once or twice a week in one modality via subcutaneous administration (for example, in a modality in the range of dose from 0.1 to 2.5, preferably at 25 mg/m2 / week, preferably at 250 mg/m2 / week). Due to superior cytotoxicity activities of the antibody according to the invention, it can be administered at least in the same range of clinical dose range (or even smaller) compared to conventional monospecific antibodies or conventional bispecific antibodies which are not cell bispecific T (ie does not bind CD3 in one branch). It is anticipated that for an antibody according to the invention, subcutaneous administration is preferred in clinical settings (eg in the dose range of 0.1 to 250 mg /m2/week). Furthermore, in patients with elevated serum APRIL and BAFF levels (eg, patients with multiple myeloma), it is not necessary to increase the dose of an antibody in accordance with this invention as it may not be affected by ligand competition. In contrast, doses for other ligand-blocking/competent anti-BCMA antibodies may need to be increased in these patients. Another advantage of the antibody according to the invention is an elimination half-life of about 4 to 12 days which allows at least once or twice a week for administration.
[000119] In one embodiment, the antibody according to the invention in the case of triggered ADCC/unconjugated monospecific antibodies is an antibody with properties that allow treatment once/twice a week intravenously, but preferably through subcutaneous administration (for example , a dosage in the range of 200 -2000 mg/m/week for 4 weeks). It is anticipated that, for an antibody according to the invention, subcutaneous administration is possible and preferred in clinical settings (eg in the dose range of 200-2000 mg/m2/week, depending on the disease indications). Furthermore, in patients with elevated levels of serum APRIL and BAFF (eg patients with multiple myeloma), it is not necessary to increase the dose of an antibody in accordance with this invention (eg blocking/competition non-ligand antibody) , as it may not be affected by binder competition. In contrast, doses for other ligand-blocking/competing anti-BCMA antibodies may need to be increased in these patients, making subcutaneous administration technically more challenging (eg, pharmaceutical). Another advantage of the antibody according to the invention is based on the inclusion of an Fc portion, which is associated with an elimination half-life of 4 to 12 days and allows administration at least once or twice a week.
[000120] Another preferred embodiment of the invention is a diagnostic composition comprising an antibody according to the invention. DESCRIPTION OF THE FIGURES
[000121] Figure 1. Bivalent bispecific antibodies comprising only the Fab fragments (specific for CD3 and BCMA) and the Fc part as specified: (A) Fab BCMA (RK/EE) -Fc-Fab CD3; (B) Fab BCMA-Fc-Fab CD3 (RK/EE). aa substitutions for RK/EE introduced in CL-CH1 to reduce mismatch of LC/by-products in production. The Fab CD3 includes a VL-VH timer to reduce mismatch of LC and by-products.
[000122] Figure 2. Preferred bispecific trivalent antibodies comprising only the Fab fragments (specific for CD3 and BCMA) and the Fc part as specified: (A) Fab BCMA (RK/EE) - Fc-Fab CD3-Fab BCMA (RK/ AND IS); (B) Fab BCMA-Fc-Fab CD3 (RK/EE)-Fab BCMA; (C) Fab BCMA (RK/EE) -Fc-Fab BCMA (RK/EE) -Fab CD3; (D) Fab BCMA-Fc-Fab BCMA-Fab CD3 (RK/EE). aa substitutions for RK/EE introduced in CL-CH1 to reduce mismatch of LC/by-products in production. Preferably, the CD3 Fab includes a VL-VH timer to reduce LC mismatch and by-products. Preferably, Fab CD3 and Fab BCMA are linked together with flexible linkers.
[000123] Figure 3. Bivalent bispecific antibodies comprising only the Fab fragments (specific for CD3 and BCMA) and the Fc part as specified: (A) Fc-Fab CD3-Fab BCMA (RK/EE); (B) Fc-Fab CD3 (RK/EE) -Fab BCMA; (C) Fc-Fab BCMA (RK/EE) -Fab CD3; (D) Fc-Fab BCMA-Fab CD3 (RK/EE). Preferably, CD3 Fabs include a VL-VH timer to reduce LC mismatch and by-products. The Fab CD3 and Fab BCMA are linked together with flexible linkers.
[000124] Figure 4. Redirected T cell lysis of H929 MM cells induced by bispecific anti-BCMA/anti-CD3 T cell antibodies as measured by LDH release. Concentration response curves for lysis of H929 MM cells induced by 21-TCBcv (closed circle), 22-TCBcv (closed triangle), 42-TCBcv (closed square) compared to 83A10-TCBcv (open circle, line) dotted). There was a concentration-dependent killing of H929 cells for all bispecific anti-BCMA/anti-CD3 T cell antibodies, whereas no killing with the control-TCB was observed. Experiments were performed with PBMC donor 1 (A), donor 3 (B), donor 4 (C), donor 5 (D) using an effector cell to target tumor cell ratio (E:T) of 10 PBMCs to 1 MM cell (see example 8).
[000125] Figure 5. Redirected T cell lysis of L363 MM cells induced by bispecific anti-BCMA/anti-CD3 T cell antibodies as measured by LDH release. Concentration response curves for lysis of L363 MM cells induced by 21-TCBcv (closed circle), 22-TCBcv (closed triangle), 42-TCBcv (closed square) compared to 83A10-TCBcv (open circle, line) dotted). A concentration of L363 cells was observed for all bispecific anti-BCMA/anti-CD3 T cell antibodies, whereas no killing was observed with the control-TCB. Experiments were performed with PBMC donor 1 (A), donor 2 (B), donor 3 (C), donor 4 (D), donor 5 (E) using an E:T ratio of 10 PBMCs to 1 MM cell ( see example 9).
[000126] Figure 6. Redirected T cell lysis of RPMI-8226 MM cells induced by bispecific anti-BCMA/anti-CD3 T cell antibodies as measured by LDH release. Concentration response curves for lysis of RPMI-8226 MM cells induced by 21-TCBcv (closed circle), 22-TCBcv (closed triangle), 42-TCBcv (closed square) compared to 83A10-TCBcv (open circle) , dotted line). A concentration of RPMI-8226 cells was observed for all bispecific anti-BCMA/anti-CD3 T cell antibodies, whereas no killing was observed with the control-TCB. Experiments were performed with PBMC donor 2 (A), donor 3 (B), donor 4 (C), donor 5 (D) using an E:T ratio of 10 PBMCs to 1 MM cell (see example 10) .
[000127] Figure 7. Redirected T cell lysis of JJN-3 MM cells induced by bispecific anti-BCMA/anti-CD3 T cell antibodies as measured by flow cytometry. Concentration-dependent substitution of JJN-3 MM cells with 22-TCBcv (closed triangle), 42-TCBcv (closed square) compared to 83A10-TCBcv (open circle, dotted line). The percentage of annexin-V positive JJN-3 cells (A, C) and tumor cell lysis (B, D) were determined and plotted. The percentage of JJN-3 cell lysis induced by a specific concentration of anti-BCMA/anti-CD3 T cell bispecific antibody determined as the following: the absolute count of annexin-V-negative JJN-3 cells at a given concentration of TCB and subtracting it from the absolute JJN-3 annexin-V-negative cell count without TCB; divided by the absolute count of annexin-V-negative JJN-3 cells without TCB. Experiments were performed with 2 PBMC donors: donor 1 (A, B) and donor 2 (C, D) using an E:T ratio of 10 PBMCs to 1 MM cell (see example 11).
[000128] Figure 8. Redirected T cell lysis of bone marrow myeloma plasma cells from multiple myeloma patient in the presence of autologous bone marrow infusion T cells induced by bispecific anti-BCMA/anti-T cell antibodies CD3 as measured by multiparametric flow cytometry. The percentage of annexin-V positive myeloma plasma cells was determined and plotted against TCB concentrations. Concentration-dependent specific lysis of the patient's myeloma plasma cells was observed while T cell, B cell, and NK cell lysis was not observed based on an 8-color multiparametric panel. No induction of myeloma plasma cell cell death with control-TCB at the highest concentration of TCB antibodies tested. Compared to 83A10-TCBcv (A), 42-TCBcv (B) and 22-TCBcv (C) were more potent in inducing the death of the patient's bone marrow myeloma plasma cells (see example 13).
[000129] Figure 9. Redirected T cell lysis of bone marrow myeloma plasma cells from multiple myeloma patient in the presence of autologous bone marrow infusion T cells induced by bispecific anti-BCMA/anti-T cell antibodies CD3 as measured by flow cytometry. The percentage of annexin-V negative myeloma plasma cells was determined and plotted against TCB concentrations. Specific and concentration-dependent lysis of the patient's myeloma plasma cells was observed while lysis of non-median bone marrow cells was not observed (data not shown). No induction of myeloma plasma cell cell death observed with TCB-control at the highest concentration of TCB antibodies tested (data not shown). Compared to 83A10-TCBcv, 42-TCBcv and 22-TCBcv were more potent in inducing bone marrow myeloma plasma cell death, as reflected by the concentration-dependent reduction of viable plasma cells (annexin-V negative) of myeloma. Representative experiences in patient 001 (A) and patient 007 (B) (see example 13).
[000130] Figure 10. Redirected T cell lysis of bone marrow myeloma plasma cells from multiple myeloma patient in the presence of autologous bone marrow infusion T cells induced by bispecific anti-BCMA/anti-T cell antibodies CD3 as measured by flow cytometry. The percentage of propidium iodide negative myeloma plasma cells was determined and the percentage of viable bone marrow plasma cells relative to the mean control (MC) was plotted against TCB concentrations. Specific concentration-dependent lysis of the patient's myeloma plasma cells was observed (A - G), whereas lysis of the bone marrow microenvironment (BMME) was not observed (H). No induction of myeloma plasma cell cell death observed with control-TCB at the highest concentration of TCB antibodies tested. Compared with 83A10-TCBcv, 42-TCBcv and 22-TCBcv were more potent in inducing bone marrow myeloma plasma cell death, as reflected by concentration-dependent reduction of viable plasma cells (propidium negative iodide) of myeloma . An effect was considered statistically significant if the P-value of its corresponding statistical test was <5% (*), <1% (**) or <0.1% (***). Experiments performed with bone marrow aspirate samples collected from patient 1 (A), patient 2 (B), patient 3 (C), patient 4 (D), patient 5 (E), patient 6 (F) and patient 7 (G, H) (see example 13).
[000131] Figure 11. Activation of bone marrow cell cells from myeloma patients in the presence of plasma bone marrow cells (whole bone marrow aspirates) induced by bispecific anti-BCMA/anti-CD3 T cell antibodies as measured by multiparametric flow cytometry (8-color staining panel). The magnitude of T cell activation was compared between 83A10-TCBcv (A), 42-TCBcv (B) and 22-TCBcv (C) (see example 14).
[000132] Figure 12. Concentrations of 83A10-TCBcv measured from serum samples (closed symbols with complete lines) and bone marrow samples (open symbols with dotted lines) after single intravenous (IV) injection in cynomolgus monkeys at 0.003, 0.03 and 0.1 mg/kg of 83A10-TCBcv. Serum sample collection was performed pre-dose and 30, 90, 180 min, 7, 24, 48, 96, 168, 336, 504 h after administration. Bone marrow samples were collected pre-dose and 96 and 336 h after administration (see example 16).
[000133] Figure 13. Peripheral T cell redistribution observed in cynomolgus monkeys after a single IV injection of 83A10-TCBcv (0.003, 0.03 and 0.3 mg/kg). Animals A and B, C and D and E and F received respectively an intravenous injection of 0.003, 0.03 and 0.3 mg/kg of 83A10-TCBcv. Whole blood T cell counts (CD2+ cells per µL of blood) were plotted against time after treatment (see example 16).
[000134] Figure 14. Reduction of blood plasma cells observed in cynomolgus monkeys after a single IV injection of 83A10-TCBcv (0.3 mg/kg) measured by multiparameter flow cytometry. Plasma cells (PCs) were identified based on a 6-color staining panel and the percentages of PCs on lymphocytes were measured and plotted in contour plots (A). The kinetics of blood plasma cell depletion after treatment with 83A10-TCBcv 0.3 mg/kg in cynomolgus monkeys was plotted (B) (see example 16).
[000135] Figure 15. Anti-tumor activity induced by anti-BCMA/anti-CD3 T cell bispecific antibody of 83A10-TCBcv in the H929 human myeloma xenograft model using PBMC-humanized NOG mice. Immunodeficient NOD/Shi-scid IL2rgamma (null) (NOG) received on day 0 (d0) H929 human multiple myeloma cells as a subcutaneous (SC) injection into the right dorsal flank. On day 15 (d15), NOG mice received a single intraperitoneal (IP) injection of human PBMCs. The mice were carefully randomized into the different treatment and control groups (n = 9/group) and a statistical test was performed to test for homogeneity between groups. The experimental groups were the untreated controlled group, the TCB control treated group, 83 A10-TCBcv 2.6 nM/kg of treated group and BCMA50-BiTE® (BCMAxCD3 (scFv)2) 2.6 nM/kg of group treated. Antibody treatment administered by tail vein injection began on day 19 (d19), ie, 19 days after SC injection of H929 tumor cells. The TCB antibody treatment regimen consisted of once-weekly IV administration for up to 3 weeks (ie, 3 total TCB antibody injections). Tumor volume (VT) was measured by forceps during the study and progress assessed by VT intergroup comparison. TV (mm3) plotted against day post tumor injection. On day 19, the first day of treatment, the mean tumor volume reached 300 ± 161 mm3 for the vehicle-controlled control group (A), 315 ± 148 mm3 for the group treated with a TCB control of 2.6 nM/kg ( A), 293 ± 135 mm3 for the 2.6 nM/kg 83A10-TCBcv group (B) and 307 ± 138 mm3 for the 2.6 nM/kg BCMA50-BiTE® group (C). VT of each mouse individually per experimental group was plotted against day post tumor injection: (A) control groups including vehicle control (full line) and TCB control (dotted line), (B) 83A10-TCBcv group ( 2.6 nM/kg) and (C) BCMA50-BiTE® (2.6 nM/kg). Black arrows show TCB treatment given by intravenous injection. In the 83A10-TCBcv group (2.6 nM/kg), 6 of the 9 mice (67%) had their tumor regressed even below the VT recorded on day 19, that is, the first TCB treatment and tumor regression was maintained until the end of the study. The 3 mice in the 83A10-TCBcv treated group (2.6 nM/kg) that did not show tumor regression had their VT equal to 376, 402 and 522 mm 3 respectively on day 19. In contrast, none of the 9 mice (0%) treated with an equimolar dose of BCMA50-BiTE® (2.6 nM/kg) on a once weekly schedule for 3 weeks had their tumor regressed at any time point (see example 17).
[000136] Figure 16. Percent tumor growth (TG) calculated for day 19 to day 43 and compared between 83A10-TCBcv (2.6 nM/kg) and BCMA50-BiTE® (2.6 nM/kg). Percent tumor growth defined as TG (%) was determined by calculating TG (%) = 100 x (mean VT of analyzed group)/(mean VT of control vehicle-treated group). For ethical reasons, mice were euthanized when the VT reached at least 2000 mm3. TG (%) is consistently and significantly reduced in the 83A10-TCBcv group (2.6 nM/kg), as well as the TG (%) is always lower when compared to BCMA50-BiTE® (2.6 nM/kg) ( see example 17).
[000137] Figure 17. Surface plasmon resonance (SPR) of 70 selected ELISA clones. All experiments were carried out at 25°C using PBST as running buffer (10 mM PBS, pH 7.4 and 0.005% (v/v) Tween®20) with a ProteOn XPR36 biosensor equipped with GLC sensor chips and GLM and coupling reagents. Immobilizations were performed at 30 µl/min on a GLM chip. pAb (goat) anti hu IgG, F(ab) 2 Specific Ab (Jackson) was coupled in the vertical direction using a standard amine coupling procedure: all six ligand channels were activated for 5 minutes with an EDC mixture (200 mM) and sulfo-NHS (50 mM). Immediately after activation of the surfaces, pAb (goat) specific antibodies anti hu IgG, F(ab) 2 (50 µg/ml, 10 mM sodium acetate, pH 5) were injected into all six channels for 5 min. Finally, the channels were blocked with a 5 min injection of 1 M ethanolamine HCl (pH 8.5). Final immobilization levels were similar across all channels, ranging from 11000 to 11500 RU. Fab variants were captured from e.coli supernaturants by simultaneous injection along five of the entire horizontal channels separated (30 µl/min) for 5 min and resulted in levels ranging from 200 to 900 RU, depending on Fab concentration in the supernatant; the conditioned medium was injected along the sixth channel to provide an "in-line" white space for double reference purposes. One-shot kinetic measurements were performed by injecting a dilution series of human and cyno BCMA (50, 10, 2, 0.4, 0.08, 0 nM, 50 µl/min) for 3 min along the channels. vertical. Dissociation was monitored for 5 min. Kinetic data was analyzed in ProteOn Manager v. 2.1. Processing the reaction point data involved the application of an interspot reference and a double reference step using a blank buffer line (Myszka, 1999). The processed data from replicated one-shot injections were fitted to a simple 1:1 Langmuir binding model without mass transport (O'Shannessy et al., 1993).
[000138] Figure 18. Binding affinity of BCMA antibodies in HEK-huBCMA cells, measured by flow cytometry. Anti-BCMA antibodies were used as the first antibody, then a secondary PE-labeled anti-human Fc was used as the detection antibody. It was found that the binding of antibodies Mab 21, Mab 22, Mab 27, Mab 39 and Mab 42 to huBCMA on HEK cells was not significantly better than binding of Mab 83A10 to huBCMA-HEK cells.
[000139] Figure 19. 42-TCBcv concentrations measured in serum and bone marrow after a single IV or SC injection in cynomolgus monkeys. Animals received a single IV or SC injection of 42-TCBcv) and blood samples per time point were collected through the peripheral vein for PK assessments at pre-dose, 30, 90, 180 min, 7, 24, 48, 96, 168, 336, 504 h after administration. Blood samples were allowed to clot in serum separation tubes for 60 minutes at room temperature. The clot was centrifuged by centrifugation. The resulting serum was stored directly at -80°C until further analysis. Bone marrow samples for PK assessments were also collected from the femur under anesthesia/analgesic treatment at pre-dose, 96 and 336 h after administration. Bone marrow samples were allowed to clot in serum separation tubes for 60 minutes at room temperature. The clot was centrifuged by centrifugation. The resulting bone marrow was stored directly at -80°C until further analysis. Analysis and evaluation of PK data was performed. A non-compartmental pattern analysis was performed using the Watson package (v.7.4, Thermo Fisher Scientific Waltman, MA, USA) or the Phoenix WinNonlin system (v.6.3, Certara Company, USA). Effective concentration range of 42-TCBcv in bone marrow aspirates from multiple myeloma patient corresponding to 10 pm to 10 nM (grey area). Concentrations in parentheses are in nM.
[000140] Figure 20. Redirected T cell lysis of plasma cell leukemia bone marrow leukemic cells in the presence of autologous T cells or bone marrow infiltrated T cells induced by bispecific anti-BCMA T cell antibodies/ anti-CD3 as measured by flow cytometry. The percentage of propidium iodide negative myeloma plasma cells was determined and the percentage of viable leukemic bone marrow cell plasma cells relative to the mean control (MC) was plotted against TCB concentrations. Concentration-dependent specific lysis of leukemic cells from the patient's plasma cells was observed (A, B), whereas lysis of the bone marrow microenvironment (BMME) was not observed (data not shown). No induction of myeloma plasma cell cell death observed with control-TCB at the highest concentration of TCB antibodies tested. 42-TCBcv was very potent in inducing leukemic cell death from bone marrow plasma cells as reflected by the concentration-dependent reduction of viable plasma myeloma cells (propidium and negative iodide). An effect was considered statistically significant if the P-value of its corresponding statistical test was <5% (*), <1% (**) or <0.1% (***). The figure shows the results obtained from bone marrow samples from patient 1 (A) and patient 2 (B) (see also example 20).
[000141] DETAILED DESCRIPTION OF THE INVENTION
[000142] The term "BCMA, BCMA target, human BCMA" as used herein refers to the human B cell maturation antigen, also known as BCMA; TR17_HUMAN, TNFRSF17 (UniProt Q02223), which is a member of the tumor necrosis receptor superfamily that is preferentially expressed in differentiated plasma cells. The extracellular domain of BCMA consists of UniProt from amino acids 1 to 54 (or 5-51). The term "anti-BCMA antibody, anti-BCMA antibody" as used in this document refers to an antibody that specifically binds to the extracellular domain of BCMA.
[000143] "Binding specifically to BCMA or binding to BCMA" refers to an antibody that is capable of binding the target BCMA with sufficient affinity such that the antibody is useful as a therapeutic agent in directing BCMA. In some embodiments, the extent of binding of an anti-BCMA antibody to an unrelated non-BCMA protein is about 10-fold, preferably >100-fold less than antibody binding to BCMA, as measured, e.g. , by surface plasmon resonance (SPR), eg Biacore®, enzyme-linked immunosorbent (ELISA) or flow cytometry (FACS). In one embodiment the BCMA binding antibody has a dissociation constant (Kd) of 10-8 M or less, preferably 10-8 M to 10-13 M, preferably 10-9 M to 10-13 M. In one embodiment, the anti-BCMA antibody binds to an epitope of BCMA that is conserved among BCMA from different species, preferably between human and cynomolgus, and additionally preferably also mouse and rat BCMA. "Bispecific antibody that specifically binds to CD3 and BCMA, bispecific antibody against CD3 and BCMA" refers to a respective definition of binding to both targets. An antibody that specifically binds to BCMA (or BCMA and CD3) does not bind to other human antigens. Therefore, in an ELISA, the OD values for such unrelated targets will be equal to or less than the detection limit of the specific assay, preferably >0.3 ng/mL, or values equal to or less than the OD of control samples without plaque - BCMA or with non-transfected HEK293 cells.
Preferably, the anti-BCMA antibody is specifically linked to a group of BCMA, consisting of human BCMA and BCMA of non-human mammal origin, preferably BCMA from cynomolgus, mouse and/or rat. "Human cyno/gap"refers to the affinity ratio KD cynomolgus BCMA [M]/KD human BCMA [M] (details see example 3). "Cino/human CD3 Mab gap", as used herein, refers to the affinity ratio KD cynomolgus CD3 [M]/KD human CD3 [M]. In one embodiment, the bispecific anti-BCMA/anti-CD3 antibody of the invention shows a cino/human CD3 Mab range between 1.25 and 5 or between 0.8 and 1.0. The bispecific antibody according to the invention is in an embodiment characterized by specifically binding to cynomolgus CD3. In one embodiment, the bispecific anti-BCMA/anti-CD3 antibody of the invention shows a cino/human CD3 Mab range between 1.25 and 5 or between 0.8 and 1.0. Preferably, the cyno/human gap is in the same range for anti-BCMA- and anti-CD3 antibody.
The term "APRIL" as used herein refers to recombinant truncated murine APRIL (amino acids 106-241; NP_076006). APRIL can be produced as described in Ryan, 2007 (Mol Cancer Ther; 6 (11): 3009-18).
[000146] The term "BAFF" as used herein refers to truncated, recombinant human BAFF (UniProt Q9Y275 (TN13B_HUMAN) which can be produced as described in Gordon, 2003 (Biochemistry; 42 (20): 5977-5983) Preferably, a His-tagged BAFF is used in accordance with the invention. Preferably, the His-tagged BAFF is produced by cloning a DNA fragment encoding BAFF residues 82-285 into a vector of expression, creating a fusion with an N-terminal His-tag followed by a thrombin cleavage site, expressing said vector and cleaving the recovered protein with thrombin.
[000147] Anti-BCMA antibodies are analyzed by ELISA for binding to human BCMA using plate-bound BCMA. For this assay, an amount of BCMA bound to the plate is preferably 1.5 µg/ml and concentration(s) ranging from 0.1 pM to 200 nM of anti-BCMA antibody.
[000148] The term "NF-KB" as used herein refers to recombinant NF-KB p50 (accession number (P19838) NF-kB activity can be measured by a binding ELISA a DNA from an extract of NCI-H929 MM cells (CRL-9068 ™) NCI-H929 MM cells, untreated or treated with 0.1 μg/ml TNF-α, 1000 ng/ml TR-truncated-BAFF thermally extracted, 1000 ng/ml truncated-BAFF, isotype control from 0.1 pM to 200 nM and with or without anti-BCMA antibodies from 0.1 pM to 200 nM are incubated for 20 min. be tested using a functional ELISA that detects the chemiluminescent signal from p65 linked to the NF-kB consensus sequence (US6150090).
[000149] The term "additional target" as used herein preferably means CD3e. The term "first target and second target" means CD3 as first target and BCMA as second target or means BCMA as first target and CD3 as second target.
[000150] The term "CD3ε or CD3" as used in this document refers to the human CD3ε described in UniProt P07766 (CD3E_HUMAN). The term "anti-CD3 antibody, anti-CD3 antibody" refers to an antibody specifically bound to CD3ε. In one embodiment, the antibody comprises a variable domain VH comprising the heavy chain CDRs of SEQ ID NO:1, 2 and 3 as respectively the heavy chain CDR1, CDR2 and CDR3 and a variable domain VL comprising the light chain CDRs of SEQ ID NO: 4, 5 and 6 as light chain CDR1, CDR2 and CDR3 respectively. In one embodiment, the antibody comprises the variable domains of SEQ ID NO: 7 (VH) and SEQ ID NO: 8 (VL).
[000151] The term "antibody" as used herein refers to a monoclonal antibody. An antibody consists of two pairs of a "light chain" (LC) and a "heavy chain" (HC) (such light chain (LC)/heavy chain pairs are abbreviated herein as LC/HC). The light chains and heavy chains of such antibodies are polypeptides that consist of several domains. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises the heavy chain CH1, CH2 and CH3 constant domains (IgA, IgD and IgG antibody classes) and, optionally, the CH4 heavy chain constant domain (IgE and IgM antibody classes). Each light chain comprises a light chain variable domain VL and a light chain constant domain CL. The VH and VL variable domains can be further subdivided into regions of hypervariability, named complementarity determining regions (CDR), interspersed with regions that are no longer conserved, named structural regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The "constant domains" of the heavy chain and light chain are not directly involved in the binding of an antibody to a target, but they do exhibit several effector functions. The term "antibody" as used herein also refers to that portion of an antibody that is required at least for specific binding to the CD3 resp antigen. BCMA. Accordingly, such an antibody (or antibody portion) may in one embodiment be a Fab fragment, if said antibody portion is comprised of a bispecific antibody according to the invention. The antibody according to the invention may also be a Fab', F(ab')2, an scFv, a di-scFv or a bispecific T cell engager (BiTE).
[000152] The term "antibody" includes, for example, mouse antibodies, human antibodies, chimeric antibodies, humanized antibodies and genetically modified antibodies (variant or mutant antibodies), as long as their characteristic properties are maintained. Especially preferred are human or humanized antibodies, especially as human or humanized recombinant antibodies. Other modalities are heterosexual antibodies (bispecific, specifically trispecific) and other conjugates, for example, with cytotoxic small molecules.
[000153] The term "bispecific antibody" as used herein refers in one embodiment to an antibody in which one of two pairs of heavy chain and light chain (HC/LC) specifically binds to CD3 and the other is specifically linked to BCMA. The term also refers to other bispecific antibody formats according to the state of the art, in an embodiment of bispecific single chain antibodies.
The term "TCB" as used herein refers to a bispecific antibody that specifically binds to BCMA and CD3. The term "83A10-TCBcv" as used herein refers to a bispecific antibody that specifically binds BCMA and CD3 as specified by its heavy and light chain combination of SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 47 (2x) and SEQ ID NO: 48, and as shown in Figure 2A and described in EP14179705. The terms "21-TCBcv, 22-TCBcv, 42-TCBcv", as used herein, refer to the respective bispecific Mab21 antibodies, as specified by their heavy and light chain combination of SEQ ID NO: 48, SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51 (2x), Mab 22 as specified by their heavy and light chain combinations of SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO: 53 and SEQ ID NO: 54 (2x) and Mab42 as specified by their heavy and light chain combination of SEQ ID NO: 48 of SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57- ( 2x).
[000155] The term "naked antibody" as used herein refers to an antibody that specifically binds to BCMA, which comprises an Fc part and is not conjugated to a therapeutic agent, for example, to a cytotoxic agent or radiolabel. . The term "conjugated antibody, drug conjugate" as used herein refers to an antibody that specifically binds BCMA and is conjugated to a therapeutic agent, for example, a cytotoxic agent or radiolabel.
[000156] The term "bispecific single-chain antibody" as used herein refers to a single polypeptide chain comprising in one embodiment two binding domains, one that specifically binds to BCMA and the other in one embodiment specifically connected to CD3. Each binding domain comprises a variable region from an antibody heavy chain ("VH region"), wherein the VH region of the first binding domain specifically binds to the CD3 molecule and the VH region of the second binding domain binds specifically to BCMA. The two binding domains are optionally linked together by a short polypeptide spacer. A non-limiting example for a polypeptide spacer is Gly-Gly-Gly-Gly-Ser (GGGGS) and its repeats. Each binding domain may further comprise a variable region of an antibody light chain ("VL region"), the VH region and the VL region within each of the first and second binding domains are linked together through a linker. polypeptide, long enough to allow the VH region and VL region of the first binding domain and the VH region and VL region of the second binding domain to pair with each other so that together they can to specifically bind to the respective first and second binding domains (see, for example, EP0623679). Bispecific single-chain antibodies are also mentioned, for example, in Choi BD et al., Expert Opin Biol Ther. 2011 Jul; 11(7):843-53 and Wolf E. et al., Drug Discov Today. 2005 September 15th; 10 (18): 1237-44.
[000157] The term "diabody" as used herein refers to a small bivalent and bispecific antibody fragment comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) therein polypeptide chain (VH-VL) linked by a peptide linker that is too short to allow pairing between the two domains on the same chain (Kipriyanov, Int. J. Cancer 77 (1998), 763-772). This forces pairing with the complementary domains of another chain and promotes the assembly of a dimeric molecule with two functional antigen-binding sites. To construct bispecific diabodies of the invention, the V domains of an anti-CD3 antibody and an anti-BCMA antibody are fused to create the two chains VH(CD3)-VL(BCMA), VH(BCMA)-VL(CD3). Each chain by itself is not capable of binding its respective antigen, but recreates the functional antigen binding sites of anti-CD3 antibody and anti-BCMA antibody by pairing with the other chain. The two scFv molecules, with a linker between the heavy chain variable domain and the light chain variable domain that is too short for intramolecular dimerization, are co-expressed and self-assembled to form bispecific molecules with the two binding sites at opposite ends. By way of example, the variable regions encoding the binding domains for BCMA and CD3, respectively, can be amplified by PCR from DNA constructs obtained as described, so that they can be cloned into a vector such as pHOG , as described in Kipiriyanov et al., J. Immunol, Methods, 200, 69-77 (1997a).
[000158] The two scFV constructs are then combined into an expression vector in the desired orientation, whereby the VH-VL linker is shortened to avoid forwarding the strings themselves. The DNA segments are separated by a STOP codon and a ribosome binding site (RBS). RBS allows the transcription of mRNA as a bicistronic message, which is translated by ribosomes into two non-covalently interacting proteins to form the diabody molecule. Diabodies, like other antibody fragments, have the advantage of being expressed in bacteria (E. coli) and yeasts (Pichia pastoris) in a functional form and with high yields (up to Ig/l).
[000159] The term "scFVs in tandem", as used herein, refers to a single-chain Fv molecule (i.e., a molecule formed by association of the variable domains of the immunoglobulin heavy and light chain, VH and VL, respectively) as described, for example, in WO 03/025018 and WO 03/048209. Such Fv molecules, which are known as TandAbs®, comprise four antibody variable domains, where (i) the first two or the last two of the four variable domains intramolecularly bind to each other within the same chain forming an antigen-binding scFv in the VH/VL or VL/VH orientation (ii) the other two domains cross-link with the corresponding VH or VL domains of another chain to form antigen-binding VH/VL pairs. In a preferred embodiment, as mentioned in WO 03/025018, the monomers of such an Fv molecule comprise at least four variable domains of which two neighboring domains of a monomer form a VH-VL or VL-VH scFv binding unit. antigen.
[000160] The term "DARPins", as used herein, refers to a bispecific ankyrin repeat molecule as described, for example, in US 2009082274. Such molecules are derived from natural ankyrin proteins, which can be found in human genome and are one of the most abundant types of binding proteins. A DARPin library module is defined by natural ankyrin repeat protein sequences, using 229 ankyrin repeats for initial design and another 2200 for subsequent refinement. Modules serve as building blocks for the DARPin libraries. The library modules resemble human genome sequences. A DARPin is made up of 4 to 6 modules. As each module is approx. 3.5 kDa, the size of an average DARPin is 16-21 kDa. Selection of agglutinants is done by display ribosome, which is completely cell-free and is described in He M and Taussig MJ., Biochem Soc Trans. 2007, Nov;35(Pt 5):962-5.
[000161] The terms "bispecific T cell attachment" are fusion proteins consisting of two single chain variable fragments (scFvs) of different antibodies, or amino acid sequences of four different genes, into a single peptide chain of about 55 kilodaltons . One of the scFvs binds to T cells via the CD3 receptor and the other to a BCMA.
[000162] There are five types of mammalian antibody heavy chains denoted by the Greek letters: α, δ, ε, Y and μ (Janeway CA, Jr et al (2001). Immunobiology. 5th edition, Garland Publishing). The type of heavy chain chain defines the antibody class; these chains are found in IgA, IgD, IgE, IgG and IgM antibodies, respectively (Rhoades RA, Pflanzer RG (2002). Human Physiology, 4th ed., Thomson Learning). Distinct heavy chains differ in size and composition; α and y contain approximately 450 amino acids, while μ and ε have approximately 550 amino acids.
[000163] Each heavy chain has two regions, the constant region and the variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of a different isotype. The y, α and δ heavy chains have a constant region composed of three constant domains CH1, CH2 and CH3 (in one row) and a hinge region for greater flexibility (Woof J, Burton D Nat Rev Immunol 4 (2004) 89- 99); μ and ε heavy chains have a constant region composed of four constant domains CH1, CH2, CH3 and CH4 (Janeway CA, Jr et al (2001). Immunobiology. 5th edition, Garland Publishing). The variable region of the heavy chain differs in antibodies produced by different B cells, but it is the same for all antibodies produced by a single B cell or clone of B cells. The variable region of each heavy chain is approximately 110 amino acids long and is composed of a single antibody domain.
[000164] In mammals there are only two types of light chain, which are called lambda (À) and kappa (K). A light chain has two successive domains: a constant domain CL and a variable domain VL. The approximate length of a light chain is 211 to 217 amino acids. In one embodiment, the light chain is a kappa (k) light chain, and the constant domain CL is in one embodiment derived from a kappa (K) light chain (the constant domain CK).
[000165] "Aa substitution", as used herein, refers to independent amino acid substitution in the constant domain CH1 at amino acid at positions 147 and 213 by glutamic acid (E) or aspartic acid (D) and in the constant domain CL o amino acid position 124 is replaced by lysine (K), arginine (R) or histidine (H). In one embodiment, additionally, in the constant domain CL the amino acid at position 123 is independently substituted with lysine (K), arginine (R) or histidine (H). In one embodiment, amino acid 124 is K, amino acid 147 is E. , amino acid 213 is E and amino acid 123 is R. The aa substitutions are in the CD3 Fab or in one or two BCMA Fabs. Bispecific antibodies against BCMA and CD3 as charge variants are described in EP14179705, disclosed by reference (hereinafter referred to as "charge variants and charge variant switching").
[000166] All amino acid numbering here is in accordance with Kabat (Kabat, EA et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242).
[000167] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of a single amino acid composition.
[000168] The "antibodies" according to the invention can be of any class (eg IgA, IgD, IgE, IgG and IgM, preferably IgG or IgE) or subclass (eg IgG1, IgG2, IgG3, IgG4 , IgA1 and IgA2, preferably IgG1), in which both antibodies, from which the bispecific bivalent derivative according to the invention is derived, have an Fc part of the same subclass (e.g. IgG1, IgG4 and the like, of preferably IgG1), preferably of the same allotype (eg Caucasian).
[000169] An "Fc part of an antibody" is a term well known to one of skill in the art and defined on the basis of the papain cleavage of antibodies. Antibodies according to the invention contain as Fc part, in one embodiment an Fc part derived from human origin and preferably all other parts of the human constant regions. The Fc part of an antibody is directly involved in complement activation, C1q binding, C3 activation, and Fc receptor binding. Although the influence of an antibody on the complement system is dependent on certain conditions, binding to C1q is caused by defined binding sites on the Fc part. Such binding sites are known in the art and described, for example, by Lukas, TJ., et al., J. Immunol. 127 (1981) 2555-2560; Brunhouse, R., and Cebra, J.J., MoI. Immunol. 16 (1979) 907-917; Burton, D.R., et al., Nature 288 (1980) 338-344; Thommesen, J.E., et al., MoI. Immunol. 37 (2000) 995-1004; Idusogie, E.E., et al., J. Immunol. 164 (2000) 41784184; Hezareh, M., et al., J. Virol. 75 (2001) 12161-12168; Morgan, A., et al., Immunology 86 (1995) 319-324; and EP 0 307 434.
[000170] Such binding sites are, for example, L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to the EU index of Kabat). Antibodies of the IgG1, IgG2 and IgG3 subclass generally show complement activation, C1q binding and C3 activation, whereas IgG4 does not activate the complement system, does not bind C1q and does not activate C3. In one modality, the Fc part is a human Fc part.
[000171] In one embodiment, an antibody according to the invention comprises an Fc variant of a wild-type human IgG Fc region, said Fc variant comprising an amino acid substitution at position Pro329 and at least one other substitution of amino acid, wherein the residues are numbered according to the EU index of Kabat, and wherein said antibody exhibits a reduced affinity for the human FcARIIIA and/or FcRIIA and/or FcRI compared to an antibody comprising the region Wild-type IgG Fc, and wherein the ADCC induced by said antibody is reduced to at least 20% of the ADCC induced by the antibody comprising a wild-type human IgG Fc region. In a specific embodiment, the Pro329 of a wild-type human Fc region in the antibody according to the invention is replaced with glycine or arginine or an amino acid residue large enough to destroy the proline sandwich within the Fc/Fcy interface of the receptor, which is formed between the proline329 of Fc residues and tryptophan Trp 87 and Dica 110 of FcRIII (Sondermann et al.: Nature 406, 267-273 (20 July 2000)). In another aspect of the invention, the at least one other amino acid substitution in the Fc variant is S228P, E233P, L234A, L235A, L235E, N297A, N297D or P331S and in yet another embodiment, said at least one other amino acid substitution is L234A and L235A from the human IgG1 Fc region or S228P and L235E from the human IgG4 Fc region. Such Fc variants are described in detail in WO2012130831.
[000172] By "effector function", as used herein, is meant a biochemical event that results from the interaction of an antibody Fc region with an Fc receptor or a ligand. Effector functions include, but are not limited to ADCC, ADCP, and CDC. By "effector cell", as used herein, is meant a cell of the immune system that expresses one or more Fc receptors and mediates one or more effector functions. Effector cells include but are not limited to monocytes, macrophages, neutrophils, dendritic cells, eosinophils, mast cells, platelets, B cells, large granular lymphocytes, Langerhans cells, natural killer (NK) cells and Yδ T cells, and may be from any organism, including but not limited to humans, mice, rats, rabbits and monkeys. By "library" herein is meant a set of Fc variants of any form, including but not limited to a list of nucleic acid or amino acid sequences, a list of nucleic acid or amino acid substitutions at variable positions, a physical library comprising nucleic acids that encode library sequences, or a physical library that comprises the Fc variant proteins, either in purified form or in unpurified form.
[000173] By "Fc gamma receptor" or "FcYR" as used herein means any member of the family of proteins that bind to the Fc region of the IgG antibody and are substantially encoded by the FcYR genes. In humans, this family includes, but is not limited to FcYRI (CD64), including FcYRIa, FcYRIb and FcYRIc isoforms; FcYRII (CD32), including FcYRlla isoforms (including H131 and R131 allotypes), FcYRllb (including FcYRllM and FcYRllb-2) and FCYRIIC; and FCYRIII (CD16), including FcYRlla (including V158 and F158 allotypes) and FcyRllb (including FcyRlllb-NA1 and FcyRlllb-NA2 allotypes) isoforms (Jefferis et al., 2002, Immunol Lett 82:57-65), as well as any FcyRs or undiscovered human FcyR isoforms or allotypes. An FcyR can be from any organism, including but not limited to humans, mice, rats, rabbits and monkeys. Mouse FcyRs include, but are not limited to, FcRIRI (CD64), FcIRII (CD32), FcRIII (CD16), and FcRIII-2 (CD16-2), as well as any unknown isoforms or allotypes of mouse FcRRs or FcRR.
[000174] "Fc variant with increased effector function", as used herein, means an Fc sequence that differs from that of an original Fc sequence by virtue of at least one amino acid modification or refers to other modifications as glycosylation alteration , for example, with Asn279 that increase the effector functions. Such modifications are for example mentioned in Duncan et al., 1988, Nature 332:563-564; Lund et al., 1991, J Immunol 147:26572662; Lund et al., 1992, Mol Immunol 29:53-59; Alegre et al., 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl Acad Sci US A 92:11980-11984; Jefferis et al., 1995, //77muno/Lett 44:111117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al., 1996, Immunol Lett 54:101-104; Lund et al., 1996, J Immunol 157:4963-4969; Armor et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al., 2000, J Immunol 164:4178-4184; Reddy et al., 2000, J Immunol 164:19251933; Xu et al., 2000, Cell Immunol 200: 16-26; Idusogie et al., 2001, J Immunol 166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferis et al., 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490; US5624821; US5885573; US6194551; WO200042072; WO199958572. Such Fc modifications also include, according to the invention, the engineered glycoforms of the Fc part. By "engineered glycoside" as used herein is meant a carbohydrate composition that is covalently linked to an Fc polypeptide, wherein said carbohydrate composition chemically differs from that of a parent Fc polypeptide. Engineered glycoforms can be generated by any method, for example, using variant or modified expression strains, by co-expression with one or more enzymes, for example D1-4-N-acetylglucosaminyltransferase III (GnTIII), expressing an Fc polypeptide in various organisms or cell lines from various organisms, or by modifying carbohydrates after the Fc polypeptide has been expressed. Methods for generating engineered glycoforms are known in the art and mentioned in Umana et al., 1999, Nat Biotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473) US6602684; WO200061739; WO200129246; WO200231140; WO200230954; Potelligent™ technology (Biowa, Inc., Princeton, N.J.); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland)). Thickened glycoform typically refers to a different composition of carbohydrates or oligosaccharides than the original Fc polypeptide.
Antibodies according to the invention comprising an Fc variant with increased effector function show high binding affinity to the Fc gamma receptor III (FCYRIII, CD 16a). High binding affinity for FCYRIII indicates that binding is increased for CD16a/F158 at least 10-fold over the parent antibody (95% fucosylation) as reference expressed in CHO host cells, such as CHO DG44 or CHO K1 cells, or/ and binding is increased for CD16a/V158 at least 20-fold over the parent antibody measured by Surface Plasma Resonance (SPR) using immobilized CD16a at an antibody concentration of 100 nM. The binding of FCYRIII can be increased by methods according to the state of the art, for example by modifying the amino acid sequence of the Fc part or the glycosylation of the Fc part of the antibody (see, for example, EP2235061). Mori, K et al., Cytotechnology 55 (2007)109 and Satoh M, et al., Expert Opin Biol Ther. 6 (2006) 1161-1173 refer to a CHO knockout line of FUT8 (α-1,6-fucosyltransferase) for the generation of afucosylated antibodies.
[000176] The term "chimeric antibody" refers to an antibody comprising a variable region, i.e. binding region, from a source or species and at least a portion of a constant region derived from a source or species different, usually prepared by means of recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are preferred. Other preferred forms of "chimeric antibodies" encompassed by the present invention are those in which the constant region has been modified or altered from the original antibody to generate the properties according to the invention, especially with respect to C1q binding and/or Fc binding. receiver (FcR). Such chimeric antibodies are also referred to as "class switched antibodies". Chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions. Methods for producing chimeric antibodies involving conventional recombinant DNA techniques and gene transfection are well known in the art. See, for example, Morrison, SL, et al., Proc. Natl. Academic Sci. USA 81 (1984) 6851-6855; Patent Nos. 5,202.238 and 5,204,244.
[000177] The term "humanized antibody" refers to antibodies in which the structure or "complementarity determining regions" (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity compared to that of the matrix immunoglobulin . In a preferred embodiment, a murine CDR is grafted onto the framework region of a human antibody to prepare the "humanized antibody". See, for example, Riechmann, L., et al., Nature 332 (1988) 323-327; and Neuberger, MS, et al., Nature 314 (1985) 268-270. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant region has been further modified or altered from the original antibody to generate the properties according to the invention, especially with respect to C1q binding and/or Fc binding of the receiver (FcR).
[000178] The term "human antibody" as used herein is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, MA and van de Winkel, JG, Curr. Opin. Chem. Biol. 5 (2001) 368-374). Human antibodies can also be produced in transgenic animals (eg mice) that are capable, after immunization, of producing a complete repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production. human germline immunoglobulin in such germline mutant mice will result in the production of human antibodies (see, for example, Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90 (1993) 2551-2555; Jakobovits, A., et al., Nature 362 (1993) 255-258; Bruggemann, M., et al., Year Immunol. 7 (1993) 33-40). Human antibodies can also be produced in phage display libraries (Hoogen-boom, HR and Winter, G., J. MoI). Biol. 227 (1992) 381-388; Marks, J.D., et al., J. MoI. Biol. 222 (1991) 581-597). The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); and Boerner, P., et al., J. Immunol. 147 (1991) 86-95). As already mentioned for chimeric and humanized antibodies according to the invention, the term "human antibody", as used herein, also comprises such antibodies which are modified in the constant region to generate the properties according to the invention, especially in in relation to C1q binding and/or FcR binding, e.g. by "class switching", i.e. changing or mutating Fc parts (eg from IgG1 to IgG4 and/or IgG1/IgG4 mutation).
[000179] The term "recombinant human antibody" as used herein is intended to include all human antibodies that are prepared, expressed, raised or isolated by recombinant means, such as antibodies isolated from such a host cell. as an NSO or CHO cell or from an animal (eg, a mouse) that is transgenic for human immunoglobulin genes or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in a rearranged fashion. Recombinant human antibodies according to the invention have been subjected to somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of recombinant antibodies are sequences which, although derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.
[000180] The "variable domain" (variable domain of a light chain (VL), variable region of a heavy chain (VH)), as used herein, indicates each of the light and heavy chains that is directly involved in joining the antibody according to the invention. The human variable heavy and light chain domains have the same general structure and each domain comprises four framework regions (FR) whose sequences are largely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β-sheet conformation and the CDRs can form loops connecting the β-sheet structure. The CDRs in each strand are held in their three-dimensional structure by the framework regions and form together with the CDRs in the other strand of the binding site. The CDR3 regions of the antibody heavy and light chain play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide another object of the invention.
The terms "hypervariable region" or "target binding portion of an antibody" when used herein refer to the amino acid residues of an antibody that are responsible for binding to the target. The hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs". "Framework" or "FR" regions are variable domain regions other than hypervariable region residues as defined herein. Therefore, the light and heavy chains of an antibody comprise from the N to C termini of the FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 domains. The CDRs in each chain are separated by these framework amino acids. Especially, the heavy chain CDR3 is the region that contributes the most to target binding. The CDR and FR regions are determined according to the standard definition of Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991). The terms "CDR1H, CDR2H and CDR3H" as used herein refer to the respective heavy chain CDRs located in the VH variable domain. The terms "CDR1L, CDR2L and CDR3L" as used herein refer to the respective light chain CDRs located in the variable domain VL.
[000182] The CH1 constant heavy chain domain by which the CH3 heavy chain domain is substituted can be of any Ig class (eg IgA, IgD, IgE, IgG and IgM), or subclass (eg IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2). The constant light chain domain CL by which the heavy chain CH3 domain is substituted can be of the lambda () or kappa (K) type, preferably the kappa (K) type.
[000183] The term "target" or "target molecule" as used herein is used interchangeably and refers to human BCMA. In relation to bispecific antibodies, the term refers to BCMA and the second target. Preferably with respect to bispecific antibodies, the term refers to BCMA and CD3.
[000184] The term "epitope" includes any determinant polypeptide capable of binding a specific antibody. In certain embodiments, the epitope determining includes chemically active surface groupings of molecules, such as amino acids, sugar, phosphoryl or sulfonyl side chains and, in certain embodiments, may have specific three dimensional structural characteristics and/or specific charge characteristics. An epitope is a region of a target that is bound by an antibody.
[000185] In general, there are two vectors that encode the light chain and the heavy chain of an antibody according to the invention. In relation to a bispecific antibody, there are two vectors encoding the light chain and the heavy chain of said antibody which specifically binds to the first target, and two other vectors which encode the light chain and heavy chain of said antibody which specifically binds to the second target. One of the two vectors is encoding the respective light chain and the other of the two vectors is encoding the respective heavy chain. However, in an alternative method for preparing an antibody according to the invention, only a first vector encoding the antibody light chain and heavy chain specifically binds to the first target and only a second vector encoding the light chain and the heavy chain of the antibody that specifically binds to the second target can be used to transform the host cell.
[000186] The term "nucleic acid" or "nucleic acid molecule" as used herein is intended to include both DNA molecules and RNA molecules. A nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA.
[000187] As used herein, the terms "cell", "cell line" and "cell culture" are used interchangeably and all these designations include progeny. Thus, the words "transformants" and "transformed cells" include the primary cell in question and cultures derived from it, without taking into account the number of transfers. It is also understood that not all offspring need be exactly identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same biological function or activity as sorted or selected in the originally transformed cell are included in this document. When distinct designations are intended, it will be clear from the context.
[000188] The term "transformation" as used herein refers to the process of transferring vectors/nucleic acid into a host cell. If cells without formidable cell wall barriers are used as host cells, transfection is carried out, for example, by the calcium phosphate precipitation method as described by Graham and Van der Eh, Virology 52 (1978) 546ff. However, other methods for introducing DNA into cells, such as nuclear injection or by protoplast fusion, can also be used. If prokaryotic cells or cells that contain substantial cell wall constructs are used, for example, one method of transfection is calcium treatment using calcium chloride as described by Cohen SN, et al, PNAS 1972, 69 (8): 21102114.
[000189] Recombinant production of antibodies using transformation is well known in the art and described, for example, in review articles by Makrides, S. C., Protein Expr. Purification 17 (1999) 183-202; Geisse, S., et al., Protein Expr. Purification 8 (1996) 271-282; Kaufman, RJ., MoI. Biotechnol. 16 (2000) 151-161; Werner, RG, et al., Arzneimittelforschung 48 (1998) 870-880, as well as in US6331415 and US4816567.
[000190] As used herein, "expression" refers to the process by which a nucleic acid is transcribed into mRNA and/or the process by which the transcribed mRNA (also referred to as transcription) is subsequently translated into peptides, polypeptides or proteins . Transcripts and encoded polypeptides are collectively referred to as gene products. If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may include mRNA splicing.
[000191] A "vector" is a particularly self-replicating nucleic acid molecule that transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily for inserting DNA or RNA into a cell (eg, chromosomal integration), replication vectors that function primarily for DNA or RNA replication, and expression vectors of that function for transcription and/or translation of DNA or RNA. Also included are vectors that provide more than one of the functions as described.
[000192] An "expression vector" is a polynucleotide that, when introduced into a suitable host cell, can be transcribed and translated into a polypeptide. An "expression system" generally refers to a suitable host cell comprised of an expression vector that can function to produce a desired expression product.
[000193] Antibodies according to the invention are preferably produced by recombinant means. Such methods are widely known in the art and comprise expression of the protein in prokaryotic and eukaryotic cells, with the subsequent isolation of the antibody polypeptide and usually purification to a pharmaceutically acceptable degree of purity. For protein expression, nucleic acids encoding light and heavy chains or fragments thereof are inserted into expression vectors by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells such as CHO cells, NSO cells, SP2/0 cells, HEK293 cells, COS cells, yeast, or E. coli cells and the antibody is recovered from the of cells (supernatant or cells after lysis). Bispecific antibodies may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Purification is carried out to eliminate other cellular components or other contaminants, for example, other nucleic acids or nuclear proteins, by standard techniques, including alkaline/SDS treatment, column chromatography and others well known in the art. See Ausubel, F., et al., Ed., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).
[000194] NS0 expression in cells is described by, for example, Barnes, LM, et al., Cytotechnology 32 (2000) 109-123; and Barnes, LM, et al., Biotech. Bioeng. 73 (2001) 261 -270. Transient expression is described by, for example, Durocher, Y., et al., Nucl. Acids Res. 30 (2002) E9. Cloning of variable domains is described by Orlandi, R., et al., Proc. Natl. Academic Sci. USA 86 (1989) 3833-3837; Carter, P., et al., Proc. Natl. Academic Sci. USA 89 (1992) 4285-4289; and Norderhaug, L., et al., J. Immunol. Methods 204 (1997) 77-87. A preferred transient expression system (HEK293) is described by Schlaeger, E.-J. and Christensen, K., in Cytotechnology 30 (1999) 71-83 and by Schlaeger, E.-J., in J. Immunol. . Methods 194 (1996) 191-199.
[000195] Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. It is known that eukaryotic cells use promoters, enhancers and polyadenylation signals.
[000196] Antibodies are suitably separated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. The DNA or RNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional procedures. Hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA can be inserted into expression vectors, which are then transfected into host cells such as HEK293 cells, CHO cells or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of immunoglobulin. recombinant monoclonal antibodies in host cells.
[000197] Amino acid sequence variants (or mutants) of an antibody of the invention are prepared by introducing appropriate nucleotide changes into the DNA antibodies, or by peptide synthesis. Such modifications can be carried out, however, only in a very limited range, for example as described above. For example, the modifications do not change the characteristics of antibodies mentioned above, such as IgG isotype and target binding, but may improve the yield of recombinant production, protein stability or facilitate purification.
[000198] The invention provides in one embodiment an isolated or purified nucleic acid sequence encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen recognition unit directed against BCMA, a transmembrane portion and a T cell activating portion, characterized in that the antigen recognition portion is an antibody according to the invention (here not the bispecific antibody). The encoded antibody may also be an antigen-binding fragment thereof as specified. The structures and generation of such "BCMA CARs" are described, for example, in WO2013154760, WO2015052538, WO2015090229 and WO2015092024.
[000199] In one embodiment, the invention comprises a chimeric antigen receptor (CAR) comprising: (i) a B cell maturation antigen recognition (BCMA) portion; (ii) a spacer domain; and (iii) a transmembrane domain; and (iv) an intracellular T cell signaling domain,
[000200] characterized in that the BCMA recognition unit is a monoclonal antibody that specifically binds to BCMA, characterized in that it comprises a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO: 20 and a combination of CDR1H region , CDR2H, CDR1L and CDR2L selected from the group of a) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:23 and CDR2L region of SEQ ID NO:24, b) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22, CDR1L region of SEQ ID NO: 25 and CDR2L region of SEQ ID NO: 26, c) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22, CDR1L region of SEQ ID NO: 27 and CDR2L region of SEQ ID NO: 28, d) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30, CDR1L region of SEQ ID NO : 31 and CDR2L region of SEQ ID NO: 32, e) CDR1H region of SEQ ID NO: 34 and CDR2H region of SEQ ID NO: 35, CDR1L region of SEQ ID NO: 31 and CDR2L region of SEQ ID NO: 32, and f) CDR1H region of SEQ ID NO: 36 and CDR2H region d a SEQ ID NO: 37, CDR1L region of SEQ ID NO: 31 and CDR2L region of SEQ ID NO: 32.
[000201] The T cell activation moiety can be any suitable moiety derived or obtained from any suitable molecule. In one embodiment, for example, the T cell activating portion comprises a transmembrane domain. The transmembrane domain can be any transmembrane domain derived or obtained from any molecule known in the art. For example, the transmembrane domain can be obtained or derived from a CD8a molecule or a CD28 molecule. CD8 is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR) and is mainly expressed on the surface of cytotoxic T cells. The most common form of CD8 exists as a dimer composed of a CD8 alpha and CD8 beta chain. CD28 is expressed on T cells and provides costimulatory signals necessary for T cell activation. CD28 is the receptor for CD80 (B7.1) and CD86 (B7.2). In a preferred embodiment, CD8 alpha and CD28 are human. In addition to the transmembrane domain, the T cell activation unit further comprises an intracellular (i.e., cytoplasmic) T cell signaling domain. The intercellular T cell signaling domain can be obtained or derived from a CD28 molecule, a CD3 zeta molecule or its modified versions, a human Fc receptor gamma chain (FcRy), a CD27 molecule, an OX40 molecule, a 4-IBB molecule, or other intracellular signaling molecules known in the art. As discussed above, CD28 is an important T cell marker in T cell costimulation. CD3 zeta associates with TCRs to produce a signal and contains immunoreceptor tyrosine-based activation motifs (ITAMs). 4-1BB, also known as CD137, transmits a potent costimulatory signal to T cells, promoting differentiation and enhancing the long-term survival of T lymphocytes. In one modality, CD28, CD3 zeta, 4-1BB, OX40 and CD27 are human.
[000202] The invention provides, in one embodiment, an isolated or purified nucleic acid sequence encoding a chimeric antigen receptor (CAR) as specified above.
[000203] Bispecific T cell binders (TCB) have an occupation-dependent potency of tumor cell/tumor cell receptors on cell death (eg EC50 in in vitro cell death assays in the sub or low picomolar range; Dreier et al. al. Int J Cancer 2002), the bispecific T-cell binder (TCB) is administered at much lower doses than conventional monospecific antibodies. For example, blinatumab (CD19xCD3) is given at a continuous intravenous dose of 5 to 15 μg/m2/day (ie, only 0.35 to 0.105 mg/m2/week) for the treatment of acute lymphocytic leukemia or 60 μg/m2/day for the treatment of non-Hodgkin's lymphoma and serum concentrations at these doses are in the range of 0.5 to 4 ng/ml (Klinger et al., Blood 2012; Topp et al., J Clin Oncol 2011; Goebeler et al. Ann Oncol 2011). As low doses of TCB can exert high efficacy in patients, it is anticipated that, for an antibody according to the invention, subcutaneous administration is possible and preferred in clinical settings (preferably in the dose range of 0 0.1 to 2.5, preferably from 25 mg/m2/week, preferably 250 mg/m2/week). Even at these low concentrations/doses/receptor occupancy, TCB can cause considerable adverse events (Klinger et al., Blood 2012). Therefore, it is essential to control the occupation/coverage of tumor cells. In patients with elevated and variable levels of APRIL and BAFF serum (eg, patients with multiple myeloma, Moreaux et al. 2004; Blo-od 103 (8): 3148-3157) number of TCB linked to tumor cells resp. tumor cell occupancy can be considerably influenced by APRIL/BAFF. But, using said antibody of this invention, tumor cell occupancy, respectively efficacy/safety, it may not be necessary to increase the dose for an antibody according to this invention, as said antibody cannot be affected by competition from APRIL/BAFF binders. Another advantage of the antibody according to the invention is based on the inclusion of an Fc portion, which increases the elimination half-life to about 4 to 12 days and allows at least once or twice a week administrations compared to TCB without an Fc portion (eg, blinatumomab) that should be administered intravenously and continuously with a patient-carried pump.
[000204] The biological properties of the antibodies according to the invention, respectively, their anti-BCMA/anti-CD3 TCB antibodies were investigated in several studies in comparison with 83A10-TCBcv. The potency to induce T-cell redirected cytotoxicity of, for example, anti-BCMA/anti-CD3 antibodies TCB 21-TCBcv, 22-TCBcv, 42-TCBcv compared to 83A10-TCBcv was measured in cell line H929 MM (Example 8, Table 12, Figure 4). The antibodies of this invention were studied and analysis showed that concentration-dependent killing of H929 resp. EC50 values were higher than the EC50 values determined for 83A10-TCBcv; suggesting that anti-BCMA antibodies according to the invention as TCBs are less potent in inducing H929 MM cell death than Mab 83A10 as TCB. Surprisingly, a turnover was observed when T-cell redirected cytotoxicity was measured in the RPMI-8226 MM cell line and also in the JJN-3 cell line (respectively, examples 10 and 11, Tables 13 and 14 and 15, Figures 6 and 7): antibodies according to the invention as TCBs had lower EC50 and, therefore, greater potency than 83A10-TCBcv. To the surprise of the inventors, antibodies according to the invention as TCBs showed several advantages in direct comparison with 83A10 TCBcv in bone marrow aspirates freshly collected from patients with MM (note: to obtain the best possible comparison, in all aspirates from bone marrow, always all bispecific T cell antibodies (TCB) were tested at the same concentrations);
[000205] - Maximum killing capacity of myeloma cells, ie even % kill already at lower concentrations than with 83A10-TCBcv respectively left-shifted concentration response curves for killing (Example 13, Tables 18, 19 and 20, Figures 8, 9 and 10). Already at a concentration of 1 nM of antibodies as TCBs according to the invention in seven different patients with reduced bone marrow aspirate in relation to control of propidium iodide negative viable multiple myeloma cancer cells was between 77.1 and 100%. With 1 nM 83A10-TCBcv in the same seven bone marrow aspirations, reductions of only 37.1 to 98.3% were achieved (Tables 20 and 21).
[000206] - Greater maximum kill compared to 83A10- TCBcv was achieved at the highest concentration tested (10 nM) in the same experiment with the seven (7) bone marrow aspirates for antibodies like TCB according to the invention (Tables 20 and 21 ).
[000207] - Non-responders to 83A10-TCBcv can be turned into responders if 22-TCBcv/42-TCBcv are used: In two (2) bone marrow patient samples in which no killing response to 83A10-TCBcv was observed, surprisingly killing could be found with antibodies like TCB according to the invention (Figures 9A and 9B).
[000208] The BCMAxCD3 TCB of this invention binds to cynomolgus monkeys and humans (cyno) BCMA and BCMA from mice and rats, suitable for toxicological examination in cynomolgus monkeys if the CD3 agglutinative also binds to cynomolgus CD3 or in mouse /mouse if the CD3 binder also binds to mouse/mouse BCMA.. Surprisingly, the binding affinity to cyno BCMA is very close to the binding affinity to human BCMA. SPR has been used to measure binding affinities to human and cyno BCMA (Example 2, Table 4). Cyno/human ranges (affinity ratio for cyno to human BCMA, KD) was calculated from the measured affinity data by dividing affinity for cyno BCMA by affinity for human BCMA (Example 3, Table 5). For 83A10 a cyno/human range of 15.3 was found (ie, 15.3 times lower binding affinity to cyno than to human BCMA). To the surprise of the inventors, the antibodies according to the invention showed cyno/human ranges between 15.4 and 1.7, which is similar or mostly to the cyno/human range greater than that of 83A10 (Table 5 ). As the CD3 binder used in BCMAxCD3 TCB according to the invention is reactive for cynomolgus monkey CD3, pharmacokinetic and pharmacodynamic surveys can be obtained from cynomolgus monkeys (see Example 16). Also toxicological investigations in cynomolgus monkeys are predictive of pharmacological and toxicological effects in humans and the cross-reactivity characteristic for cynomolgus monkeys is beneficial for patients. The BCMA antibodies of this invention also bind murine BCMA (e.g. Kd from clones 22 and 42 measured by SPR as 0.9 nM and 2.5 nM) see table 2D in Example 1.1.1A.4). The CD3 binder of BCMAxCD3 TCB is not reactive for murine CD3.
[000209] In summary, the potency and efficacy advantages for killing low BCMA expressing MM cell lines like RPMI-8226 and JJN-3 and especially for killing MM cells in patient bone marrow aspirates and, in addition, the The very favorable cyan/human range in binding affinities to BCMA makes the antibodies of this invention and respective TCBs essentially promising for the treatment of patients with MM. Furthermore, the anti-BCMAxCD3TCB of the present invention has, like 83A10-TCBcv, favorable properties such as long elimination half-life, efficacy of once-weekly administration (intravenous, subcutaneous), low or no tendency to aggregation and it can be manufactured with high purity and good yield. Table 1A: Antibody Sequences








[000210] Note: SEQ ID NO: 20 and SEQ ID NO: 33 are identical Table 1B: Antibody sequences (short list)
Table 2A: Additional Constructs
Table 2B: Additional Constructs

[000211] To make the following TCBs anti-BCMA/anti-CD3 (2+1) Fc, the respective construct/sequence identifications mentioned in table 2B above were used: 83A10-TCBcv: 45, 46, 47 (x2), 48 (Figure 2A) 21-TCBcv: 48, 49, 50, 51 (x2) (Figure 2A) 22-TCBcv: 48, 52, 53, 54 (x2) (Figure 2A) 42-TCBcv: 48, 55, 56 , 57 (x2) (Figure 2A)
[000212] In the following specific embodiments of the invention are listed: 1. A monoclonal antibody that specifically binds to BCMA, characterized in that it comprises a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO: 20 and a combination of CDR1H, CDR2H, CDR1L and CDR2L region selected from the group of a) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22, CDR1L region of SEQ ID NO: 23 and CDR2L region of SEQ ID NO: 24, b) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22, CDR1L region of SEQ ID NO: 25 and CDR2L region of SEQ ID NO: 26, c) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22, CDR1L region of SEQ ID NO: 27 and CDR2L region of SEQ ID NO: 28, d) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30, CDR1L region of SEQ ID NO:31 and CDR2L region of SEQ ID NO:32, e) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, CDR1L region of SEQ ID NO:31 and CDR2L region of SEQ ID NO: 32, and f) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37, CDR1L region of SEQ ID NO:31 and CDR2L region of SEQ ID NO:32.
[000213] 2. Monoclonal antibody that specifically binds to BCMA, characterized in that it comprises a VH region comprising a CDR1H region of SEQ ID NO: 21, a CDR2H region of SEQ ID NO: 22 and a CDR3H region of SEQ ID NO: 17 and a VL region comprising a CDR3L region of SEQ ID NO: 20 and a combination of the CDR1L and CDR2L regions selected from the group of
[000214] 3. The antibody according to modality 1 or 2, characterized in that it comprises as VL region a VL region selected from the group consisting of VL regions of SEQ ID NO: 12, 13 and 14.
[000215] 4. The antibody according to any one of the modalities 1 to 3, characterized in that it comprises as VH region a VH region of SEQ ID NO: 10 and as VL region a VL region of SEQ ID NO: 12.
[000216] 5. The antibody according to any one of the modalities 1 to 3, characterized in that it comprises as VH region a VH region of SEQ ID NO: 10 and as VL region a VL region of SEQ ID NO: 13.
[000217] 6. The antibody according to any one of the modalities 1 to 3, characterized in that it comprises as VH region a VH region of SEQ ID NO: 10 and as VL region a VL region of SEQ ID NO: 14.
[000218] 7. The antibody according to modality 1 or 2, characterized in that amino acid 49 of the VL region is selected from the group of amino acids tyrosine (Y), glutamic acid (E), serine (S) and histidine (H ).
[000219] 8. The antibody according to embodiment 7, characterized in that amino acid 74 of the VL region is threonine (T) or alanine (A).
[000220] 9. A monoclonal antibody that specifically binds BCMA, characterized in that it comprises a VH region comprising a CDR3H region of SEQ ID NO: 17 and a VL region comprising a CDR1L region of SEQ ID NO: 31, a region CDR2L of SEQ ID NO: 32 and a CDR3L region of SEQ ID NO: 20 and a combination of CDR1L and CDR2L region selected from the group of a) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30 , b) CDR1H region of SEQ ID NO:34 and CDR2H region of SEQ ID NO:35, or c) CDR1H region of SEQ ID NO:36 and CDR2H region of SEQ ID NO:37.
[000221] 10. An antibody according to embodiment 9, characterized in that it comprises a VL region of SEQ ID NO: 12 and a VH region selected from the group comprising the VH regions of SEQ ID NO: 38, 39 and 40.
[000222] 11. The antibody according to modality 9 or 10, characterized in that it comprises amino acid 49 of the VL region being selected from the group of amino acids tyrosine (Y), glutamic acid (E), serine (S) and histidine ( H).
12. The antibody according to modality 9 or 10, characterized in that amino acid 74 of the VL region is threonine (T) or alanine (A).
13. The antibody according to any one of embodiments 1 to 12, characterized in that it also specifically binds to BCMA cynomolgus and comprises an additional Fab fragment which specifically binds to CD3ε.
14. The antibody according to any one of embodiments 1 to 13, characterized in that it is an antibody with an Fc or without an Fc part.
[000226] 15. A bispecific antibody that specifically binds BCMA and CD3ε, characterized in that it comprises a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO: 20 and a combination of region CDR1H, CDR2H, CDR1L and CDR2L selected from the group of a) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22, CDR1L region of SEQ ID NO: 23 and CDR2L region of SEQ ID NO: 24, b) CDR1H region of SEQ ID NO: 21 and CDR2H region from SEQ ID NO: 22, CDR1L region from SEQ ID NO: 25 and CDR2L region from SEQ ID NO: 26, c) CDR1H region from SEQ ID NO: 21 and CDR2H region from SEQ ID NO : 22, CDR1L region of SEQ ID NO: 27 and CDR2L region of SEQ ID NO: 28, d) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30, CDR1L region of SEQ ID NO: 31 and CDR2L region of SEQ ID NO: 32, e) CDR1H region of SEQ ID NO: 34 and CDR2H region of SEQ ID NO: 35, CDR1L region of SEQ ID NO: 31 and CDR2L region of SEQ ID NO: 32, and f) region CDR1H of SEQ ID NO: 36 and CDR2H region of SEQ ID NO: 37, CDR1L region of SEQ ID NO :31 and CDR2L region of SEQ ID NO:32.
[000227] 16. A bispecific antibody that specifically binds to the two targets that are the extracellular domain of human BCMA (also called "BCMA") and human CD3 (also called "CD3"), characterized in that it comprises a VH region comprising a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ ID NO:22 and a CDR3H region of SEQ ID NO:17, and a VL region comprising a CDR3L region of SEQ ID NO:20 and a combination of the CDR1L regions and CDR2L selected from the group of a) CDR1L region of SEQ ID NO: 23 and CDR2L region of SEQ ID NO: 24, b) CDR1L region of SEQ ID NO: 25 and CDR2L region of SEQ ID NO: 26, or c ) CDR1L region of SEQ ID NO:27 and CDR2L region of SEQ ID NO:28.
[000228] 17. The bispecific antibody according to modality 15 or 16, characterized in that it comprises as VH region a VH region of SEQ ID NO: 10.
[000229] 18. The bispecific antibody according to any one of embodiments 15 to 16, characterized in that the BCMA VL is selected from the group consisting of VL regions of SEQ ID NO: 12, 13 and 14.
[000230] 19. The bispecific antibody according to any one of embodiments 14 to 18, characterized in that it comprises as BCMA VH region a VH region of SEQ ID NO: 10, and as VL region a VL region of SEQ ID NO: 12; or as BCMA VH region a VH region from SEQ ID NO:10, and as VL region a VL region from SEQ ID NO:13; or as a BCMA VH region a VH region from SEQ ID NO:10, and as a VL region a VL region from SEQ ID NO:14.
[000231] 20. The bispecific antibody according to either modality 15 or 19, characterized in that it comprises amino acid 49 of the VL region being selected from the group of amino acids tyrosine (Y), glutamic acid (E), serine (S ) and histidine (H).
[000232] 21. The bispecific antibody according to any one of embodiments 15 to 20, characterized in that amino acid 74) of the VL region is threonine (T) or alanine (A).
[000233] 22. A monoclonal antibody that specifically binds BCMA and CD3 characterized in that it comprises a VH region comprising a CDR3H region of SEQ ID NO: 17 and a VL region comprising a CDR1L region of SEQ ID NO: 31, a CDR2L region of SEQ ID NO: 32 and a CDR3L region of SEQ ID NO: 20 and a combination of CDR1L and CDR2L region selected from the group of a) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30, b) CDR1H region of SEQ ID NO: 34 and CDR2H region of SEQ ID NO: 35, or c) CDR1H region of SEQ ID NO: 36 and CDR2H region of SEQ ID NO: 37.
[000234] 23. A bispecific antibody according to embodiment 22, characterized in that it comprises a VL region of SEQ ID NO: 12 and a VH region selected from the group comprising the VH regions of SEQ ID NO: 38, 39 and 40.
[000235] 24. The bispecific antibody according to modality 22 or 23, characterized in that it comprises amino acid 49 of the VL region being selected from the group of amino acids tyrosine (Y), glutamic acid (E), serine (S) and histidine (H).
[000236] 25. The bispecific antibody according to any one of embodiments 22 to 24, characterized in that amino acid 74 of the VL region is threonine (T) or alanine (A).
[000237] 26. The bispecific antibody according to any one of embodiments 15 to 25, characterized in that it comprises an anti BCMA antibody according to the invention and an anti CD3 antibody, wherein a) light chain and heavy chain of an antibody of according to any one of modalities 1 to 7; and b) the light chain and heavy chain of an antibody that specifically binds to CD3, wherein the variable domains VL and VH or the constant domains CL and CH1 are substituted for one another.
[000238] 27. Bispecific antibody, according to any one of embodiments 15 to 26, characterized in that it comprises no more than one Fab fragment of an anti-CD3 antibody portion, no more than two Fab fragments of a portion of anti-BCMA antibody and no more than one Fc part.
[000239] 28. Bispecific antibody, according to any one of embodiments 15 to 27, characterized in that it comprises an Fc part linked with its N-terminus to the C-terminus of said Fab fragment of CD3 antibody and to the C-terminus of one of said fragments BCMA antibody Fab.
[000240] 29. The bispecific antibody according to any one of embodiments 15-28, characterized in that it comprises a second Fab fragment of said anti-BCMA antibody (BCMA antibody portion) linked with its C-terminus to the N-terminus of said fragment Fab of said anti-CD3 antibody (CD3 antibody portion) of said bispecific antibody.
[000241] 30. Bispecific antibody, according to modality 29, characterized in that the VL domain of said anti-CD3 antibody Fab fragment is linked to the CH1 domain of said second anti-BCMA antibody Fab fragment.
[000242] 31. Bispecific antibody, according to any one of modalities 15 to 30, characterized by the fact that the VH variable domain of the anti-CD3 antibody portion (further called "CD3 VH") is the heavy chain CDRs of the SEQ ID NO:1, 2 and 3, as well as heavy chain CDR1, CDR2 and CDR3 respectively, and the variable domain VL of the anti-CD3 antibody portion (further called "CD3 VL") comprises the light chain CDRs of SEQ ID NO: 4, 5 and 6 as well as light chain CDR1, CDR2 and CDR3 respectively.
32. The bispecific antibody according to any one of embodiments 15 to 31, characterized in that the variable domains of the anti-CD3 antibody portion are of SEQ ID NO: 7 and 8.
33. A bispecific antibody that specifically binds to the two targets that are the extracellular domain of human BCMA and human CD3ε, characterized in that it comprises a) the first light chain and the first heavy chain of a first antibody according to either of claims 1 to 7; and b) the second light chain and the second heavy chain of a second antibody which specifically binds to CD3, and wherein the VL and VH variable domains in the second light chain and second heavy chain of the second antibody are substituted for each other; and c) where in the constant domain CL of the first light chain under which a) the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and where , in the CH1 constant domain of the first heavy chain under which a) the amino acid at position 147 and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat ) (see for example Figures 1A, 2A, 2C, 3A, 3C).
[000245] 34. Bispecific antibody specifically according to claim 33, characterized in that it additionally comprises a Fab fragment of said first antibody (also called "BCMA-Fab") and in the constant domain CL of said BCMA-Fab, the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat), and wherein, in the constant domain CH1 of said BCMA-Fab, the amino acid at positions 147 and the amino acid at position 213 are independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat) (see, for example, Figures 2A and 2C).
[000246] 35. A bispecific antibody that specifically binds to the two targets that are the extracellular domain of human BCMA and human CD3ε, characterized in that it comprises a) the first light chain and the first heavy chain of a first antibody according to either of claims 1 to 7; and b) the second light chain and the second heavy chain of a second antibody which specifically binds to CD3, and wherein the VL and VH variable domains in the second light chain and second heavy chain of the second antibody are substituted for each other; and wherein c) in the constant domain CL of the second light chain under which b) the amino acid at position 124 is independently substituted by lysine (K), arginine (R) or histidine (H) (numbering according to Kabat) and in that, in the CH1 constant domain of the second heavy chain under which b) the amino acid at position 147 and the amino acid at position 213 is independently substituted by glutamic acid (E) or aspartic acid (D) (numbering according to Kabat).
[000247] 36. A bispecific antibody that specifically binds to two targets which are the extracellular domain of human BCMA and human CD3ε, characterized in that it comprises a set of heavy and light chains selected from the group consisting of polypeptides
[000248] SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51 (2x); (set 1 TCB of antibody 21),
[000249] SEQ ID NO: 48, SEQ ID NO: 52, SEQ ID NO: 53 and SEQ ID NO: 54 (2x) (set 2 TCB antibody 22), and
[000250] SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 (2x) (set 3 TCB of antibody 42).
[000251] 37. Method for the preparation of an antibody, according to any one of claims 1 to 36, characterized in that it comprises the steps of a) transforming a host cell with b) vectors comprising nucleic acid molecules encoding the light chain and heavy chain of an antibody according to any one of claims 1 to 36, c) culturing the host cell under conditions which permit the synthesis of said antibody molecule; and d) recovering said antibody molecule from said culture.
38. A host cell comprising vectors comprising nucleic acid molecules encoding an antibody according to any one of claims 1 to 36.
A pharmaceutical composition comprising an antibody according to any one of claims 1 to 36 and a pharmaceutically acceptable excipient.
[000254] A pharmaceutical composition comprising an antibody according to any one of claims 1 to 36 for use as a medicine.
[000255] 41. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 36 for use as a medicament in the treatment of plasma cell disorders.
[000256] A pharmaceutical composition comprising an antibody according to any one of claims 1 to 36 for use as a medicament in the treatment of multiple myeloma or plasma cell leukemia or AL-amyloidosis
[000257] 43. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 36 for use as a medicament in the treatment of systemic lupus erythematosus.
44. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 36, including a monospecific antibody, an improved naked ADCC antibody, an antibody-drug conjugate or a bispecific antibody for use as a a drug in the treatment of antibody-mediated rejection.
[000259] 45. Chimeric antigen receptor (CAR), comprising: an antigen recognition unit directed against BCMA and a T cell activation unit, characterized in that the antigen recognition unit is a monoclonal antibody or antibody fragment according to any one of embodiments 1 to 14.
[000260] 46. A chimeric antigen receptor (CAR) according to modality 45, characterized in that it comprises: (i) a B cell maturation antigen recognition (BCMA) portion; (ii) a spacer domain; and (iii) a transmembrane domain; and (iv) an intracellular T cell signaling domain.
[000261] 47. A chimeric antigen receptor (CAR) according to modality 45 or 46, characterized in that the antigen recognition portion is a monoclonal antibody or antibody fragment specifically linked to BCMA, characterized in that it comprises a CDR3H region of SEQ ID NO: 17 and a CDR3L region of SEQ ID NO: 20 and a combination of CDR1H, CDR2H, CDR1L and CDR2L region selected from the group of a) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22 , CDR1L region of SEQ ID NO:23, and CDR2L region of SEQ ID NO:24, b) CDR1H region of SEQ ID NO:21 and CDR2H region of SEQ ID NO:22, CDR1L region of SEQ ID NO:25 and region CDR2L of SEQ ID NO: 26, c) CDR1H region of SEQ ID NO: 21 and CDR2H region of SEQ ID NO: 22, CDR1L region of SEQ ID NO: 27 and CDR2L region of SEQ ID NO: 28, d) CDR1H region of SEQ ID NO: 29 and CDR2H region of SEQ ID NO: 30, CDR1L region of SEQ ID NO: 31 and CDR2L region of SEQ ID NO: 32, e) CDR1H region of SEQ ID NO: 34 and CDR2H region of SEQ ID NO: 35, region the CDR1L of SEQ ID NO: 31 and CDR2L region of SEQ ID NO: 32, and f) CDR1H region of SEQ ID NO: 36 and CDR2H region of SEQ ID NO: 37, CDR1L region of SEQ ID NO: 31 and CDR2L region of SEQ ID NO: 32.
[000262] 48. An isolated or purified nucleic acid sequence encoding a chimeric antigen receptor (CAR) according to any one of embodiments 45 to 47.
[000263] 49. Method of generating a monoclonal antibody that specifically binds to BCMA, which depletes as a bispecific antibody according to any one of modalities 15 to 36, from human malignant plasma cells in bone marrow aspirates of Multiple Myeloma MM in a manner of at least 80% after a 48 hour treatment at a concentration between 10 nM and 1 fM, and of anti-BCMA antibody, characterized by the fact that a) moving a chain phage display library variable heavy (VH) of SEQ ID NO: 9 with BCMA from 1 to 50 nM of BCMA from cynomolgus monkeys in 1-3 rounds and select a variable heavy chain which, when combined with the variable light chain of SEQ ID NO: 11 for a bispecific antibody according to any one of embodiments 15 to 36, depletes such human malignant plasma cells thus, b) moving a variable light chain (VL) phage display library of SEQ ID NO: 11 with 1- 50 nM of BCMA from cynomolgus monkeys in 1-3 rounds and b) selecting a variable light chain which, when combined with the variable heavy chain of SEQ ID NO: 9 for a bispecific antibody according to any one of modalities 15 to 36, depletes such human malignant plasma cells in this way , and
[000264] combine said selected variable heavy chain and selected variable light chain up to a bispecific antibody according to any one of embodiments 4 to 16, which depletes these human malignant plasma cells in this way.
[000265] 50. Pharmaceutical composition, characterized in that it comprises an antibody according to any one of claims 1 to 36 and 45 to 47 for use as a medicine in the treatment of multiple myeloma or systemic lupus erythematosus or cell leukemia plasmatic or AL-amyloidosis.
[000266] In an antibody binding modality according to the invention is not reduced by 100 ng/ml of APRIL for more than 20%, measured in an ELISA assay as OD at 405 nm compared to the binding of said antibody to the Human BCMA without APRIL does not alter APRIL-dependent NF-kB activation by more than 20% compared to APRIL, and does not alter NF-kB activation without APRIL by more than 20% compared to without said antibody .
[000267] In one embodiment, antibody binding at a concentration of 6.25 nM is not reduced by 140 ng/ml murine APRIL by more than 10%, preferably not reduced by more than 1% as measured in an ELISA assay as OD at 450 nm compared to binding of said antibody to human BCMA without APRIL. Binding of said antibody at a concentration of 50 nM is not reduced by 140 ng/ml murine APRIL for more than 10%, measured in an ELISA assay as OD at 450 nm, compared to binding of said antibody to human BCMA without APRIL.
[000268] In one embodiment, the binding of said antibody is not reduced by 100 ng/ml APRIL and not reduced by 100 ng/ml BAFF by more than 20%, measured in an ELISA assay as OD at 405 nm compared to binding of said antibody to human BCMA without APRIL or BAFF, respectively, the antibody does not alter APRIL-dependent NF-kB activation by more than 20%, compared to APRIL alone, does not alter NF-kB activation BAFF-dependent by more than 20% compared to BAFF alone, and does not alter NF-kB activation without BAFF and APRIL by more than 20% compared to without said antibody.
[000269] In one embodiment, binding of said antibody to human BCMA is not reduced by 100 ng/ml APRIL by more than 15% as measured in said ELISA, not reduced by 1000 ng/ml APRIL by more than 20%, measured in said ELISA, and not reduced by 1000 ng/ml APRIL by more than 15% as measured in said ELISA.
[000270] In one embodiment, the binding of said antibody to human BCMA is not reduced by 100 ng/ml APRIL and not reduced by 100 ng/ml BAFF by more than 15% as measured in said ELISA, not reduced by 1000 ng /ml APRIL and not reduced by 1000 ng/ml BAFF, by more than 20%, measured in the above-mentioned ELISA, not reduced by 1000 ng/ml APRIL and not reduced by 1000 ng/ml BAFF by more than 15%, measured in said ELISA.
[000271] In one embodiment, the antibody according to the invention does not change APRIL-dependent NF-kB activation by more than 15%, does not change BAFF-dependent NF-kB activation by more than 15%, and does not change NF-KB activation without APRIL and BAFF by more than 15%.
[000272] In one embodiment, antibody binding to BCMA is not reduced by APRIL, not reduced by BAFF by more than 25%, not more than 20% and not more than 10%, measured as the binding of said antibody in a concentration of 5 nM, preferably 50 nM and 140 nM for NCI-H929 cells (ATCC® CRL-9068 ™) in the presence or absence of APRIL or respectively BAFF at a concentration of 2.5 µg/ml compared to binding of said antibody to NCI-H929 cells lacking APRIL or BAFF, respectively.
[000273] In one embodiment the following examples, sequence listing and figures are provided to aid understanding of the present invention, the true scope of which is defined in the appended claims. It is understood that modifications can be made to the presented procedures without departing from the spirit of the invention. MATERIALS AND GENERAL METHODS Recombinant DNA Techniques
Standard methods were used to manipulate the DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to manufacturer's instructions. General information on the nucleotide sequences of human immunoglobulin light and heavy chains is given in: Kabat, E.A. et al., (1991) Sequences of Proteins of Immunological Interest, 5th ed., NIH Publication No. 91-3242. Antibody chain amino acids were numbered and referenced according to Kabat, E.A., et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, (1991). Gene synthesis a) Desired gene segments were prepared from oligonucleotides made by chemical synthesis. The 600 to 1800 bp long gene segments, which were bound by unique restriction endonuclease cleavage sites, were assembled by annealing and ligation of oligonucleotides including PCR amplification and subsequently cloned through the indicated restriction sites, e.g., Kpnl /Sad or Ascl/Pacl in a pPCRScript (Stratagene) based on the pGA4 cloning vector. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing. The gene synthesis fragments were ordered according to certain specifications in Geneart (Regensburg, Germany). b) The desired gene segments, where necessary, were generated by PCR using appropriate templates or were synthesized by Geneart AG (Regensburg, Germany) from synthetic oligonucleotides and PCR products by automated gene synthesis. Gene segments flanked by unique restriction endonuclease cleavage sites were cloned into standard expression vectors or sequencing vectors for further analysis. Plasmid DNA was purified from transformed bacteria using commercially available plasmid purification kits. Plasmid concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. The gene segments were designed with suitable restriction sites to allow subcloning into the respective expression vectors. If necessary, protein-coding genes were designed with a 5'-end DNA sequence encoding a leader peptide that targets proteins for secretion in eukaryotic cells. DNA Sequence Determination
[000275] DNA sequences were determined by double-stranded sequencing. DNA and protein sequence analysis and sequence data management
[000276] The Clone Manager software package (Scientific and Educational Software) version 9.2 was used for sequence mapping, analysis, annotation and illustration. Expression vectors a) Fusion genes comprising the antibody chains described as described below were generated by PCR and/or gene synthesis and assembled with known recombinant methods and techniques by ligating the appropriate nucleic acid segments, for example, using unique restriction sites in the respective vectors. Subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfections, larger amounts of plasmids are prepared by preparing plasmids from transformed E. coli cultures (Nucleobond AX, Macherey-Nagel). b) For the generation of anti-BCMA antibody expression vectors, the variable regions of heavy and light chain DNA sequences were subcloned in frame with human IgG1 constant heavy chain or pre-human IgG1 constant light chain. - inserted into the respective generic receptor expression vector optimized for expression in mammalian cell lines. Antibody expression is driven by a chimeric MPSV promoter comprising a CMV enhancer and an MPSV promoter followed by a 5' UTR, an intron and an Ig kappa MAR element. Transcription is terminated by a synthetic polyA signal sequence at the 3' end of the CDS. All vectors have a 5'-end DNA sequence that encodes a leader peptide that targets proteins for secretion into eukaryotic cells. In addition, each vector contains an EBV OriP sequence for episomal plasmid replication in EBNA EBV expressing cells. c) For the generation of BCMAxCD3 bispecific antibody vectors, IgG1-derived bispecific molecules consist of at least two antigen-binding moieties capable of specifically binding to two distinct antigenic determinants CD3 and BCMA. Antigen-binding portions are Fab fragments composed of a heavy and a light chain, each comprising a variable region and a constant region. At least one of the Fab fragments was a "Crossfab" fragment, in which VH and VL were exchanged. The exchange of VH and VL within the Fab fragment ensures that Fab fragments of different specificity do not have identical domain arrangements. The design of the bispecific molecule was monovalent for CD3 and bivalent for BCMA when a Fab fragment was fused to the N-terminus of the internal CrossFab (2+1). The bispecific molecule contained an Fc part so that the molecule has a long half-life. A schematic representation of the constructs is given in Figure 2; the preferred sequences of the constructs are shown in SEQ ID NOs 39 to 52. The molecules were produced by co-transfection of HEK293 EBNA cells grown in suspension with the mammalian expression vectors using polymer-based solution. For the preparation of CrossFab-IgG 2+1 constructs, cells were transfected with the corresponding expression vectors at a 1:2:1:1 ratio ("Fc(knob)":"vector light chain": "Vector Light Chain CrossFab": "Vector Light Chain-CrossFab"). Cell culture techniques
[000277] Standard cell culture techniques are used as described in Current Protocols in Cell Biology (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M. (eds.), John Wiley & Sons, Inc. Transient expression in HEK293 cells (HEK293-EBNA system)
[000278] Bispecific antibodies were expressed by transient co-transfection of the respective mammalian expression vectors in HEK293-EBNA cells, which were cultured in suspension, using a polymer-based solution. One day before transfection, HEK293-EBNA cells were seeded into viable cells of 1.5 Mio/ml in Ex-Cell medium, supplemented with 6 mM L-glutamine. For each ml of final production volume, 2.0 Mio of viable cells were centrifuged (5 minutes at 210 x g). The supernatant was aspirated and cells were resuspended in 100 µL of CD CHO medium. The DNA for each mL of final production volume was prepared by mixing 1 μg of DNA (ratio of heavy chain: modified heavy chain: light chain: modified light chain = 1: 1: 2: 1) in 100 μL of CD medium CHO. After addition of 0.27 µL of polymer-based solution (1 mg/mL), the mixture was vortexed for 15 seconds and left at room temperature for 10 minutes. After 10 minutes, the resuspended cells and the DNA/polymer-based solution mixture were pooled and then transferred to an appropriate container which was placed on a shaking device (37°C, 5% CO2). After an incubation period of 3 hours, 800 μL of Ex-Cell medium, supplemented with 6 mM L-Glutamine, 1.25 mM valproic acid and 12.5% Pepsoy (50 g/L) were added for each mL of final production volume. After 24 hours, 70 µL of feed solution was added for each mL of final production volume. After 7 days or when cell viability was equal to or less than 70%, cells were separated from the supernatant by centrifugation and sterile filtration. Antibodies were purified by an affinity step and one or two polishing steps, namely: cation exchange chromatography and size exclusion chromatography. When necessary, an additional polishing step was used. Recombinant human anti-BCMA antibody and bispecific antibodies were produced in suspension by co-transfecting HEK293-EBNA cells with mammalian expression vectors using a polymer-based solution. The cells were transfected with two or four vectors, depending on the format. For the human plasmid IgG1 encoded the heavy chain and the other plasmid the light chain. For bispecific antibodies, four plasmids were co-transfected. Two of them encoded the two different heavy chains and the other two encoded the two different light chains. One day before transfection, HEK293-EBNA cells were seeded into viable cells of 1.5 Mio/ml in F17 medium, supplemented with 6 mM L-glutamine. Protein determination
[000279] The determination of the antibody concentration was carried out by measuring the absorbance at 280 nm, using the theoretical value of the absorbance of a 0.1% solution of the antibody. This value was based on the amino acid sequence and calculated by the GPMAW (Lighthouse data) software. SDS-PAGE
[000280] The NuPAGE® Pre-Cast gel system (Invitrogen) is used according to the manufacturer's instructions. In particular, 10% or 412% of NuPAGE® Novex® Bis-TRIS precast gels (pH 6.4) and a NuPAGE® MES (reduced gels, with NuPAGE® Anti-oxidant buffer additive) or MOPS running buffer (non gels reduced) is used. protein purification By protein A affinity chromatography
[000281] For the affinity step, the supernatant was loaded onto a protein A column (HiTrap Protein A FF, 5 mL, GE Healthcare) equilibrated with 6 CV of 20 mM sodium phosphate, 20 mM sodium citrate mM, pH 7.5. After a washing step with the same buffer, the antibody was eluted from the column elution with 20 mM sodium phosphate, 100 mM sodium chloride and 100 mM glycine, pH 3.0. The portions with the desired antibody were immediately neutralized by 0.5 M sodium phosphate, pH 8.0 (1:10), pooled and concentrated by centrifugation. The concentrate was sterilized and further processed by cation exchange chromatography and/or size exclusion chromatography. By cation exchange chromatography
[000282] For the cation exchange chromatography step, the concentrated protein was diluted 1:10 with the elution buffer used for the affinity step and loaded onto a cation exchange column (Poros 50 HS, Applied Biosystems) . After two washing steps with the equilibration buffer and a wash buffer, respectively. 20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, pH 5.0 and 20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100 mM sodium chloride, pH 5.0. Protein was eluted with a gradient using 20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100 mM sodium chloride, pH 8.5. Portions containing the desired antibody were pooled, concentrated by centrifugation, sterile filtered and processed further to a size exclusion step. By analytical exclusion chromatography
[000283] For the size exclusion step, the concentrated protein was injected into an XK16/60 HiLoad Superdex 200 column and in 20 mM histidine, 140 mM sodium chloride, pH 6.0 with or without Tween20 as a buffer. formulation. The portions containing the monomers were pooled, concentrated by centrifugation and sterile filtered in a sterile flask. Measurement of purity and monomer content
[000284] The purity and monomer content of the final protein preparation were determined by CE-SDS (Caliper LabChip GXII system (Caliper Life Sciences)). HPLC (TSKgel G3000 SW XL (Tosoh) analytical size exclusion column) in a 25 mM potassium phosphate, 125 mM sodium chloride, 200 mM L-arginine monohydrochloride, 0.02% (w/ v) sodium azide, pH 6.7 buffer. Molecular weight confirmation by LC-MS analysis Deglycosylation
[000285] To confirm the homogeneous preparation of the molecules, the final protein solution was analyzed by LC-MS analyses. To remove heterogeneity introduced by carbohydrates, constructs are treated with PNGaseF (ProZyme). Thus, the pH of the protein solution was adjusted to pH 7.0 by adding 2 µl of 2 M Tris to 20 µg of protein at a concentration of 0.5 mg/ml. 0.8 µg PNGaseF was added and incubated for 12 h at 37°C. LC-MS Analysis - Online Detection
[000286] The LC-MS method was performed on an Agilent 1200 HPLC coupled to a TOF 6441 mass spectrometer (Agilent). Chromatographic separation was performed on a Macherey Nagel Polysterene column; RP1000-8 (8 µm particle size, 4.6 x 250 mm; Cat. No. 719510). Eluent A was 5% acetonitrile and 0.05% (v/v) formic acid in water, eluent B was 95% acetonitrile, 5% water and 0.05% formic acid. The fluid rate was 1 ml/min, separation was performed at 40°C and 6 μg (15 μl) of a protein sample obtained with a treatment as described above.

[000287] During the first 4 minutes, the eluate was directed to the waste to protect the mass spectrometer from salt contamination. The ESI source was operating with a drying gas flow of 12 L/min, a temperature of 350°C and a nebulizer pressure of 60 psi. MS spectra were obtained using a fragmenter voltage of 380 V and a mass range of 700 to 3200 m/z in the positive ion usage mode. MS data were acquired by instrument software from 4 to 17 minutes. Isolation of human PBMCs from blood
Peripheral blood mononuclear cells (PBMCs) were prepared by Histopaque density centrifugation from enriched lymphocyte preparations (buffy shells) obtained from local blood banks or fresh blood from healthy human donors. Briefly, blood was diluted with sterile PBS and carefully layered over a Histopaque gradient (Sigma, H8889). After centrifugation for 30 minutes at 450 xg at room temperature (brake off), part of the plasma above the PBP containing interference was discarded. The PBMCs were transferred to new 50 ml Falcon tubes and the tubes were filled with PBS to a total volume of 50 ml. The mixture was centrifuged at room temperature for 10 minutes at 400 xg (brake on). The supernatant was discarded and the PBMC pellet washed twice with sterile PBS (centrifugation steps at 4°C for 10 minutes at 350 xg). The resulting PBMC population was automatically counted (ViCell) and stored in RPMI1640 medium, containing 10% FCS and 1% L-alanyl-L-glutamine (Biochrom, K0302) at 37°C, 5% CO2 in the incubator until the beginning of the test. Isolation of primary cynomolgus PBMC from he-parinized blood
[000289] Peripheral blood mononuclear cells (PBMCs) were prepared by density centrifugation from fresh blood from healthy cynomolgus donors as follows: heparinized blood was diluted 1:3 with sterile PBS and Lymphoprep medium ( Axon Lab #1114545) was diluted to 90% with sterile PBS. Two volumes of the diluted blood were layered over one volume of the diluted density gradient and the PBMC portion was separated by centrifugation for 30 min at 520 xg, without brake, at room temperature. The PBMC band was transferred to a new 50 ml Falcon tube and washed with sterile PBS by centrifugation for 10 min at 400 xg at 4°C. A low speed centrifugation was performed to remove platelets (15 min at 150 xg, 4°C) and the resulting population of PBMC was automatically counted (ViCell) and immediately used for further assays. EXAMPLES Example 1: Generation of anti-BCMA antibodies Example 1.1: Production of tool antigens and reagents Example 1.1.1: Recombinant, soluble and human BCMA extracellular domain
[000290] The extracellular domains of human, cynomolgus and murine BCMA that were used as antigens for phage display selections were transiently expressed as N-terminal monomeric Fc-fusion in HEK cells EBNA and in vivo specifically biotinylated via co -expression of biotin BirA binds to the avi-marker recognition sequence located at the C-terminus of the Fc portion carrying the receptor chain (Fc button chain). The extracellular domains of human and cynomolgus BCMA comprised methionine 4 to asparagine 53 and methionine 4 to asparagine 52, respectively. These were N-terminally fused to the hinge of a human IgG1 that allows heterodimerization with an IgG1 Fc portion without human fusion (hole chain) by knobs-in-holes technology. Example 1.1.1A: Generation of anti-BCMA antibodies by maturation 1.1.1A.1 Libraries and Selections
[000291] Two libraries were constructed based on the 83A10 antibody. These libraries are randomized into light chain CDR1 and CDR2 (83A10 L1/L2) or heavy chain CDR1 and CDR2 (83A10 H1/H2), respectively. Each of these libraries was built by 2 subsequent steps of amplification and assembly. Final assembly products were NcoI/BsiWI digested to 83A10 L1/L2 library, MunI and NheI to 83A10 H1/H2 library, along with similarly treated acceptor vectors based on plasmid preparations from clone 83A10. The following amounts of digested randomized V (vascular) domains and digested acceptor(s) vector(s) were linked to the respective libraries (μg V domain/μg vector): am83A10 L1/L2 library (3/10) , 83A10 H1/library H2 (3/10). Purified ligations from the 83A10 L1/L2 and 83A10 H1/H2 libraries were pooled, respectively, and used for 15 E. coli TG1 cell transformations for each of the 2 libraries, to obtain the final library sizes of 2.44 x 1010 for the 83A10 L1/L2 library and 1.4 x 1010 for the am83A10 H1/H2 library. Phagemid particles displaying these Fab libraries were rescued and purified. 1.1.1A.2 Clone Selections
[000292] Selections were performed against the ectodomain of the human or cyno B cell maturation antigen (BCMA) to which they were cloned upstream of an Fc and an avi-tag. Prior to selections, the Fc depletor was coated onto neutrovidin plates at a concentration of 500 nM. Selections were made according to the following pattern:
[000293] binding of ~1012 phagemid particles from the library. 83A10 L1/L2 library or 83A10 H1/H2 library to Fc cluster immobilized for 1h, 2) transfer of unbound phagemid particles from library.83A10 L1/L2 library or 83A10 H1/H2 library to 50nM, 25nM, 10nM or 2nM human or cyno BCMA (depending on library and selection round) for 20min, 3) addition of streptavidin magnetic beads for 10min, 4) washing of magnetic streptavidin beads using 10 x 1 ml PBS/Tween® 20 and 10 x 1 ml of PBS, 5) elution of phage particles by addition of 1 ml of 100 mM TEA (triethylamine) for 10 min and neutralization by addition of 500 µl of 1 M Tris®/HCl pH 7.4 and 6) reinfection of log-phase E. coli TG1 cells, infection with Helperphage VCSM13 and subsequent PEG/NaCl precipitation of phagemid particles to be used in subsequent rounds of selection.
[000294] Selections were performed over 3 rounds and conditions were fitted into 5 rationalizations for each of the 2 libraries individually. In detail, the selection parameters were:
[000295] Streamline 1 (huBCMA 50nM for 1 cycle, cynoBCMA 25nM for cycle 2, 10 nm huBCMA for cycle 3), Streamline 2 (huBCMA 50nM for cycle 1, 10 nm huBCMA for cycle 2, huB-CMA 2nM for cycle 3), Streamline 3 (50 km huBCMA for cycle 1, 25nM CMNBCBC for cycle 2, 10nM cynoBCMA for cycle 3), Streamline 4 (50nM cinBCMA for cycle 1, 25nM cynoBCMA for cycle cycle 2, 10nM cynoBCMA for cycle 3), Streamline 5 (50nM cynoBCMA for cycle 1, 25nM cyanoBCMA for cycle 2, 10nM cyanoBCMA for cycle 3).
[000296] The heavy chains of Mab 21, Mab 22, Mab 33 and Mab 42 BCMA antibodies were derived from Streamline 5, which used only cynoBCMA. 1.1.1A.3 Screening method
Individual clones were bacterially expressed as 1 ml cultures in 96-well format and the supernatants were screened by ELISA. Specific binders were defined as signals greater than 5 x background for humans and cyno BCMA and signals less than 3 x background for Fc depletion. Neutravidin 96-well strip plates were coated with 10nM huBCMA, 10nM cyBCMA or 50nM Fc depletion followed by addition of bacterial supernatants containing Fab and detection of binding Fabs specifically via their Flag-tags using an antibody anti-Flag/HRP secondary. ELISA positive clones were bacterially expressed as 1 ml cultures in 96-well format and supernatants were subjected to a ProteOn kinetic sorting experiment. 500 positive clones were identified, most with similar affinity. 1.1.1A.4 surface plasma resonance screen with soluble Fabs and IgGs
[000298] 70 clones were tested by SPR. All experiments were performed at 25°C using PBST as running buffer (10 mM PBS, pH 7.4 and 0.005% (v/v) Tween®20). A ProteOn XPR36 biosensor equipped with GLC and GLM sensor chips and coupling reagents (10 mM sodium acetate, pH 4.5, sulfo-N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl) hydrochloride - carbodiimide [EDC] and ethanolamine) was purchased from BioRad Inc. (Hercules, CA). Immobilizations were performed at 30 µl/min on a GLM chip. pAb (goat) anti hu IgG, F(ab) 2 Specific Ab (Jackson) was coupled in the vertical direction using a standard amine coupling procedure: all six ligand channels were activated for 5 minutes with an EDC mixture (200 mM) and sulfo-NHS (50 mM). Immediately after activation of the surfaces, pAb (goat) specific antibodies anti hu IgG, F(ab) 2 (50 µg/ml, 10 mM sodium acetate, pH 5) were injected into all six channels for 5 min. Finally, the channels were blocked with a 5 min injection of 1 M ethanolamine HCl (pH 8.5). Final immobilization levels were similar across all channels, ranging from 11000 to 11500 RU. Fab variants were captured from e.coli supernaturants by simultaneous injection along five of the entire horizontal channels separated (30 μl/min) for 5 min and resulted in levels ranging from 200 to 900 RU, depending on concentration. of Fab in the supernatant; the conditioned medium was injected along the sixth channel to provide an "in-line" blank for dual reference purposes. One-shot kinetic measurements were performed by injecting a dilution series of human, cyno and mouse BCMA (50, 10, 2, 0.4, 0.08, 0 nM, 50 μl/min) for 3 min over of the vertical channels. Dissociation was monitored for 5 min. Kinetic data was analyzed in ProteOn Manager v. 2.1. Processing the reaction point data involved the application of an interspot reference and a double reference step using a blank buffer line (Myszka, 1999). The processed data from replicated one-shot injections were fitted to a simple 1:1 Langmuir binding model without mass transport (O'Shannessy et al., 1993).
[000299] For IgG measurements from supernatants of HEK productions in 6-well format, IgG variants were captured from HEK293 supernatants by simultaneous injection over five of the separate, entire horizontal channels (30 μl /min) for 5 min and resulted in levels ranging from 200 to 400 RU; the conditioned medium was injected along the sixth channel to provide an "in-line" blank for dual reference purposes. One-shot kinetic measurements were performed by injecting a dilution series of human, cyno and mouse BCMA (25, 5, 1, 0.2, 0.04, 0 nM, 50 µl/min) for 3 min over of the vertical channels. Dissociation was monitored for 5 min. Kinetic data were analyzed as described above. The OSK measurements are summarized in Table 2D; i/m, measurement inconclusive. Affinity with huBCMA was found between about 50 µm to 5 nM. Affinity with cynoBCMA was found between about 2 nM to 20 nM (few clones fall outside the range, see Figure 17). 1.1.1A5. Additional selection of HC and LC clones
[000300] Due to their experience, the inventors selected these 70 clones in addition to 27 clones based on their binding properties to huBCMA, cynoBCMA, murine CMMA and ratio, measured in different assays. Out of these clones, clones 4VH and 9VL were selected, resulting in 34 VH/VL combinations. The binding affinity in HEK-huBCMA cells was measured (Figure 18 and Table 2E). It was found that the binding of antibodies Mab 21, Mab 22, Mab 27, Mab 39 and Mab 42 to huBCMA on HEK cells was not significantly better than binding of Mab 83A10 to huBCMA-HEK cells. However, Mab21, Mab 22, Mab27, Mab33, Mab39 and Mab42 were selected because of their general properties such as affinity for huBCMA, cynoBCMA, binding as bispecific antibody to multiple myeloma cell lines positive to BCMA H929, L363 and RPMI-8226 by flow cytometry, killing potency of H929, L363 and RPMI-8226 myeloma cells, from patient bone marrow aspirate viable myeloma plasma cells and pharmacokinetics (PK)) and pharmacodynamic data (BCMA cell death) in cynomolgus monkeys. Table 2C: Antibody relationship with streamlines
Table 2D: One-shot-kinetic affinity measurements for human, cynomolgus and mouse BCMA
Table 2E: Binding of IgG variants in HEK-huBCMA cells
Example 1.2: BCMA Expressing Cells as Tools Example 1.2.1: Human Myeloma Cell Lines Expressing BCMA on Their Surface and Quantifying the Number of BCMA Receptors on the Cell Surface
[000301] BCMA expression was evaluated in five human myeloma cell lines (NCI-H929, RPMI-8226, U266B1, L-363 and JJN-3) by flow cytometry. NCI-H929 cells ((H929) ATCC® CRL-9068™) were grown in 80-90% RPMI 1640 with 10 to 20% heat-inactivated FCS and may contain 2 mM L-glutamine, 1 mM pyruvate sodium and 50 μM mercaptoethanol. RPMI-8226 cells ((RPMI) ATCC® CCL-155™) were grown in a medium containing 90% RPMI 1640 and 10% heat-inactivated FCS. U266B1 ((U266) ATCC® TIB-196TM) were grown in RPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose and 1500 mg/l L sodium bicarbonate and 15% heat-inactivated FCS Cell line L-363 (Leibniz Institute DSMZ - German collection of microorganisms and cell cultures, DSMZ No. ACC 49) was grown in 85% RPMI 1640 and 15% FCS disabled by heat. The JJN-3 cell line (DSMZ No. ACC 541) was grown in 40% Dulbecco MEM + 40% Iscove MDM + 20% heat deactivated FBS. Briefly, cells were collected, washed, counted for viability, resuspended at 50,000 cells/well of a 96-well round baseplate and incubated with anti-human BCMA antibody (Ab-cam, # ab54834, mouse IgG1) at 10 μg/ml for 30 min at 4°C (to avoid internalization). A mouse IgG1 was used as an isotype control (BD Biosciences, #554121). Cells were then centrifuged (5 min at 350 xg), washed twice and incubated with FITC-conjugated anti-mouse secondary antibody for 30 min at 4°C. At the end of the incubation time, cells were centrifuged (5 min at 350 xg), washed twice with FACS buffer, resuspended in 100 ul of FACS buffer and analyzed in a CantoII device with FACS Diva software. Relative quantification of BCMA receptor number on the surface membrane of myeloma cell lines H929, RPMI-8226, and U266B1 was assessed by QIFIKIT analysis (Dako, # K0078, following manufacturer's instructions). H929 cells expressed human BCMA with the highest density, up to 5-6 times greater than other myeloma cell lines. H929 is considered to be a myeloma cell line that expresses high BCMA compared to U266 and L363, which are myeloma cells that express medium/low BCMA, RPMI-8226, which are myeloma cells that express low BCMA and JJN-3, which are myeloma cells that express very low BCMA. Table 3 summarizes the relative number of BCMA receptor on the cell surface of the human multiple myeloma cell lines from each experiment (n = 5). Table 3: Quantification of BCMA receptor number on the membrane surface of human myeloma cell lines H929, L363, RPMI-8226, U266B1 and JJN-3
Example 2: BCMA binding assays: surface plasma resonance
[000302] Assessment of binding of anti-BCMA antibodies to recombinant BCMA by surface plasma resonance (SPR) as follows. All SPR experiments were performed in a Biacore T200 at 25°C with HBS-EP as a buffer run (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, Surfac- P20 at 0.005%, Biacore, Freiburg/Germany). The avidity of the interaction between anti-BCMA antibodies and Fc (kih) of recombinant BCMA (human and cynomolgus) was determined. Biotinylated recombinant humans and cynomolgus BCMA Fc(kih) were directly coupled to an SA chip following the instructions (Biacore, Freiburg/Germany). The level of immobilization ranged from 200 to 700 RU. Anti-BCMA antibodies were passed at a 2-fold concentration range (1.95 to 500 nM) with a flow of 30 µL/minute through the flow cells over 120 seconds. Dissociation was monitored for 180 seconds. Refractive index differences in mass were corrected by subtracting the response obtained in the reference flow cell. Here, anti-BCMA antibodies were flown onto an empty surface previously activated and deactivated as described in the standard amine coupling kit. Apparent kinetic constants were generated using the Biacore T200 Evaluation Software (vAA, Biacore AB, Upp-sala/Sweden), to fit the ratio equations for Langmuir binding to 1:1 by numerical integration, despite the bivalence of the interaction for purposes comparatives. The affinity of the interaction between anti-BCMA antibodies and recombinant human BCMA Fc(kih) was also determined. Anti-human Fab antibody (GE Healthcare) was coupled directly to a CM5 chip at pH 5.0 using the standard amine coupling kit (Biacore, Freiburg/Germany). The level of immobilization was around 6500 RU. Anti-BCMA antibody was captured for 90 seconds at 25 nM. Recombinant human BCMA Fc(kih) was passed at a 4-fold concentration range (1.95 to 500 nM) with a flow rate of 30 µL/minute through the flow cells over 120 seconds. Dissociation was monitored for 120 seconds. Refractive index differences in mass were corrected by subtracting the response obtained in the reference flow cell. Here, the recombinant BCMA was flown onto a surface with immobilized anti-human Fab antibody, but the HBS-EP was injected instead of the anti-BCMA antibody. Kinetic constants were generated using the Biacore T100 Evaluation Software (vAA, Biacore AB, Uppsala/Sweden) to fit the ratio equations for Langmuir binding to 1:1 by numerical integration (Table 4). Table 4. Affinity constants determined by fit rate equations for 1:1 Langmuir binding

Example 3: human/cynomolgus (hu/cyno) affinity range
[000303] Based on the affinity values described in Example 2, the affinity of the anti-BCMA antibodies to the human BCMA compared to the cynomolgus BCM was compared and the cino/hu affinity ratio (range) values were calculated ( Table 5). The cino/hu affinity phosphate was calculated as the antibody's affinity to the cynomolgo BCMA divided by the affinity to the human BCMA and means that the BCMA antibody binds to the human BCMA with an affinity binding affinity of x times more than the BCMA of cynomolgo, where x = value of the range of cyno/hu. The results are shown in Table 5. Table 5: Affinity of anti-BCMA antibodies to human BCMA compared to cynomolgus BCMA and hu/kino range values
Example 4: Generation of bispecific anti-BCMA/anti-CD3 T cell antibodies
[000304] Bispecific anti-BCMA/anti-CD3 T cell antibodies were generated according to WO2014/122144, which is incorporated by reference. Example 4.1: anti-CD3 antibodies
[000305] The term "CD3ε or CD3", as used herein, refers to the human CD3ε described in UniProt P07766 (CD3E_HUMAN). The term "antibody to CD3, antibody to CD3" refers to a binding of the antibody to CD3ε. Preferably, the antibody comprises a variable domain VH comprising the heavy chain CDRs of SEQ ID NO:1, 2 and 3 as respectively the heavy chain CDR1, CDR2 and CDR3 and a variable domain VL comprising the light chain CDRs of SEQ ID NO: 4, 5 and 6 as light chain CDR1, CDR2 and CDR3 respectively. Preferably, the antibody comprises the variable domains of SEQ ID NO: 7 (VH) and SEQ ID NO: 8 (VL). The anti-CD3 antibody as described above was used to generate the bispecific T cell antibodies that were used in the examples below. Example 4.2: Generation of bispecific anti-BCMA/anti-CD3 T cell antibodies of 2+1 format containing Fc
The cDNAs encoding the complete heavy and light chains of the corresponding anti-BCMA IgG1 antibodies, as well as the anti-CD3 VH and VL cDNAs were used as starting materials. For each bispecific antibody, four protein chains comprising the corresponding anti-BCMA antibody heavy and light chains and the above-described anti-CD3 antibody heavy and light chains, respectively, were involved. In order to minimize the formation of by-products with inappropriate heavy chains, for example with two heavy chains of the anti-CD3 antibody, a modified heterodimeric Fc region bearing "pin-in-hole mutations" and a modified disulfide bond is used, as described in WO2009080251 and in WO2009080252. In order to minimize the formation of by-products with inappropriate light chains, for example with two anti-BCMA antibody light chains, a constant kappa x CH1 cross is applied to the anti-CD3 antibody heavy and light chains using the methodology described in WO2009080251 and in WO2009080252. a) A bispecific anti-BCMA/anti-CD3 T cell antibody with a 2+1 format, ie a bispecific antibody (Fab)2 x (Fab) that is bivalent to BCMA and monovalent to CD3 would have advantages in potency, predictability of efficacy and safety, because it preferentially binds to the BCMA of the tumor target and avoids the dissipation of CD3 antibodies, therefore, there is a greater probability of exposure to the drug focusing on the tumor.
Bispecific anti-BCMA/anti-CD3 T cells of the 2+1 format (i.e., the antibody (Fab)2 x (Fab) bivalent to BCMA and monovalent to CD3 with Fc) were produced for the previously selected human BCMA antibodies. cDNAs encoding the complete Fabs (heavy chain VH and CH1 domains plus light chain VL and CL domains) of the corresponding anti-BCMA IgG1 antibodies as well as the anti-CD3 VH and VL cDNAs were used as starting materials. as starting materials. For each bispecific antibody, four protein chains comprising the corresponding anti-BCMA antibody heavy and light chains and the above-described anti-CD3 antibody heavy and light chains, respectively, were involved with Fc regions.
[000308] In summary, each bispecific antibody is produced by simultaneous co-transfection of four mammalian expression vectors that encode, respectively: a) the complete light chain cDNA of the corresponding BCMA antibody, b) a fusion cDNA generated by methods conventional molecular biology techniques such as superposition-extension PCR, encoding a fusion protein made from the secretory leader sequence (in NC-terminal order), Fab (VH followed by CH1 domains) of the corresponding anti-BCMA antibody described above, a flexible glycine(Gly)-serine(Ser) linker with the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser, Fab (VH followed by CH1 domains of the anti antibody -Corresponding BCMA described above, a flexible glycine(Gly)-serine(Ser) linker with the sequence Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser, the VH of the described anti-CD3 antibody above and the constant kappa domain of a human light chain cDNA, c) a fusion cDNA generated by conventional methods of b molecular iology, such as the PC splice-overlay-extension package, the encoding of a fusion protein made from the secretory leader sequence (in NC order), the VL of the anti-CD3 antibody described above, the constant CH1 domain of a human IgG1 cDNA. Co-transfection of mammalian cells and antibody production and purification using the methods described above for the production of human or humanized IgG1 antibodies, with one modification: for antibody purification, the first cap-tura step is not done using protein A, but is made using an affinity chromatography column packed with a resin bond to the human kappa light chain constant region, such as KappaSelect (GE Healthcare Life Sciences). Furthermore, a disulfide can be included to increase stability and yields as well as additional residues that form ionic bridges and increase heterodimerization yields (EP 1870459A1).
[000309] For the generation of BCMAxCD3 bispecific antibody vectors, IgG1-derived bispecific molecules consist of at least two antigen-binding moieties capable of specifically binding to two distinct antigenic determinants CD3 and BCMA. The antigen-binding portions were Fab fragments composed of a heavy and a light chain, each comprising a variable region and a constant region. At least one of the Fab fragments was a "Crossfab" fragment, in which the Fab heavy and light chain constant domains were exchanged. The exchange of heavy and light chain constant domains within the Fab fragment ensures that Fab fragments of different specificity do not have identical domain arrays and therefore do not exchange light chains. The design of the bispecific molecule was monovalent for CD3 and bivalent for BCMA when a Fab fragment was fused to the N-terminus of the internal CrossFab (2+1). The bispecific molecule contained an Fc portion in order to have a longer half-life. A schematic representation of the constructions is given in Figures 1-3; the sequences of preferred constructs are shown in Table 2A. The molecules were produced by co-transfecting HEK293 EBNA cells grown in suspension with mammalian expression vectors using polymer-based solution. For the preparation of CrossFab-IgG 2+1 constructs, cells were transfected with the corresponding expression vectors at a 1:2:1:1 ratio ("Fc(knob) vector":"vector light chain ":"Vector Light Chain CrossFab": "Vector Light Chain-CrossFab"). Example 4.3: Generation of bispecific anti-BCMA/anti-CD3 T cell antibodies for comparison
[000310] The generation of bispecific anti-BCMA/anti-CD3 T cell antibody BCMA50-sc (Fv)2 (also known as BCMA50-BiTE®) and the amino acid sequences used were in accordance with WO2013072406 and WO2013072415. Example 5: Production and purification of bispecific T cell antibodies containing anti-BCMA/anti-CD3 Fc (2+1) with charge variants
[000311] Bispecific anti-BCMA/anti-CD3 T cell antibodies were produced and purified according to WO2014/122144, which is incorporated by reference.
[000312] For the production of bispecific antibodies, bispecific antibodies were expressed by transient co-transfection of the respective mammalian expression vectors in HEK293-EBNA cells, which were cultured in suspension, using a polymer-based solution. One day before transfection, HEK293-EBNA cells were seeded into viable cells at 1.5 Mio/ml in Ex-Cell medium, supplemented with 6 mM L-glutamine. For each ml of final production volume, 2.0 Mio of viable cells were centrifuged (5 minutes at 210 x g). The supernatant was aspirated and cells were resuspended in 100 µL of CD CHO medium. The DNA for each mL of final production volume was prepared by mixing 1 μg of DNA (ratio of heavy chain: modified heavy chain: light chain: modified light chain = 1: 1: 2: 1) in 100 μL of CD medium CHO. After addition of 0.27 µL of polymer-based solution (1 mg/mL), the mixture was vortexed for 15 seconds and left at room temperature for 10 minutes. After 10 minutes, the resuspended cells and the DNA/polymer-based solution mixture were pooled and then transferred to an appropriate container which was placed on a shaking device (37°C, 5% CO2). After an incubation period of 3 hours, 800 μL of Ex-Cell medium, supplemented with 6 mM L-Glutamine, 1.25 mM valproic acid and 12.5% Pepsoy (50 g/L) were added for each mL of final production volume. After 24 hours, 70 µL of feed solution was added for each mL of final production volume. After 7 days or when cell viability was equal to or less than 70%, cells were separated from the supernatant by centrifugation and sterile filtration. Antibodies were purified by an affinity step and one or two polishing steps, namely: cation exchange chromatography and size exclusion chromatography. When necessary, an additional polishing step was used.
[000313] For the affinity step, the supernatant was loaded onto a protein A column (HiTrap Protein A FF, 5 ml, GE Healthcare) equilibrated with 6 CV of 20 mM sodium phosphate, 20 mM sodium citrate, pH 7.5. After a washing step with the same buffer, the antibody was eluted from the column elution with 20 mM sodium phosphate, 100 mM sodium chloride and 100 mM glycine, pH 3.0. Fractions with the desired antibody were immediately neutralized by 0.5 M sodium phosphate, pH 8.0 (1:10), pooled and concentrated by centrifugation. The concentrate was sterilized and further processed by cation exchange chromatography and/or size exclusion chromatography.
[000314] For the cation exchange chromatography step, the concentrated protein was diluted 1:10 with the elution buffer used for the affinity step and loaded onto a cation exchange column (Poros 50 HS, Applied Biosystems) . After two wash steps with the equilibration buffer and a wash buffer, respectively. 20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, pH 5.0 and 20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100 mM sodium chloride, pH 5.0. Protein was eluted with a gradient using 20 mM sodium phosphate, 20 mM sodium citrate, 20 mM TRIS, 100 mM sodium chloride, pH 8.5. Fractions containing the desired antibody were pooled, concentrated by centrifugation, sterile filtered and further processed to a size exclusion step.
[000315] For the size exclusion step, the concentrated protein was injected onto an XK16/60 HiLoad Superdex 200 column and into 20 mM histidine, 140 mM sodium chloride, pH 6.0 with or without Tween20 as a buffer. formulation. The portions containing the monomers were pooled, concentrated by centrifugation and sterile filtered in a sterile flask.
[000316] The determination of the antibody concentration was performed by measuring the absorbance at 280 nm, using the theoretical value of the absorbance of a 0.1% solution of the antibody. This value was based on the amino acid sequence and calculated by the GPMAW (Lighthouse data) software.
[000317] The purity and monomer content of the final protein preparation were determined by CE-SDS (Caliper LabChip GXII system (Caliper Life Sciences)). HPLC (TSKgel G3000 SW XL (Tosoh) analytical size exclusion column) in a 25 mM potassium phosphate, 125 mM sodium chloride, 200 mM L-arginine monohydrochloride, 0.02% (w/ v) sodium azide, pH 6.7 buffer.
[000318] To check the molecular weight of the final protein preparations and confirm the homogeneous preparation of the final protein solution of the molecules, liquid chromatography mass spectrometry (LC-MS) was used. A first deglycosylation step was carried out. To remove the heterogeneity introduced by carbohydrates, the constructs were treated with PNGaseF (ProZyme). Thus, the pH of the protein solution was adjusted to pH 7.0 by adding 2 µl of 2 M Tris to 20 µg of protein at a concentration of 0.5 mg/ml. 0.8 μg PNGaseF was added and incubated for 12 h at 37 °C. Online detection of LC-MS was then performed. The LC-MS method was performed on an Agilent 1200 HPLC coupled to a TOF 6441 mass spectrometer (Agilent). Chromatographic separation was performed on a Macherey Nagel Polysterene column; RP1000-8 (8 µm particle size, 4.6 x 250 mm; Cat. No. 719510). The A eluent was 5% acetonitrile and 0.05% (v/v) formic acid in water, the B eluent was 95% acetonitrile, 5% water and 0.05% formic acid. The fluid rate was 1 ml/min, separation was performed at 40 °C and 6 μg (15 μl) of a protein sample obtained with a treatment as described above.
[000319] During the first 4 minutes, the eluate was directed to the waste to protect the mass spectrometer from salt contamination. The ESI source was operating with a drying gas flow of 12 L/min, a temperature of 350 °C and a nebulizer pressure of 60 psi. MS spectra were obtained using a fragmenter voltage of 380 V and a mass range of 700 to 3200 m/z in the positive ion usage mode. MS data were acquired by instrument software from 4 to 17 minutes.
[000320] Figure 10 of EP14179705 (incorporated by reference) depicts the CE-SDS plots (unreduced) of the final protein preparations after different purification methods for the 83A10-TCB and 83A10-TCBcv antibodies. The purification steps of protein A affinity chromatography (PA) and size exclusion chromatography (SEC) applied to the 83A10-TCB antibody resulted in a purity of <30% and 82.8% monomer (A) content. When additional purification steps, including cation exchange chromatography (cIEX) steps and final size exclusion chromatography (re-SEC) steps, were added to the final protein preparations in (A), the purity was increased to 93, 4%, but the monomer content remained the same and the yield was significantly reduced to 0.42 mg/L. However, when specific charge modifications were applied to the 83A10 anti-BCMA Fab CL-CH1, i.e. the 83A10-TCBcv antibody, a superior production/purification profile of the TCB molecule, as demonstrated by a purity of 95 .3%, 100% monomer content and a yield of up to 3.3 mg/L have already been observed even when PA + cIEX + SEC purification steps were applied (C) compared to (B) a profile production/purification showing a 7.9 fold lower yield and 17.2% less monomer content despite including an additional re-SEC purification step.
[000321] A direct production run to compare the production/purification profile of the 83A10-TCB vs. 83A10-TCBcv was then performed to further evaluate the advantages of modifications of the CL-CH1 load applied to the antibodies. The 83A10-TCB and 83A10-TCBcv molecules are both molecularly shaped, as described in Figure 2a. As shown in Figure 11, the properties of the 83A10-TCB and 83A10-TCBcv antibodies were measured side by side and compared after each purification step 1) PA only affinity chromatography (A, B), 2) PA affinity chromatography, in then SEC (C, D) and 3) PA affinity chromatography, then SEC, then cIEX and re-SEC (E, F). CE-SDS (non-reduced) graphs of the final protein solutions after the respective purification methods for the 83A10-TCB and 83A10-TCBcv antibodies are shown in Figure 11 of EP14179705 (incorporated by reference). As shown in Figures 11A and 11B of EP14179705 (incorporated by reference), improvements with applying the charge variants to the TCB antibody were already seen after purification by PA affinity chromatography alone. In this direct study, the PA affinity chromatography purification step applied to the 83A10-TCB antibody resulted in a purity of 61.3%, a yield of 26.2 mg/L and 63.7% of the monomer content (11A ). In comparison, when the 83A10-TCBcv antibody was purified by PA affinity chromatography, all properties were improved with a better purity of 81.0%, a better yield of 51.5 mg/L and 68.2% of monomer content (11B). When an additional SEC purification step was applied to the final protein preparations as seen in Figures 12A and 12B of EP14179705 (incorporated by reference), 83A10-TCB achieved a purity of 69.5%, a yield of 14.1 mg /L and 74.7% monomer content (C) compared to 83A10-TCBcv with improved purity and monomer content up to 91.0% and 83.9%, respectively, and a yield of 10.3 mg/L (D). Even though the yield was slightly lower (ie 27% lower) for 83A10-TCBcv than for 83A10-TCB, in this particular experiment, the correct molecular percentage was much better for 83A10-TCBcv than for 83A10- TCB, respectively, 90% vs. 40-60% as measured by LC-MS. In the third direct comparison, the final protein preparations of 83A10-TCB and 83A10-TCBcv from Figures 11C and 11D of EP14179705 (incorporated by reference) were pooled with approximately 1L (equivalent) of the respective final protein preparations from another batch of purification (same production) after the PA affinity chromatography purification step only. The pooled protein preparations were further purified by CIEX and SEC purification methods. As illustrated in Figures 11E and 11F of EP14179705 (incorporated by reference), improvement in the production/purification profile of TCB antibody with charge variants compared to TCB antibody without charge variant was consistently observed. After several steps of purification methods (ie PA +/- SEC + cIEX + SEC) were used to purify the 83A10-TCB antibody, only 43.1% purity was achieved and 98.3% of the monomer content could be achieved, at the expense of the yield which was reduced to 0.43 mg/L. The percentage of correct molecule measured by LC-MS was still scarce, at 60-70%. In the end, the quality of the final protein preparation was not acceptable for in vitro use. In contrast, when the same multiple purification steps with the same timeline were applied to the 83A10-TCBcv antibody, 96.2% purity and 98.9% monomer content were achieved, as well as 95% of the correct molecule measured. by LC-MS. The yield, however, was also greatly reduced to 0.64 mg/L after the cIEX purification step. The results show that higher purity, higher monomer content, higher percentage of correct molecule and higher yield can be achieved with the 83A10-TCBcv antibody only after two standard purification steps, ie, by PA affinity chromatography and SEC (Figure 11D of EP14179705), although such properties could not be obtained with 83A10-TCB even when additional purification steps were applied (Figure 11E of EP14179705).
[000322] Table 12 of EP14179705 (incorporated by reference) summarizes the properties of 83A10-TCB compared to 83A10-TCVcv after the PA purification step. Table 13 of EP14179705 (incorporated by reference) summarizes the properties of 83A10-TCB compared to 83A10-TCVcv after the PA and SEC purification step. Table 14 of EP14179705 (incorporated by reference) summarizes the properties of 83A10-TCB compared to 83A10-TCVcv after the PA and SEC plus PA steps alone and then the cI-EX and re-SEC purification steps. For those of Tables 12 to 14 of EP14179705 (incorporated by reference), the values in bold highlight the superior property in the comparison between 83A10-TCB and 83A10-TCVcv. With one exception (ie the respective yield amount, see Table 13 of EP14179705 (incorporated by reference)) which may not be representative, all production/purification parameters and values resulting from the 3 direct comparison experiments were superior for 83A10-TCBcv compared to 83A10-TCB. The overall results clearly demonstrate that advantages in production/purification characteristics could be achieved by applying CL-CH1 charge modifications to TCB antibodies and that only two purification steps (ie PA affinity chromatography) and SEC) were necessary to obtain already high quality protein preparations with very good developmental properties. Improved production/purification properties of 83A10-TCBcv, 21-TCBcv, 22-TCBcv, 27-TCBcv, 33-TCBcv, 39-TCBcv and 42-TCBcv were generated with charge variants, similarly to 83A10-TCBcv . Table 6: Production/purification profile of anti-BCMA/anti-CD3 T cell bispecific antibodies after protein A affinity chromatography purification step
Table 7: Production/purification profile of bispecific anti-BCMA/anti-CD3 T cell antibodies after protein A affinity chromatography purification steps and size exclusion chromatography purification steps
Table 8: Production/purification profile of bispecific anti-BCMA/anti-CD3 T cell antibodies after steps of 1.a) purification from protein A affinity chromatography and size exclusion chromatography and 1.b) chromatography protein A affinity only pooled and after 2) cation exchange chromatography and 3) end size exclusion chromatography purification steps
Example 6: Binding of anti-BCMA/anti-CD3 T cell bispecific antibodies to BCMA positive multiple myeloma cell lines (flow cytometry)
Anti-BCMA/anti-CD3 TCB antibodies (21-TCBcv, 22-TCBcv, 42-TCBcv, 83A10-TCBcv) were analyzed by flow cytometry for binding to human BCMA in H929, L363 and RPMI-8226 cells that express BCMA. MKN45 (human gastric adenocarcinoma cell line that does not express BCMA) was used as a negative control. Briefly, cultured cells are collected, counted and cell viability was assessed using ViCell. Viable cells are then adjusted to 2 x 106 cells per ml in FACS dye buffer containing BSA (BD Biosciences). 100 µl of this cell suspension was further aliquoted per well into a 96-well round-bottom plate and incubated with 30 µl of the corresponding anti-BCMA antibodies or IgG control for 30 min at 4°C. All anti-BCMA/anti-CD3 TCB antibodies (and TCB controls) were titrated and analyzed in the final concentration range between 1 and 300 nM. The cells were then centrifuged (5 min, 350 xg), washed with 120 µl/well of FACS dye buffer (BD Biosciences), resuspended and incubated for a further 30 min at 4°C with an Fc-specific anti-human IgG Fc fragment from goat a fluorochrome-conjugated PE-conjugated AffiniPure F(ab')2 fragment (Jackson Immuno Research Lab; 109-116-170). Cells were then washed twice with a dye buffer (BD Biosciences), fixed using 100 µl of BD fixation buffer per well (#BD Biosciences, 554655) at 4°C for 20 min, resuspended in 120 µl of dye buffer. FACS and analyzed using BD FACS CantoII. Where applicable, EC50 values were calculated using Prism GraphPad (LaJolla, CA, USA) and EC50 values, indicating the antibody concentration required to achieve 50% maximal binding for anti-BCMA/anti-antibody binding. CD3 TCB for H929, L363 and RPMI-8226 cells summarized in Table 8, Table 9 and Table 10, respectively. Asterix denotes the estimated EC50 values as extrapolated and calculated by Prism software. EC50 values for the binding of 21-TCBcv cells to L363 and for the binding of 22-TCBcv cells to RPMI-8226 could not be estimated. Table 8: EC50 values for binding of anti-BCMA/anti-CD3 T cell bispecific antibodies to H929 multiple myeloma cells
Table 9: EC50 values for binding of anti-BCMA/anti-CD3 T cell bispecific antibodies to L363 multiple myeloma cells
Table 10: EC50 values for binding of anti-BCMA/anti-CD3 T cell bispecific antibodies to RMPI-8226 multiple myeloma cells
Example 7: Cytokine production from activated T cells after binding of anti-BCMA/anti-CD3 T cell bispecific antibodies to CD3 positive T cells and BCMA positive multiple myeloma cell lines (CBA assay assay cytokine release)
Anti-BCMA/anti-CD3 T cell bispecific antibodies are analyzed for their ability to induce de novo T cell-mediated cytokine production in the presence or absence of human myeloma cells expressing human BCMA (RPMI- 8226, JJN-3). In summary, human PBMCs are isolated from the buffy coat creams and 0.3 million cells per well is coated onto a 96-well round-bottom plate. Alternatively, 280 μl of whole blood from a healthy donor is plated per well of a 96-well deep plate. BCMA-positive tumor target cells are added to obtain a final E:T ratio of 10:1. Anti-BCMA/anti-CD3 TCB antibodies and controls are added to a final concentration of 0.1 pM-10 nM. After an incubation of up to 24 h at 37°C, 5% CO 2 , the assay plate is centrifuged for 5 min at 350 x g and the supernatant is transferred to a new 96-well depth plate for subsequent analysis. CBA analysis was performed on the FACS CantoII according to the manufacturer's instructions, using the Th1/Th2 Cytokine Kit II (BD # 551809) or the combination of the following CBA Flex Kits: human granzyme B (BD # 560304), Human IFN-Y Flex Kit (BD #558269), TNF-α Flex Kit (BD #558273) Human IL-10 Flex Kit (BD #558274) Human IL-6 Flex Kit (BD #558276) , Human IL-4 Flex Kit (BD #558272), Human IL-2 Flex Kit (BD #558270). Table 13 shows that 83A10-TCBcv induced a concentration-dependent increase in cytokine production and serine protease granzyme B, a marker of cytotoxic T cell function. Table 11 shows the EC50 values and the amount of cytokines/proteases secreted by anti-BCMA/anti-CD3 T cell bispecific antibody concentrations. Table 11. Secretion of cytokines and proteases induced by bispecific anti-BCMA/anti-CD3 T cell antibodies in the presence of RPMI-8226 cells
Example 8: Redirected T cell cytoplasia of H929 myeloma cells expressing elevated BCMA by anti-BCMA/anti-CD3 T cell bispecific antibodies (colorimetric LDH release assay)
[000325] Anti-BCMA/anti-CD3 TCB antibodies were analyzed for their potential to induce T cell mediated apoptosis in BCMA high expressing MM cells after cross-linking the construct by binding the antigen-binding moieties to the BCMA in cells. Briefly, high expressing human BCMA H929 multiple myeloma target cells were collected with cell dissociation buffer, washed and resuspended in RPMI supplemented with 10% fetal bovine serum (Invitrogen). Approximately 30,000 cells per well were plated in a 96-well round-bottom plate and the respective construct dilution was added to a desired final concentration (in triplicate); final concentrations ranging from 0.1 pM to 10 nM. For proper comparison, all TCB constructs and controls were fitted to the same molarity. Human PBMCs (effector cells) were added to the wells to obtain a final E:T ratio of 10:1, corresponding to an E:T ratio of approximately 3 to 5 T cells to 1 target tumor cell. Negative control groups were represented only by effector or target cells. For normalization, the maximum lysis of H929 MM target cells (= 100%) was determined by incubating the target cells with a final concentration of 1% Triton X-100, inducing cell death. Minimal lysis (= 0%) was represented by target cells co-incubated with effector cells only, i.e., no T cell bispecific antibody. After 20-24 h or 48 h incubation at 37°C, 5% CO2, LDH release from the target apoptotic/necrotic MM cells into the supernatant was then measured with the LDH detection kit (Roche Applied Science), following the manufacturer's instructions. Percentage of LDH release was plotted against anti-BCMA/anti-CD3 T cell bispecific antibody concentrations in concentration-response curves. EC50 values were measured using Prism software (GraphPad) and determined as the concentration of TCB antibodies that resulted in 50% of maximum LDH release. As shown in Figure 4, all TCB anti-BCMA/anti-CD3 antibodies (21, 22, 42, and 83 A10-TCBcv) induced a concentration-dependent killing of BCMA-positive H929 myeloma cells as measured by the release of LDH. Lysis of H929 cells was specific, since the TCB control antibody that does not bind to BCMA-positive target cells, but only to CD3 on T cells, did not induce LDH release, even at the highest concentration tested. Table 12 summarizes the EC50 values for the removal of redirected T cells from high-expressing BCMA H929 cells induced by anti-BCMA/anti-CD3 TCB antibodies. Table 12: EC50 values for removal of redirected T cells from H929 cells induced by anti-BCMA/anti-CD3 TCB antibodies
Example 9: Redirected T cell cytotoxicity of L363 myeloma cells expressed with low BCMA value induced by anti-BCMA/anti-CD3 T cell bispecific antibodies (LDH release assay)
[000326] Anti-BCMA/anti-CD3 TCB antibodies were also analyzed for their ability to induce T cell mediated apoptosis in medium/low BCMA expressing MM cells after cross-linking the construct by binding the moieties of antigen-binding to BCMA in cells. Briefly, medium/low expressing human BCMA L363 multiple myeloma target cells are collected with cell dissociation buffer, washed and resuspended in RPMI supplemented with 10% fetal bovine serum (Invitrogen). Approximately 30,000 cells per well are plated in a 96-well round-bottom plate and the respective construct dilution is added to a desired final concentration (in triplicate); final concentrations ranging from 0.1 pM to 10 nM. For proper comparison, all TCB constructions and controls are set to the same molarity. Human PBMCs (effector cells) were added to the wells to obtain a final E:T ratio of 10:1, corresponding to an E:T ratio of approximately 3 to 5 T cells to 1 target tumor cell. Negative control groups were represented only by effector or target cells. For normalization, the maximum lysis of target MM cells (= 100%) is determined by incubating the target cells with a final concentration of 1% Triton X-100, inducing cell death. Minimal lysis (= 0%) was represented by target cells co-incubated with effector cells only, i.e., no T cell bispecific antibody. After 20-24 h incubation at 37°C, 5% CO 2 , LDH release from the apoptotic/necrotic target MM cells into the supernatant was then measured with the LDH detection kit (Roche Applied Science) following the manufacturer's instructions. Percentage of LDH release was plotted against anti-BCMA/anti-CD3 T cell bispecific antibody concentrations on concentration-response curves. EC50 values were measured using Prism software (GraphPad) and determined as a concentration of TCB antibodies that results in 50% of maximal LDH release. As shown in Figure 5, all anti-BCMA/anti-CD3 TCB antibodies (21, 22, 42, and 83 A10-TCBcv) induced a concentration-dependent killing of BCMA-positive L363 myeloma cells as measured by the release of LDH. Lysis of L363 cells was specific, since the TCB control antibody that does not bind to BCMA-positive target cells, but only to CD3 on T cells, did not induce LDH release, even at the highest concentration tested. Table 13 summarizes the EC50 values for the clearance of T cells redirected from L363 cells of medium/low BCMA expression induced by anti-BCMA/anti-CD3 TCB antibodies. Table 13: EC50 values for removal of redirected T cells from H363 cells induced by anti-BCMA/anti-CD3 TCB antibodies
Example 10: Redirected T cell cytotoxicity of L8226 myeloma cells expressed with low BCMA value induced by anti-BCMA/anti-CD3 T cell bispecific antibodies (LDH release assay)
Anti-BCMA/anti-CD3 TCB antibodies were analyzed for their ability to induce T cell mediated apoptosis in medium/low BCMA expressing MM cells after cross-linking the construct by binding the binding moieties to the antigen to BCMA in cells. Briefly, medium/low expressing human BCMA L363 multiple myeloma target cells are collected with cell dissociation buffer, washed and resuspended in RPMI supplemented with 10% fetal bovine serum (Invitrogen). Approximately 30,000 cells per well are plated in a 96-well round-bottom plate and the respective construct dilution is added to a desired final concentration (in triplicate); final concentrations ranging from 0.1 pM to 10 nM. For proper comparison, all TCB constructs and controls are fitted to the same molarity. Human PBMCs (effector cells) were added to the wells to obtain a final E:T ratio of 10:1, corresponding to an E:T ratio of approximately 3 to 5 T cells to 1 target tumor cell. Negative control groups were represented only by effector or target cells. For normalization, the maximum lysis of target MM cells (= 100%) was determined by incubating the target cells with a final concentration of 1% Triton X-100, inducing cell death. Minimal lysis (= 0%) was represented by target cells co-incubated with effector cells only, i.e., no T cell bispecific antibody. After 20-24 h incubation at 37°C, 5% CO 2 , LDH release from the target apoptotic/necrotic MM cells into the supernatant was then measured with the LDH detection kit (Roche Applied Science), following the manufacturer's instructions. The percentage of LDH release was plotted against anti-BCMA/anti-CD3 T cell bispecific antibody concentrations in concentration-response curves. EC50 values were measured using Prism software (GraphPad) and determined as the concentration of TCB antibodies that resulted in 50% of maximum LDH release. As shown in Figure 6, all anti-BCMA/anti-CD3 TCB antibodies (21, 22, 42 and 83 A10-TCBcv) induced a concentration-dependent killing of BCMA-positive RPMI-8226 myeloma cells as measured by LDH release. The lysis of RPMI-8226 cells was specific, since the TCB control antibody that does not bind to BCMA-positive target cells, but only to CD3 on T cells, did not induce LDH release, even at the highest concentration tested . Table 13 summarizes EC50 values for RPMI-8226 cell-retargeted T cell clearance of medium/low BCMA expression induced by anti-BCMA/anti-CD3 TCB antibodies. Table 13: EC50 values for removal of redirected T cells from RPMI-8226 cells induced by anti-BCMA/anti-CD3 TCB antibodies
Example 11: Redirected T cell cytotoxicity of JJN-3 myeloma cells expressing low BCMA induced by anti-BCMA/anti-CD3 T cell bispecific antibodies (flow cytometry and LDH release)
[000328] Anti-BCMA/anti-CD3 TCB antibodies were analyzed for their ability to induce T cell mediated apoptosis in BCMA low expressing MM cells after cross-linking the construct by binding the antigen-binding moieties to the BCMA in cells. Briefly, low expressing human BCMA JJN-3 multiple myeloma target cells are collected with cell dissociation buffer, washed and resuspended in RPMI supplemented with 10% fetal bovine serum (Invitrogen). Approximately 30,000 cells per well are plated in a 96-well round-bottom plate and the respective construct dilution is added to a desired final concentration (in triplicate); final concentrations ranging from 0.1 pM to 10 nM. For proper comparison, all TCB constructs and controls are fitted to the same molarity. Human PBMCs (effector cells) were added to the wells to obtain a final E:T ratio of 10:1, corresponding to an E:T ratio of approximately 3 to 5 T cells to 1 target tumor cell. Negative control groups were represented only by effector or target cells. For normalization, the maximum lysis of target MM cells (= 100%) was determined by incubating the target cells with a final concentration of 1% Triton X-100, inducing cell death. Minimal lysis (= 0%) was represented by target cells co-incubated with effector cells only, ie without any T cell bispecific antibody. i) After 48 h of incubation at 37°C, 5% CO 2 , the cultured myeloma cells were collected, washed and stained with antibodies conjugated to fluorochrome and annexin-V for determination of apoptotic myeloma cells. The staining panel comprised CD138-APCC750/CD38-FITC/CD5-BV510/CD56-PE/CD19-PerCP-Cy7/CD45-V450/Annexin-V-PerCP-Cy5.5. The fluorochrome-labeled antibodies used were purchased from BD Biosciences (San Jose, CA) and Caltag Laboratories (San Francisco CA). Acquisition was performed using a multicolor flow cytometer and installed software (eg, the CantoII device running FACS Diva software or the FACSCalibur flow cytometer using CellQUEST software). The Paint-A-Gate PRO program (BD Biosciences) was used for data analysis. Annexin-V was measured in JJN-3 cells and the percentage of annexin-v-positive JJN-3 cells was plotted against the concentration of anti-BCMA/anti-CD3 T cell bispecific antibodies. The percentage of JJN-3 cell lysis induced by a specific concentration of anti-BCMA/anti-CD3 T cell bispecific antibody was also determined by measuring the absolute count of annexin-V-negative JJN-3 cells at a given concentration of TCB and subtracting it from the absolute JJN-3 annexin-V-negative cell count without TCB; divided by the absolute count of annexin-V-negative JJN-3 cells without TCB. Figure 7 shows that anti-BCMA/anti-CD3 TCB antibodies (22, 42 and 83A10-TCBcv) induced concentration-dependent killing of low-expressing BCMA JJN-3 myeloma cells as measured by flow cytometry. Lysis of JJN-3 cells was specific since the control antibody-TCB that did not bind BCMA positive target cells but only CD3 on T cells did not induce an increase in JJN-3 cells positive for annexin-v or in the lysis of JJN-3 cells, even at the highest concentration tested. Table 14 and Table 15 summarize the percentages of JJN-3 cells positive for annexin-v and the percentages of lysis of JJN-3 cells induced by anti-BCMA/anti-CD3 TCB antibodies, respectively.
[000329] Detection of LDH is also performed after 20-24 h or 48 h of incubation at 37°C, 5% CO2. LDH release from the apoptotic/necrotic MM JJN-3 target cells into the supernatant is then measured with the LDH detection kit (Roche Applied Science) following the manufacturer's instructions. The percentage of LDH release is plotted against anti-BCMA/anti-CD3 T cell bispecific antibody concentrations in concentration-response curves. EC50 values are measured using Prism software (GraphPad) and determined as the concentration of TCB antibodies that results in 50% of maximum LDH release. Table 14. Elimination of Redirected T cells from low-expressing BCMA JJN-3 cells induced by anti-BCMA/anti-CD3 TCB antibodies: percentages of positive cells for annexin-V
Table 15. Elimination of Redirected T cells from JJN-3 cells of low BCMA expression induced by anti-BCMA/anti-CD3 TCB antibodies: percentages of JJN-3 cell lysis.

Example 12: BCMA expression in bone marrow myeloma plasma cells from patients with multiple myeloma
[000330] Human cell lines expressing the tumor target of interest are very useful and practical tools for measuring the potency of the TCB antibody to induce tumor cell cytotoxicity in the presence of T cells and the determination of EC50 values and for the classification of TCB molecules. However, despite being easily accessible and practical, human myeloma cell lines have the caveat that they do not represent the heterogeneity of multiple myeloma, a very complex disease characterized by significant heterogeneity at the molecular level. Furthermore, myeloma cell lines do not express the BCMA receptor with the same intensity and density, as some cells express BCMA more strongly than others (eg H929 cells compared to RPMI-8226), and this heterogeneity at the cellular level it can also be seen between different patients. Through academic collaborations with leading authorities on multiple myeloma, the determination of BCMA expression and density in patient samples and the evaluation of anti-BCMA/anti-CD3 TCB antibodies with clinical patient samples are being investigated. Blood and bone marrow aspirates are collected from patients with multiple myeloma after informed consent, in accordance with local ethics committee guidelines and the Declaration of Helsinki. a) BCMA expression detected by multiparameter flow cytometry (mean fluorescence intensity)
[000331] To determine BCMA receptor expression on bone marrow myeloma cells, immunophenotypic analyzes were performed using freshly isolated whole bone marrow aspirates. Whole bone marrow samples anticoagulated with erythrocyte-lysate K3 (ethylenediaminetetraacetic acid) were used for immunophenotypic analyses. A total of 2 x 106 cells per tube were stained, lysed and then washed using a direct immunofluorescence and multicolor staining technique, which aimed at the specific identification and immunophenotypic characterization of malignant plasma cells identified as CD138+ CD38+ CD45+ CD19- CD56+ . Cells were then stained using a panel of fluorochrome-conjugated antibodies including at least CD38-FITC/CD56-PE/CD19-PerCP-Cy7/CD45-V450/BCMA-APC. The fluorochrome-labeled antibodies used are purchased from BD Biosciences (San Jose, CA) and Caltag Laboratories (San Francisco CA). Anti-human BCMA antibody conjugated to APC was used in the immunophenotypic analyses. Acquisition was performed using a multicolor flow cytometer and installed software (eg CantoII device running FACS Diva software or FACSCalibur flow cytometer using CellQUEST software). The Paint-A-Gate PRO program (BD Biosciences) was used for data analysis. BCMA expression was measured based on the malignant plasma cell population and mean fluorescence intensity (FMI) values were determined and compared among myeloma patients. Table 16. BCMA expression in the patient's bone marrow myeloma plasma cells as detected by multiparametric flow cytometry (mean fluorescence intensity)
Determination of BCMA-specific antigen-binding capacity (quantitative flow cytometry analysis)
[000332] The Qifikit (Dako) method was used to quantify the BCMA-specific antigen-binding capacity (SABC) on the cell surface of the patient's bone marrow myeloma plasma cells. Plasma myeloma cells isolated from whole bone marrow aspirates were stained with 50 μl of mouse anti-human BCMA IgG (BioLegend # 357502) or a mouse IgG2a isotype control (BioLegend # 401501) diluted in FACS buffer (PBS, 0.1% BSA) at a final concentration of 25 µg/ml (or at saturating concentrations) and staining was performed for 30 min at 4°C in the dark. Then, 100 µl of the calibration beads were added in separate wells and the cells as well as the beads were washed twice with the FACS buffer. Cells and granules were resuspended in 25 μl of FACS buffer containing the secondary anti-mouse antibody conjugated to fluorescein (at saturation concentrations) supplied by Qifikit. Cells and beads were stained for 45 min at 4°C in the dark. Cells were washed once and all samples were resuspended in 100 µl of FACS buffer. Samples were analyzed immediately on a multicolor flow cytometer and installed software (eg, a CantoII device running FACS Diva software or a FACSCalibur flow cytometer using CellQUEST software). Table 17. BCMA-specific antigen-binding capacity in patient bone marrow myeloma plasma cells as measured by quantitative flow cytometry analysis

Example 13: Redirected T cell cytotoxicity from bone marrow myeloma plasma cells from patients in the presence of autologous bone marrow infiltrating T cells induced by bispecific anti-BCMA/anti-CD3 T cell antibodies (multiparametric flow cytometry)
[000333] One of the most important and critical in vitro features during the preclinical evaluation of TCB antibody candidates for multiple myeloma is whether the TCB molecule could activate patients' T cells and induce the removal of redirected T cells from cells results of primary myeloma from the bone marrow of patients. To assess the effect of anti-BCMA/anti-CD3 TCB antibodies to induce the removal of redirected T cells from bone myeloma plasma cells, whole bone marrow aspirates were collected from multiple myeloma patients in EDTA-coated tubes and used immediately for cell culture assays. The ratio of effector cells to tumor cells (E:T ratio) present in all bone marrow samples was determined and measured by flow cytometry. Briefly, 200 µl of bone marrow samples were transferred to 96 deep-well plates. Anti-BCMA/anti-CD3 TCB antibody and control antibody dilutions were prepared in sterile medium and 10 µl of the preparation was added to the respective wells for final concentrations ranging from 0.1 pM to 30 nM. The bone marrow-antibody suspension is mixed by gentle shaking and then incubated at 37°C, 5% CO2 for 48 h, sealed with a paraffin film. After the incubation period, 20 µl of a corresponding FACS antibody solution prepared based on a panel of antibodies including CD138-APCC750/CD38-FITC/CD5-BV510/CD56-PE/CD19-PerCP-Cy7/CD45-V450 /BCMA-APC/Annexin-V-PerCP-Cy5.5 were added to a 96-well U-bottom plate. Fluorochrome labeled antibodies were purchased from BD Biosciences (San Jose, CA) and Caltag Laboratories ( San Francisco CA) and APC-conjugated anti-human BCCA antibody was used. Samples were then incubated for 15 minutes in the dark at room temperature and were acquired and analyzed using a multicolor flow cytometer. Cell death of myeloma cells was determined by evaluating the positive expression of closed annexin-V in CD138+ CD38+ CD45+ CD19- CD56+ myeloma cell populations. The percentage of myeloma cell death was then determined. The percentage of myeloma plasma cell lysis of the patients' bone marrow induced by a specific concentration of anti-BCMA/anti-CD3 T cell bispecific antibody was also determined by measuring the absolute count of annexin-negative myeloma plasma cells -V at a given concentration of TCB and subtracting it from the absolute count of plasma myeloma cells negative for annexin-V without TCB; divided by the absolute count of annexin-V negative plasma myeloma cells without TCB. To verify the specificity of bispecific anti-BCMA/anti-CD3 T cell antibodies, annexin-V expression was also measured in other types of bone marrow cells, such as T cells, B cells and NK cells. As shown in Figure 8, there was a specific and concentration-dependent lysis of the patient's plasma myeloma cells, whereas lysis of T cells, B cells and NK cells was not observed. Furthermore, the control-TCB that only binds to CD3 only, but not to BCMA, did not induce cell death of myeloma plasma cells at the highest concentrations of TCB antibodies. As shown in Table 18, the percentage of patient bone marrow myeloma cells with annexin-V at the highest concentration (30 nM) reached up to 52.54% and 55.72% for 42-TCBcv and 22-TCBcv, respectively , compared to 29.31% for 83A10-TCBcv, concluding that 42-TCBcv and 22-TCBcv are more potent than 83A10-TCBcv in inducing the death of the patient's bone marrow myeloma plasma cells. Table 18. Percentage of annexin-V positive plasma myeloma cells from patient bone marrow aspirates induced by bispecific anti-BCMA/anti-CD3 T cell antibodies.

[000334] In another study on bone marrow aspirates from 5 different MM patients, the percentage of viable plasma myeloma cells was determined by closing the cell population negative for annexin-V and plotted against the antibody concentration anti-BCMA/anti-CD3 T cell bispecific. EC50 values were measured and determined as the concentration of TCB antibody that results in 50% of the maximum viable plasma myeloma cells. EMAX (%) was determined as maximum viable plasma myeloma cells in the presence of anti-BCMA/anti-CD3 T cell bispecific antibody. 83A10-TCBcv was much less potent in inducing myeloma plasma cell lysis than 22-TCBcv and 42-TCBcv in most of the five bone marrow aspirate samples from the myeloma patient (Table 26; Figure 9 shows curves as an example). of concentration response for 2 of the 5 patients). A concentration-dependent reduction of viable myeloma cells was observed in 5/5 patient samples treated with 22-TCBcv or 42-TCBcv, compared to only 1/5 patient samples for 83A10-TCBcv. Table 19 shows the comparison of 83A10-TCBcv with 22-TCBcv and 42-TCBcv and the effect of anti-BCMA/anti-CD3 T cell bispecific antibodies on bone marrow myeloma plasma cell viability. The results clearly show that there were less viable bone marrow myeloma plasma cells with 22-TCBcv and 42-TCBcv (ie, more lysis of bone marrow myeloma plasma cells) in 4/5 patient samples, as demonstrated by lower values of EMAX (%) for 22-TCBcv and 42-TCBcv vs. 83A10-TCBcv in the respective patient samples. Specific and concentration-dependent lysis of the patient's myeloma plasma cells was observed while non-median bone marrow cell lysis was not observed (data not shown). Table 19. EMAX values (%) against viable annexin-V negative plasma myeloma cells from patient bone marrow aspirates in the presence of anti-BCMA/anti-CD3 T cell bispecific antibodies.

[000335] In further investigations into the novel anti-BCMA/anti-CD3 T cell bispecific antibodies of the present invention compared to 83A10-TCBcv, seven freshly taken whole bone marrow samples/aspirates from patients were stained with magnetic microgranules CD138 (Miltenyi Biotec, Bergisch Gladbach, Germany), passed through an autoMACS cell sorting column and the collected portions with a sufficient remaining number of MM plasma cells of more than 4% myeloma plasma cells were used for additional experiments. In 24-well plates, 500,000 cells/well were incubated and cultured for 48 hours. Anti-BCMA/anti-CD3 TCB antibodies and control antibody dilutions were added to the respective wells for a final TCB concentration of 0.1 pM to 10 nM. Each dose point was done in triplicate. The viability of plasma cells and cells from the bone marrow microenvironment was investigated using two-color propidium iodide/CD138-FITC with flow cytometry (FACSCalibur; Becton Dickinson). Data analysis was performed using FACSDiva software (Becton Dickinson). As illustrated in Figure 10, the bar graphs show mean values normalized to the mean over the respective medium control (MC) triplicates. For statistical analysis, a t-test was used. The maximum inhibition of MM plasma cell growth at a concentration of 10 nM (IMAX10) and the measured inhibition at 1 nM (IMAX1), respectively, were given in percentage as referred to the control medium. The maximum inhibition of the TCB control antibody (10 nM) compared to the control medium is also represented. Calculations were made using R 3.1.19 and Bioconductor 2.1310, but for the calculation of IMAX values (Microsoft Excel®; Microsoft Office Professional 2013). An effect was considered statistically significant if the P-value of its corresponding statistical test was <5% (*), <1% (**) or <0.1% (***). As shown in Figures 10A-10G, the results clearly show that there were less viable plasma bone marrow myeloma cells with 22-TCBcv and 42-TCBcv (ie, more lysis of bone marrow myeloma plasma cells) by 7/7 patient samples compared to 83A10-TCBcv. Table 20 demonstrates the percentage of viable plasma myeloma cells from patient bone marrow aspirates induced by bispecific anti-BCMA/anti-CD3 T cell antibodies relative to media control. Table 21 shows the values for IMAX10 and IMAX1. The results demonstrate that 22-TCBcv and 42-TCBcv are clearly more potent than 83A10-TCBcv in inducing the death of the patient's bone marrow myeloma plasma cells. Despite the specific lysis of bone marrow plasma cells (BMPC) induced by bispecific anti-BCMA/anti-CD3 T cell antibodies and observed in all patient bone marrow samples, the bone marrow microenvironment (BMME) does not was affected in the respective samples (Figure 10H, representative of 7 patient samples). Table 20. Relative percentage of viable myeloma cells negative for propidium iodide from patient bone marrow aspirates induced by anti-BCMA/anti-CD3 T cell bispecific antibodies.

Table 21. IMAX10 and IMAX1 values in relation to maximal inhibition of MM plasma cell growth at 10 nM (IMAX10) and inhibition at 1 nM (IMAX1) based on viable myeloma cells negative for propidium iodide aspirates of the patient's bone marrow in the presence of bispecific anti-BCMA/anti-CD3 T cell antibodies.
Example 14: T cell activation of patient bone marrow T cells induced by bispecific anti-BCMA/anti-CD3 T cell antibodies (multiparametric flow cytometry)
[000336] To assess whether anti-BCMA/anti-CD3 TCB antibodies induce patient activation with CD4+ and CD8+ T cells from the patient's myeloma (ie, bone marrow infiltrating T cells (MILs)), samples from respective treated, untreated and control groups after 48 h of incubation were also stained with a FACS antibody solution prepared based on an antibody panel including eight markers: CD8/CD69/TIM-3/CD16/CD25/CD4/ HLA-DR/PD-1. Samples were then incubated for 15 minutes in the dark at room temperature and were acquired and analyzed using a multicolor flow cytometer. T cell activation was determined by evaluating the positive expression of CD25, CD69 and/or closed HLA-DR in CD4+ and CD8+ T cell populations. Percentages of T cell activation were then measured. Figure 11 shows a concentration-dependent upregulation of CD69 and CD25 on CD4+ and CD8+ T cells infiltrating the bone marrow of patients with multiple myeloma. Table 22 summarizes the increased expression of CD69 and CD25 T cells on CD4+ and CD8+ induced by anti-BCMA/anti-CD3 TCB antibodies; data from a patient. Table 22: Activation of autologous T cell T cells from myeloma patients induced by bispecific anti-BCMA/anti-CD3 T cell antibodies in the presence of myeloma plasma cells from the patient's bone marrow

Example 15: Enhancement of T cell function (cytokine production) of patient bone marrow T cells induced by anti-BCMA/anti-CD3 T cell bispecific antibodies (multiplexed flow immunoassay/flow cytometry)
[000337] To assess whether anti-BCMA/anti-CD3 TCB antibodies (83A10-TCBcv, 22-TCBcv and 42-TCBcv) induce T cell activation and increased function of myeloma bone marrow infiltrating CD4+ and CD8+ cells From the patient, culture supernatants were collected from the respective treated, untreated and control groups after 48 hours of incubation and the content of cytokines and serine proteases was measured. Cytokine microgranule (CBA) array analysis was performed on the multicolor flow cytometer according to the manufacturer's instructions, using the Th1/Th2 Cytokine Kit II (BD # 551809) or the combination of the following Flex Kits from CBA: human granzyme B (BD #560304), Human IFN-Y Flex Kit (BD #558269), TNF-α Flex Kit (BD #558273) Human IL-10 Flex Kit (BD #558274) Flex Kit of human IL-6 (BD #558276), Human IL-4 Flex Kit (BD #558272), Human IL-2 Flex Kit (BD #558270). Example 16: pharmacokinetic/pharmacodynamic (PK/PD) study in cynomolgus monkeys
[000338] A clear advantage that an anti-BCMA/anti-CD3 TCBcv antibody could have over other bispecific antibodies such as (scFV)2 (for example, BCMAxCD3 bispecific T cell Engager BiTE®, as described in WO2013072415 and WO2013072406 ) is the much longer/lower clearance in vivo half-life, which could allow twice or once weekly SC or IV administration compared to the very short elimination half-life of (scFV)2 (eg, 1-4 hours) that requires treatment administered via a pump carried by patients for weeks to months (Topp et al. J Clin Oncol 2011; 29(18): 2493-8). An administration twice or once a week would be much more convenient for patients and also much less risky (eg pump failure, catheter problems, etc.). a) To verify the elimination/clearance half-life of the anti-BCMA/anti-CD3 antibody 83A10-TCBcv in vivo, single-dose pharmacokinetic (PK) pharmacodynamic (PD) studies with bispecific anti-T cell antibodies BCMA/anti-CD3 (83A10-TCBcv, 22-TCBcv and 42-TCBcv) were performed in accredited CRO with experienced AAA-LAC. Biologically pure adult cynomolgus monkeys about two years old and weighing approximately 3 kg were acclimated for at least 40 days and selected based on body weight, clinical observations, and clinical pathology examinations. Animals were identified by individual tattoos and color-coded cage cards. All animal procedures (including housing, health monitoring, restriction, dosage, etc.) and ethical review were carried out in accordance with the country's current legislation, applying the Directive on the protection of animals used in biomedical research. Animals were randomly assigned to the treatment group based on the most recent pre-test body weight. After excluding animals with unacceptable pretest findings, a computer program included in the Pristima® system, designed to balance pretest body weights, was used to exclude animals from both extremes of body weight and randomize the remaining animals to the treatment group. Animals were assigned to three 83A10-TCBcv treatment groups (n = 2 animals, ie 1 female and 1 male per group) with 0.003; 0.03; and 0.3 mg/kg. Animals received a single intravenous injection of 83A10-TCBcv and at least 0.8 mL of blood samples per time point was collected through the peripheral vein for PK assessments according to the following collection schedule and procedures: pre-dose , 30, 90, 180 min, 7, 24, 48, 96, 168, 336, 504 h after administration. Blood samples were allowed to clot in serum separation tubes for 60 minutes at room temperature. The clot was centrifuged by centrifugation (at least 10 minutes, 1200 g, +4°C). The resulting serum (about 300 μL) was stored directly at -80°C until further analysis. Bone marrow samples for PK evaluations were also collected from the femur under anesthetic/analgesic treatment according to the following collection schedule: pre-dose, 96 and 336 h after administration. Bone marrow samples were allowed to clot in serum separation tubes for 60 minutes at room temperature. The clot was centrifuged by centrifugation (at least 10 minutes, 1200 g, +4°C). The resulting bone marrow (about 1 mL) was stored directly at -80°C until further analysis. Analysis and evaluation of PK data is performed. A non-compartmental standard analysis is performed using the Watson package (v.7.4, Thermo Fisher Scientific Waltman, MA, USA) or the Phoenix WinNonlin system (v.6.3, Certara Company, USA). As shown in Figure 12 and Table 23, serum 83A10-TCBcv concentrations were measured by ELISA from serum samples collected at different time points after intravenous injection. Table 24 shows the concentrations of 83A10-TCBcv in bone marrow as measured by ELISA for each treatment group (BLQ means below quantification level).
[000339] Various information relevant to the potential clinical use of a bispecific antibody according to the invention can be taken from Figure 12, Table 23 and Table 24:
[000340] In bone marrow aspirates from patients with MM, concentrations of 1 nM or 10 nM of TCBs of this invention induce significant or even total clearance of MM plasma cells; at a dose of 0.03 mg/kg in the interval from injection to 168 hours (7 days), plasma concentrations between approx. 1 nM and 4 nM were obtained, showing that therapy with doses of approx. 0.03 mg/kg once a week may be feasible (200 ng/ml corresponds to about 1 nM)
[000341] Figure 12 shows that, over the range of the investigated dose range, the PK is largely linear. This means that concentrations are proportional to dose, a useful property for clinical therapy.
[000342] MM is a disease located primarily in the bone marrow. The 83A10-TCBcv concentrations detected in bone marrow are close to serum concentrations (Table 24), eg 96 h after bone marrow injection concentrations of approx. 1 and 2 nM were measured; these are TCB concentrations of this invention where significant MM plasma cell death is observed in bone marrow aspirates freshly taken from patients with MM, again demonstrating the opportunity for convenient dosing intervals such as, for example, once a year week
[000343] Between 24 and 504 hours after injection, elimination is largely first order with an elimination half-life of approx. 6 to 8 days, again showing the opportunity for, for example, a dosage of, for example, once a week. Table 23. Serum concentrations of 83A10-TCBcv after IV treatment in cynomolgus monkeys
Table 24. Bone marrow concentration of 83A10-TCBcv after single IV treatment in cynomolgus monkeys

[000344] Pharmacodynamic measurements (PD): blood samples (time points: pre-dose, 24, 48, 96, 168, 336, 504 h after administration) and bone marrow samples (time points: pre-dose, 96 and 336 h after administration) were collected in tubes containing 7.5% K3 EDTA for PD evaluation by flow cytometry to assess the effect of single-dose 83A10-TCBcv iv on blood plasma cells and bone marrow, B cells, and T cells. A direct "lysis and wash" immunofluorescence staining method of the surface markers was applied. Briefly, 100 µL of blood or bone marrow was incubated with two mixtures of antibodies including CD45/CD2/CD16/CD20/CD27/CD38 or CD45/CD2/CD16/CD4/CD25/CD8 in the dark for 30 min at +4 °C. To lyse red blood cells, 2 ml of lysis buffer solution was added to the sample and incubated for 15 min at room temperature in the dark. Cells were collected by centrifugation and washed with staining buffer (2% PBS fetal bovine serum). The stained samples were kept refrigerated and protected from light until acquisition with a cytometer on the same day. FACS data acquisition was performed with a Becton Dickinson flow cytometer equipped with 488 and 635 laser lines, BD FACS Canto II. BD FACSDiva software was used for data collection and analysis. Absolute cell number enumeration was performed with a dual platform, based on the WBC count obtained by the hematology analyzer (ADVIATM 120, Siemens). As shown in Figure 13, peripheral T cell redistribution was observed in all animals that received a single-dose IV treatment of 83A10-TCBcv, as shown by the decrease in circulating T cell counts. As shown in Figure 14A, already 24h after treatment with 83A10-TCBcv 0.3 mg/kg, a decrease in blood plasma cells (BCMA positive cells) was observed in treated animals, although there was no decrease in B cells total (BCMA negative cells). Figure 14b shows the kinetics of blood plasma cell reduction after treatment with 83A10-TCBcv 0.3 mg/kg in cynomolgus monkeys.
[000345] Blood samples were also processed for plasma collection for cytokine analysis (IL-1b, IL-2, IL-6, IL-10, TNF-α and IFN-Y), according to the following schedule collection: pre-dose, 30, 90, 180 min, 7, 24, 48, 96, 168 h after administration. Blood samples were placed in plastic tubes kept in an ice-water bath, then centrifuged (at least 10 min., 1200g, +4°C). The resulting plasma was stored directly at -80°C until analysis. Cytokine analysis is performed with Multiplex granule-based cytokine immunoassay (Luminex technology). Data are analyzed using Bio-Plex Manager 4.1 software (Bio-Rad): a five-parameter (5PL) logistic regression model is used.
[000346] b) In a later study, cynomolgus monkeys were treated with 42-TCBcv or 22-TCBcv. The animals (n = 2/group) received a single IV injection (0.01; 0.1 and 1.0 mg/kg) or SC (0.01 and 0.1 mg/kg) of 42-TCBcv or a single IV injection with 22-TCBCv (0.1 mg/kg). Blood and bone marrow samples are collected at time points following a defined collection schedule and processed according to PK and PD measurements (immunophenotyping and cytokine production).
[000347] Animals received a single IV or SC injection of 42-TCBcv or 22-TCBcv (IV only) and blood samples per time point were collected through the peripheral vein for PK assessments according to the following collection schedule and procedures: pre-dose, 30, 90, 180 min, 7, 24, 48, 96, 168, 336, 504 h after administration. Blood samples were allowed to clot in serum separation tubes for 60 minutes at room temperature. The clot was centrifuged by centrifugation (at least 10 minutes, 1200 g, +4°C). The resulting serum (about 300 μL) was stored directly at -80°C until further analysis. Bone marrow samples for PK evaluations were also collected from the femur under anesthetic/analgesic treatment according to the following collection schedule: pre-dose, 96 and 336 h after administration. Bone marrow samples were allowed to clot in serum separation tubes for 60 minutes at room temperature. The clot was centrifuged by centrifugation (at least 10 minutes, 1200 g, +4°C). The resulting bone marrow (about 1 mL) was stored directly at -80°C until further analysis. Analysis and evaluation of PK data were performed. A non-compartmental pattern analysis was performed using the Watson package (v.7.4, Thermo Fisher Scientific Waltman, MA, USA) or the Phoenix WinNonlin system (v.6.3, Certara Company, USA). As shown in Figure 19 and Table 24A-D, 42-TCBcv concentrations were measured by ELISA from serum and bone marrow samples collected at different time points after IV or SC injection. Effective concentration range of 42-TCBcv in bone marrow aspirates from multiple myeloma patient corresponding to 10 pm to 10 nM (grey area). Concentrations in parentheses are in nM. BLQ, below the quantification level; i/m, measurement inconclusive. Table 24A. Serum 42-TCBcv concentrations after IV treatment in cynomolgus monkeys

Table 24B. Bone marrow concentration of 42-TCBcv after single IV treatment in cynomolgus monkeys
Table 24C. Serum 42-TCBcv concentrations after SC treatment in cynomolgus monkeys
Table 24D. Bone marrow concentration of 42-TCBcv after single SC treatment in cynomolgus monkeys

[000348] The results in Tables 24A and 24C show an adequate serum attractive concentration profile once a week or even once every two weeks of 42-TCBcv treatment. The area under the AUC curve for serum concentrations after IV and SC administration was determined, comparing AUC values showed high bioavailability of about 100% with SC injection of 42 TCBcv. Furthermore, the results show that the concentration of 42-TCBcv in bone marrow is very similar to the serum concentrations of 42-TCBcv. The 42-TCBcv concentrations in serum could represent the 42-TCBcv concentrations available in the bone marrow, that is, in the main site where myeloma tumor cells are enriched.
[000349] Pharmacodynamic measurements (PD) are valuable information to support PK measurements. Further PD analyzes were performed. Blood cynomolgus CD20+ B cells also express BCMA on the cell surface and are significantly more frequent (higher absolute count) than blood plasma cells. Blood B cell depletion was used as a reliable pharmacodynamic effect of anti-BCMA/anti-CD3 TCBcv antibodies and to compare the in vivo efficacy between 83A10-TCBcv, 42-TCBcv and 22-TCBcv. Absolute B cell counts were calculated based on the dual platform consisting of flow cytometry and WBC count obtained with a hematology analyzer and measured at the following time points: pre-dose, 24h, 48h, 96h and 196h after IV infusion 10 min. Percent B cell exclusion was calculated as follows: = [re-dose absolute B cell count] - [absolute B cell count at time point] [re-dose absolute B cell count] * 100 Table 24E : Pharmacodynamic effects of anti-BCMA/anti-CD3 TCBcv antibodies: exclusion of B cells

[000350] 42-TCBcv and 22-TCBcv are more potent than 83A10-TCBcv to induce the exclusion of BCMA expressing B cells in cynomolgus monkeys after a single intravenous injection (see Table 24E). Since the three molecules share the same molecular and binding structure of CD3, the difference in efficacy in cynomolgus monkeys can be attributed mainly to the respective BCMA antibody.
[000351] To confirm that the exclusion of BCMA expressing B cells in cynomolgus monkeys after IV injection is a result of the mechanistic pharmacodynamic effects of anti-BCMA/anti-CD3 TCBcv antibodies, the increase in activated cytotoxic CD8+ T cells (or ie, effector cells) were measured in bone marrow enriched with BCMA positive cells (ie target cells) 4 days (96 h) and 3 weeks (336 h) after intravenous injection. Absolute counts of activated CD8+ CD25+ activated T cells were calculated based on the dual platform consisting of flow cytometry and WBC count obtained with a hematology analyzer Table 24F: Pharmacodynamic effects of anti-BCMA/anti-CD3 TCBcv antibodies: Increase in activated CD8+ CD25+ T cells

[000352] 42-TCBcv and 22-TCBcv are more potent than 83A10-TCBcv in inducing T-cell activation in cynomolgus monkeys after a single-dose intravenous injection (see Table 24F). Since the three molecules share the same molecular and binding structure of CD3, the difference in pharmacodynamic effects in cynomolgus monkeys can be attributed mainly to the respective BCMA antibody. The results indicate that the exclusion of BCMA-positive B cells in bone marrow and blood is likely the result of activation of cytotoxic T cells induced by anti-BCMA/anti-CD3 antibodies of TCBcv. Example 17: Anti-tumor activity induced by anti-BCMA/anti-CD3 T cell bispecific antibody in the H929 human myeloma xenograft model using PBMC-humanized NOG mice.
[000353] With a long elimination half-life, Fc-containing anti-BCMA/anti-CD3 TCBcv antibodies may be more effective than (scFv)2-based bispecific antibodies, such as BCMA50-BiTE®, administered in equimolar doses, on a once-a-week schedule. The in vivo effect of 83A10-TCBcv and BCMA50-BiTE® (as described in WO2013072415 and WO2013072406) was compared and evaluated in the H929 human myeloma xenograft model in PBMC-humanized NOG mice. NOG mice are suitable for humanized mouse models as they completely lack immune cells, including the resident NK cell population, and therefore are more permissive for tumor engraftment of human xenogenic cells (Ito et al. Curr Top Microbiol Immunol 2008; 324: 53-76). Briefly, on day 0 (d0) of the study, the 5x106 human myeloma cell line NCI-H929 (NCI-H929, ATCC® CRL-9068 ™) in 100 µL of RPMI 1640 medium containing 50:50 matrigel (BD Biosciences, France) were injected subcutaneously (SC) into the right dorsal flank of an immunodeficient twin NOD mouse (NOD/Shi-scid IL2rgamma (null) (NOG) from 8 to 10 weeks of age (Taconic, Ry, Denmark). Twenty-four to 72 hours before SC implantation of the H929 tumor cell, all mice received whole-body irradiation with a y-source (1.44 Gy, 60Co, BioMep, Bretenières, France). On day 15 (d15), NOG mice received a single intraperitoneal (IP) injection of 2x107 human PBMCs (in 500 μL of 1X PBS, pH 7.4). The characterization of human PBMC was performed by immunophenotyping (flow cytometry). The mice were carefully randomized into the different treatment and control groups (n = 9/group) using the Vivo® manager software (Biosystemes, Couternon, France) and a statistical test (analysis of variance) was performed to test the homogeneity between the groups. Antibody treatment started on day 19 (d19), ie 19 days after SC injection of H929 tumor cells when tumor volume reached at least 100150 mm3 in all mice, with a mean tumor volume of 300 ± 161 mm3 for the vehicle-treated control group, 315 ± 148 mm3 for the 2.6 nM/kg control-TCB group, 293 ± 135 mm3 for the 2.6 nM/kg 83A10-TCBcv group and 307 ± 138 mm3 for the 2.6 nM/kg BCMA50- (scFv)2 (BCMA50-BiTE®) group. The TCB antibody treatment schedule was based on previously obtained pharmacokinetic results with 83A10-TCBcv and consisted of a once-weekly IV administration for up to 3 weeks (ie, a total of 3 TCB antibody injections). Four days after reconstitution of the host mice with human PBMCs (d19), a first dose of the anti-BCMA/anti-CD3 antibody 83A10-TCBcv (2.6 nM/kg, respectively, 0.5 mg/kg) was given by injection into the tail vein. Blood samples were collected by puncture of the jugular/mandibular vein (under anesthesia) 1 h before each treatment, 2 h before the second treatment and at the end in mice from all groups treated with 83A10-TCBcv and control-TCBcv . Blood samples were immediately transferred to tubes containing clot activator (MG T tubes, cherry red top, Capiject®, Terumo®). Tubes were left at room temperature for 30 minutes to allow for coagulation. Then, the tubes were centrifuged at 1300 g for 5 min to separate the clot/serum. Serum aliquots were prepared, quickly frozen in liquid nitrogen and stored at -80°C until further analysis. Tumor volume (VT) was measured by forceps during the study and progress assessed by intergroup comparison of VT. Tumor growth percentage defined as TG (%) was determined by calculating TG (%) = 100 x (mean VT of analyzed group)/(mean VT of control vehicle-treated group). For ethical reasons, mice were euthanized when the VT reached at least 2000 mm3. Figure 15 shows the VT of each mouse individually by experimental group: (A) control groups including vehicle control (full line) and TCB control (dotted line), (B) 83A10-TCBcv group (2.6 nM) µg/kg) and (C) BCMA50-BiTE® (2.6 nM/kg). In the 83A10-TCBcv group (2.6 nM/kg), 6 of the 9 mice (67%) had their tumor regressed even below the VT recorded on d19, that is, the first TCB treatment and tumor regression was maintained until completion of the study. The 3 mice in the 83A10-TCBcv treated group (2.6 nM/kg) that did not show tumor regression had their VT equal to 376, 402 and 522 mm3 respectively on d19. In contrast, none of the 9 mice (0%) treated with an equimolar dose of BCMA50-BiTE® (2.6 nM/kg) on a once-weekly schedule for 3 weeks had their tumor regressed at any point in time. time. Table 25 shows the progression of tumor volumes over time in all experimental groups. Percent tumor growth was calculated for d19 to d43 and compared between 83A10-TCBcv (2.6 nM/kg) and BCMA50-BiTE® (2.6 nM/kg) (Figure 16). The results demonstrate that TG (%) is consistently and significantly reduced in the 83A10-TCBcv group (2.6 nM/kg), as well as the TG (%) is always lower when compared to BCMA50-BiTE® (2.6 nM/kg). Table 26 shows the medium tumor volume (VT) and tumor growth percentage (TG (%)) on days 19 to 43. The overall results clearly demonstrated that 83A10-TCBcv is superior to BCMA50-BiTE® for inducing activity antitumor in vivo when treatment is given at an equimolar dose on a once-weekly schedule for 3 weeks. Table 25. Progression of tumor volumes over time in mice from the control vehicle group and mice treated with equimolar doses of control TCB, 83A10-TCBcv and BCMA50- (scFv)2 (BCMA50-BiTE®)



Table 26. Medium tumor volume (VT) and tumor growth percentage (TG (%)) on days 19 to 43: 83A10-TCBcv compared to BCMA50-BiTE®.
Example 18: Anti-tumor activity induced by anti-BCMA/anti-CD3 T cell bispecific antibodies in human myeloma xenograft model RPMI-8226 in PBMC-humanized NOG mice
[000354] Alternatively to the myeloma cell line H929, the human myeloma cell line RPMI-8226 for which the expression level of superficial BCMA is lower than that of H929 and more representative of the level detected in primary myeloma cells is used as tumor xenotransplantation. Briefly, on day 0 (d0) of the study, 10x106 - 20x106 human myeloma cell line RPMI-8226 (ATCC® CCL-155™) in 200 µL of 0.9% NaCl solution containing 50:50 matrigel ( BD Biosciences, France) are injected subcutaneously (SC) into the right dorsal flank of 8-10 week old female NOD/Shiscid IL2rgamma (NOG) (NOG) mice (Taconic, Ry, Danemark). Twenty-four to 72 hours before SC implantation of the RPMI-8226 cell line, all mice received whole-body irradiation with a y source (1.44 Gy, 60Co, BioMep, Bretenières, France). NOG mice receive a single intraperitoneal (IP) injection of 2x107 human PBMCs (in 500 μL of PBS 1X pH 7.4) once between day 9 (d9) and day 45 (d45) once the tumor volumes reach at least 100-150 mm3. The characterization of human PBMC is performed by immunophenotyping (flow cytometry). Mice are carefully randomized into different treatment and control groups (n = 9/group) using the Vivo® manager software (Biosystemes, Couternon, France) and a statistical test (analysis of variance) is performed to test homogeneity between groups . Antibody treatment begins at least 24h to 48h after intraperitoneal injection of human PBMC and when tumor volume reaches at least 100-150 mm3 in all mice. The TCB antibody treatment schedule is based on pharmacokinetic results and consisted of once or twice weekly IV administration via the tail vein for up to 3 weeks (ie a total of 3 TCB antibody injections). Blood samples are collected by puncture of the jugular/mandibular vein (under anesthesia) 1 hour before each treatment, 2 hours before the second treatment and at the end. Blood samples are immediately transferred to tubes containing clot activator (MG T tubes, cherry red top, Capiject®, Terumo®). Tubes are left at room temperature for 30 minutes to allow for coagulation. Then the tubes are centrifuged at 1300 g for 5 min to separate the clot/serum. Serum aliquots are prepared, snap frozen in liquid nitrogen and stored at -80°C until further analysis. Tumor volume (VT) is measured by forceps during the study and progress assessed by VT intergroup comparison. The percentage of tumor growth defined as TG inhibition (%) is determined by calculating TG (%) = 100 x (mean VT of analyzed group)/(mean VT of control vehicle-treated group).
[000355] The development of the RPMI-8226 human myeloma xenograft model in PBMC-humanized NOG mice was first performed to ensure that the xenograft model was appropriate for testing anti-BCMA/anti-CD3 T cell bispecific antibodies. BCMAlow expressing RPMI-8226 cells were injected subcutaneously into NOG mice on day 0. On day 22, human PBMCs were injected intraperitoneally and human T cells could be detected in the blood one week later (data not shown) . As illustrated in Figure 20, tumor growth and body weight were measured until day 50. Unfortunately and unexpectedly, this xenotransplantation model proved inadequate for testing anti-tumor activity of anti-BCMA/ T cell bispecific antibodies. anti-CD3 for the following reasons: 1) Human myeloma xenograft RPMI-8226 failed to grow consistently in PBMC-humanized NOG mice; 2) PBMC-humanized NOG mice transplanted with RPMI-8226 xenotransplantation began to lose body weight soon after intraperitoneal injection of human PBMC, a sign of graft versus host disease. These mice were sacrificed for ethical reasons; 3) loss of BCMA expression observed in tumor xenotransplantation after subcutaneous injection in the sacrifice of host mice. Example 19: Plasma cell redirected T cell cytoplasia of peripheral blood mononuclear cells or bone marrow aspirates from a patient with plasma cell leukemia (PCL) in the presence of autologous T cells induced by bispecific anti-BCMA T cell antibodies /anti-CD3 as measured by flow cytometry
[000356] Plasma cell leukemia (PCL) is a leukemic variant of de novo or clinically pre-existing multiple myeloma (MM) myeloma. Current treatments available are quite limited and consist mainly of combinations of MM drugs and chemotherapy. To date, no therapy has been explicitly registered for this highly aggressive and deadly disease. BCMA plays an essential role in the survival of normal plasma cells and a bispecific anti-BCMA/anti-CD3 T cell antibody according to the invention can be used for treating plasma cell leukemia in a patient suffering from said disease. Freshly collected peripheral blood mononuclear cells (PBMC) from plasma cell leukemia, patient samples containing plasma cells >80% with high leukocyte counts are isolated by density gradient using Ficoll or other comparable methods and incubated for 24h and 48h with concentrations of bispecific anti-BCMA/anti-CD3 T cell antibody or control antibodies of 0.1 pM to 30 nM at 37°C in a humidified air atmosphere. All bone marrow aspirates from patients with plasma cell leukemia can also be used as specimens. Each dose point is done in triplicate. Apoptosis is determined by annexin/propidium iodide staining of the entire population and CD138 positive cells on a FACSCalibur using Diva (BD) software. Plasma cell viability and total PBMC population are investigated by propidium iodide/CD138-Double staining with FITC using flow cytometry (FACSCalibur; Becton Dickinson). Data analysis is performed using FACSDiva software (Becton Dickinson). The mean values are normalized to the mean over the triplicates of the respective control medium (MC). For statistical analysis, a one-sided t-test is used. The maximum inhibition of PCL cell growth at a concentration of 10 nM (IMAX10) and the measured inhibition at 1 nM (IMAX1), respectively, are given in percentage as referred to the control medium. The maximum inhibition of the TCB control antibody (10 or 30 nM) compared to the control medium is also measured. Calculations are performed using R 3.1.19 and Bioconductor 2.1310, but for the calculation of IMAX values (Microsoft Excel®; Microsoft Office Professional 2013). An effect is considered statistically significant if the P-value of its corresponding statistical test is <5% (*), <1% (**) or <0.1% (***). BCMA expression is also measured in PBMC CD138+ plasma cells from plasma cell samples from patients with leukemia, and the ratio of effector cells to tumor cells (E:T) is determined. As shown in Figure 20, the results clearly show that there was a significant reduction in viable bone marrow plasma cell leukemic cells with 42-TCBcv (ie, more lysis of bone marrow plasma cell leukemic cells) in two plasma cell samples. - cases of patients with leukemia, as compared to a means of control. Table 27 demonstrates the maximum leukemic cell inhibition percentage of plasma cells from bone marrow aspirates or peripheral blood induced induced by 10 nM (IMAX10) and 1 nM (IMAX1) of anti-BCMA/anti-CD3 T cell bispecific antibodies in relation to the means of control. The results demonstrate that 42-TCBcv is very potent to induce leukemic cell death from bone marrow plasma cells. Despite the specific lysis of leukemic cells from bone marrow plasma cells induced by bispecific anti-BCMA/anti-CD3 T cell antibodies and observed bone marrow samples (PCL 1 patient), the bone marrow microenvironment (BMME) was not affected in the respective samples (data not shown). Table 27. IMAX10 and IMAX1 values in relation to maximal inhibition of plasma cell leukemic cell growth at 10 nM (IMAX10) and inhibition at 1 nM (IMAX1) based on iodide-negative viable plasma cell leukemic cells of propidium from patient bone marrow aspirates in the presence of anti-BCMA/anti-CD3 T cell bispecific antibodies.
Example 20: Redirected T cell cytotoxicity of plasma cells from bone marrow of patient with AL amyloidosis in the presence of autologous T cells induced by bispecific anti-BCMA/anti-CD3 T cell antibodies as measured by flow cytometry [000357] AL amyloidosis is a rare disease caused by a bone marrow disorder, which usually affects people between the ages of 50 and 80 and two thirds of patients are men. Amyloidosis is reflected by an abnormal production of antibody/immunoglobin protein by plasma cells. In AL amyloidosis, the antibody light chains (CL) are defectively folded and the result of the defective LC protein is amyloid formation. These defective amyloid proteins are deposited in tissues, nerves and organs. As amyloid builds up in an organ, nerve, or tissue, it gradually causes damage and affects its function. Patients with AL amyloidosis are often affected with more than one organ. Since BCMA plays an essential role in the survival of normal plasma cells, it is highly warranted to evaluate the effect of anti-BCMA/anti-CD3 T cell bispecific antibodies on plasma cell death in AL amyloidosis. Freshly taken whole bone marrow samples/aspirates taken with AL amyloidosis are directly exposed to anti-BCMA/anti-CD3 TCB antibodies or stained with CD138 magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany), passed through a autoMACS cell separation column and the harvested portions with sufficient number of remaining AL amyloidosis plasma cells of generally > 4% are used for further experiments. In 24-well plates, 500,000 cells/well are incubated and cultured for 48 hours. Anti-BCMA/anti-CD3 TCB antibodies and control antibody dilutions are added to the respective wells for a final TCB concentration of 0.1 pM to 30 nM. Each dose point is done in triplicate. The viability of plasma cells and cells in the bone marrow microenvironment is investigated by means of propidium iodide/CD138-FITC of two dyes with flow cytometry (FACSCalibur; Becton Dickinson). Data analysis is performed using FACSDiva software (Becton Dickinson). The mean values are normalized to the mean over the triplicates of the respective control medium (MC). For statistical analysis, a one-sided t-test is used. The maximum inhibition of PCL cell growth at a concentration of 10 nM (IMAX10) and the measured inhibition at 1 nM (IMAX1), respectively, are given in percentage as referred to the control medium. The maximum inhibition of the TCB control antibody (10 or 30 nM) compared to the control medium is also measured. Calculations are performed using R 3.1.19 and Bioconductor 2.1310, but for the calculation of IMAX values (Microsoft Excel®; Microsoft Office Professional 2013). An effect is considered statistically significant if the P-value of its corresponding statistical test is <5% (*), <1% (**) or <0.1% (***). BCMA expression is also measured in CD138+ plasma bone marrow cells from samples from patients with AL amyloidosis, and the ratio of effector cells to tumor cells (E:T) is determined.

权利要求:
Claims (7)
[0001]
1. Monoclonal antibody that specifically binds to human B cell maturation antigen (BCMA), characterized in that it comprises: (a) a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ ID NO:22, a CDR3H region of SEQ ID NO: 17, a CDR1L region of SEQ ID NO: 27, a CDR2L region of SEQ ID NO: 28 and a CDR3L region of SEQ ID NO: 20, (b) a CDR1H region of SEQ ID NO: : 21, a CDR2H region of SEQ ID NO: 22, a CDR3H region of SEQ ID NO: 17, a CDR1L region of SEQ ID NO: 23, a CDR2L region of SEQ ID NO: 24 and a CDR3L region of SEQ ID NO: 20, (c) a CDR1H region of SEQ ID NO:21, a CDR2H region of SEQ ID NO:22, a CDR3H region of SEQ ID NO:17, a CDR1L region of SEQ ID NO:25, a CDR2L region of SEQ ID NO: 26 and a CDR3L region of SEQ ID NO: 20, (d) a CDR1H region of SEQ ID NO: 29, a CDR2H region of SEQ ID NO: 30, a CDR3H region of SEQ ID NO: 17, a region CDR1L of SEQ ID NO: 31, a CDR2L region of SEQ ID NO: 32 and a CDR3L region of SEQ ID: 20, (e) a CDR1H region of SEQ ID NO: 34, a CDR2H region of SEQ ID NO: 35, a CDR3H region of SEQ ID NO: 17, a CDR1L region of SEQ ID NO: 31, a CDR2L region of SEQ ID NO: 32 and a CDR3L region of SEQ ID NO: 20, or (f) a CDR1H region of SEQ ID NO: 36, a CDR2H region of SEQ ID NO: 37, a CDR3H region of SEQ ID NO: 17, a CDR1L region of SEQ ID NO:31, a CDR2L region of SEQ ID NO:32 and a CDR3L region of SEQ ID NO:20.
[0002]
2. Antibody according to claim 1, characterized in that it comprises, as VH region, a VH region of SEQ ID NO:10 and, as VL region, a VL region selected from the group consisting of VL regions of SEQ ID NO: 12, 13 and 14.
[0003]
3. Bispecific antibody that specifically binds BCMA and CD3, characterized in that it comprises a heavy chain and light chain set selected from the group consisting of the polypeptides: (i) SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO:50 and SEQ ID NO:51 (2x), (ii) SEQ ID NO:48, SEQ ID NO:52, SEQ ID NO:53 and SEQ ID NO:54 (2x), and (iii) ) SEQ ID NO: 48, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 (2x).
[0004]
4. Pharmaceutical composition, characterized in that it comprises an antibody as defined in any one of claims 1 to 3 and a pharmaceutically acceptable excipient.
[0005]
5. Pharmaceutical composition, characterized in that it comprises an antibody as defined in any one of claims 1 to 3 for use as a medicine, optionally wherein the pharmaceutical composition is for use as a medicine in the treatment of plasma cell disorders.
[0006]
6. Pharmaceutical composition, characterized in that it comprises an antibody as defined in any one of claims 1 to 3 for use as a medicine in the treatment of multiple myeloma or systemic lupus erythematosus, plasma cell leukemia or AL-amyloidosis.
[0007]
7. Use of the pharmaceutical composition as defined in any one of claims 4 to 6, characterized in that it is in the preparation of a drug in the treatment of multiple myeloma or systemic lupus erythematosus, plasma cell leukemia or AL-amyloidosis.

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法律状态:
2019-11-12| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-03-10| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2020-12-15| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-03-23| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-05-11| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 03/08/2016, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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EP15179549.9|2015-08-03|

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EP15179549|2015-08-03|

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