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    abstract " single chain trail receptor agonist proteins " specific trail receptor agonist proteins, nucleic acids that encode them, and methods for treating a subject with a trail-associated disease or disorder are provided in this report. the trail receptor agonist proteins provided in this report comprise three soluble trail domains and an fc fragment. trail receptor agonist proteins are substantially non-aggregating and suitable for therapeutic, diagnostic and / or research applications.
    公开号:BR112016024515B1
    申请号:R112016024515-6
    申请日:2015-04-23
    公开日:2020-03-31
    发明作者:Oliver Hill；Christian Gieffers；Meinolf Thiemann；Fritz G. Buchanan；Darren C. Phillips；Susan E. Lappe
    申请人:Apogenix Ag；Abbvie Inc.；
    IPC主号:
    专利说明:

    “TRAIL RECEPTOR AGONIST PROTEINS”
    RELATED APPLICATIONS [001] This application claims the benefit of priority to US Provisional Patent Application No. 61 / 983,152, filed 23 April 2014, which is fully incorporated by reference herein.
    FIELD OF THE INVENTION [002] The present invention provides specific TRAIL receptor agonist proteins comprising three soluble TRAIL domains and an Fc fragment, nucleic acid molecules encoding the TRAIL receptor agonist proteins and uses thereof. TRAIL receptor agonist proteins are substantially non-aggregating and suitable for therapeutic, diagnostic and / or research applications.
    BACKGROUND OF THE INVENTION [003] It is known that the trimerization of the cytokines of the TNF superfamily (TNFSF) is required for effective receptor binding and activation. Trimeric cytokine complexes of the TNF superfamily, however, are difficult to prepare from recombinant monomer units.
    [004] WO 01/49866 and WO 02/09055 disclose recombinant fusion proteins comprising a TNF cytokine and a multimerization component, particularly a protein from the C1q protein family or a collectin. A disadvantage of these fusion proteins is, however, that the trimerization domain usually has a large molecular weight and / or that trimerization is ineffective.
    [005] Schneider et al. (J Exp Med 187 (1989), 1205 to 1213) describe that TNF cytokine trimers are stabilized for N-terminally stabilized reasons. In CD95L, stabilization of the trimer of the receptor binding domain is presumably caused by the N amino acid domains
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    2/64 terminals that are located close to the cytoplasmic membrane.
    [006] Shiraishi et al. (Biochem Biophys Res Commun 322 (2004), 197 to 202) describe that the binding domain to the CD95L receptor can be stabilized by the spiral-wrapped (leucine zipper) α-helical artificial N-terminally motifs. It has been found, however, that the orientation of the polypeptide chains to each other, for example, parallel or antiparallel orientation, can hardly be predicted. In addition, the ideal number of hepta-repetitions in the spiral-wound zipper pattern is difficult to determine. In addition, spiral-wound structures tend to form macromolecular aggregates after changing the pH and / or ionic concentration.
    WO 01/25277 refers to single chain oligomeric polypeptides that bind to a cell receptor extracellular ligand binding domain, wherein the polypeptide comprises at least three receptor binding sites of which at least one is able to bind to a cell receptor ligand binding domain and at least one is unable to effectively bind to a cell receptor ligand binding domain, whereby single-chain oligomeric polypeptides are capable of binding connect to the receiver, but unable to activate the receiver. For example, monomers are derived from cytokine ligands of the TNF family, particularly from TNF-α.
    [008] WO 2005/103077 discloses single chain fusion polypeptides comprising at least three monomers of a member of the TNF linker family and at least two peptide linkers that link the monomers of the members of the TNF linker family to each other. Recent experiments, however, have shown that these single chain fusion polypeptides exhibit unwanted aggregation.
    [009] WO 2010/010051 discloses single chain fusion polypeptides comprising three soluble TNF cytokine domains and at least two
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    3/64 peptide linkers. The described fusion polypeptides are substantially non-aggregating.
    [010] In addition, previous work, including that by Papadopoulos et al. (Cancer Chemother Pharmacol, 2015, DOI 10.1007 / s00280-015-2712-0), has demonstrated that overgrouping the TRAIL receptor can result in toxicity.
    [011] Consequently, there is a need in the art for new TRAIL receptor agonists that exhibit high biological activity, high stability, low toxicity and enable effective recombinant manufacturing.
    SUMMARY OF THE INVENTION [012] The present invention provides specific TRAIL receptor agonist proteins which exhibit low proteolytic degradation, long half-life and low TRAIL receptor supergrouping in vivo (along with concomitant toxicity).
    [013] The TRAIL receptor agonist proteins of the present invention, in general, comprise: (i) a first soluble TRAIL cytokine domain; (ii) a first peptide linker; (iii) a second soluble TRAIL domain; (iv) a second peptide linker; (v) a third soluble TRAIL domain; and (vi) an antibody Fc fragment.
    [014] In one aspect, the present invention provides a single chain fusion polypeptide comprising: (i) a first soluble TRAIL domain, (ii) a first peptide linker, (iii) a second soluble TRAIL domain, (iv) a second peptide linker, (v) a third soluble TRAIL domain and (vi) an antibody Fc fragment. In one embodiment, the antibody Fc fragment (vi) is located N-terminal to the first TRAIL domain (i) and / or Cterminal to the third TRAIL domain (v). In another embodiment, the antibody Fc fragment is located C-terminally to the third TRAIL (v) domain. In one embodiment, the polypeptide is substantially not
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    4/64 aggregator. In another embodiment, the second and / or third soluble TRAIL domains are an N-terminally shortened domain that optionally comprises amino acid sequence mutations.
    [015] In one embodiment, at least one among the soluble TRAIL domains, particularly, at least one between the soluble TRAIL domains (iii) and (v), is a soluble TRAIL domain with an N-terminal sequence that starts between the human TRAIL amino acid Gln120 and Val122 where Arg121 can be replaced by a neutral amino acid, for example, Ser or GIy. In another embodiment, at least one among the soluble TRAIL domains, particularly, at least one between the soluble TRAIL domains (iii) and (v), is a soluble TRAIL domain with an N-terminal sequence selected from ( a) Arg121 - Val122 - Ala123 and (b) (Gly / Ser) 121 - Val122 - Ala123. In one embodiment, the soluble TRAIL domain ends with the human TRAIL amino acid Gly281 and / or optionally comprises a mutation at positions R130, G160, H168, R170, H177, Y189, R191, Q193, E195, N199, K201, Y213 , T214, S215, H264, I266, D267 or D269 or in two or more between said positions. In one embodiment, the soluble TRAIL domain (i) consists of the human TRAIL amino acids Gln120 - Gly281, according to SEQ ID NO: 1, and the soluble TRAIL domains (iii) and (v) consist of the amino acids Arg121 - Human TRAIL gly281, according to SEQ ID NO: 1.
    [016] In one embodiment, the first and second peptide linkers (ii) and (iv), independently, have a length of 3 to 8 amino acids, particularly a length of 3, 4, 5, 6, 7 or 8 amino acids, and are preferably glycine / serine linkers, optionally comprising an asparagine residue that can be glycosylated. In one embodiment, the first and second peptide linkers (ii) and (iv) consist of the amino acid sequence, according to SEQ ID NO: 2. In another embodiment, the
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    The polypeptide additionally comprises a N-terminal signal peptide domain, for example, of SEQ ID NO: 12, which may comprise a protease cleavage site and / or which additionally comprises a C-terminal element that can understand and / or connect to a recognition / purification domain, for example, a Strep tag, according to SEQ ID NO: 13.
    [017] In one embodiment, the antibody Fc fragment (vi) is fused to the soluble TRAIL domain (i) and / or (v) via a hinge linker, preferably of SEQ ID NO: 11. In in another embodiment, the antibody Fc fragment (vi) consists of the amino acid sequence shown in SEQ ID NO: 10 or 17. In one embodiment, the polypeptide comprises the amino acid sequence of SEQ ID NO: 14, 15 or 18.
    [018] In another aspect, the present invention provides a TRAIL receptor agonist protein comprising a polypeptide that has the amino acid sequence shown in SEQ ID NO: 19, 20 or 21.
    [019] In another aspect, the present invention provides a TRAIL receptor agonist protein comprising a polypeptide that has the amino acid sequence shown in SEQ ID NO: 26, 27, 28, 29 or 30.
    [020] In another aspect, the present invention provides a TRAIL receptor agonist protein comprising two polypeptides having the amino acid sequence shown in SEQ ID NO: 19. In one embodiment, the two polypeptides are covalently linked through three disulfide bonds between chains formed between cysteine residues 513, 519 and 522 of each polypeptide.
    [021] In one embodiment, one or more asparagine residues at positions 168 and 337 of the polypeptides are N-glycosylated. In another embodiment, the asparagine residues at positions 168 and 337 of the polypeptides are N-glycosylated.
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    6/64 [022] In another embodiment, polypeptides are still post-institutionally modified. In another embodiment, the post-translational modification comprises the N-terminal glutamine modified in pyroglutamate.
    [023] In another aspect, the present invention provides a pharmaceutical composition comprising a TRAIL receptor agonist protein disclosed in this report and one or more pharmaceutically acceptable carriers, diluents, excipients and / or adjuvants.
    [024] In another aspect, the present invention provides a nucleic acid molecule that encodes the TRAIL receptor agonist protein. In another embodiment, the present invention provides an expression vector comprising the nucleic acid molecule. In another embodiment, the present invention provides a cell comprising the nucleic acid molecule. In another embodiment, the cell is a eukaryotic cell. In another embodiment, the cell is a mammalian cell. In another embodiment, the cell is a Chinese Hamster Ovary (CHO) cell. In other embodiments, the cell is selected from the group consisting of CHO-DBX11, CHO-DG44, CHO-S and CHO-K1 cells. In other embodiments, the cell is selected from the group consisting of cells Vero, BHK, HeLa, COS, MDCK, HEK-293, NIH-3T3, W138, BT483, Hs578T, HTB2, BT20, T47D, NS0, CRL7030, HsS78Bst, PER.C6, SP2 / 0-Agl4 and hybridoma.
    [025] In another aspect, the present invention provides a method for treating a subject who has a TRAIL-associated disease or disorder, the method comprising administering to the subject an effective amount of the TRAIL receptor agonist protein. In one embodiment, the TRAIL receptor agonist protein is administered alone. In another embodiment, the TRAIL receptor agonist protein is administered before, concurrently or after the administration of a second agent. In another embodiment,
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    7/64 the disease or disorder is selected from the group consisting of: tumors, infectious diseases, inflammatory diseases, metabolic diseases, autoimmune disorders, degenerative diseases, diseases associated with apoptosis and transplant rejections. In one embodiment, the tumors are solid tumors. In one embodiment, tumors arise from the group of cancers that consist of sarcoma, oesophageal cancer and gastric cancer. In another embodiment, the tumors arise from Ewing's sarcoma or fibrosarcoma. In another embodiment, tumors arise from the group of cancers consisting of non-small cell lung carcinoma (NSCLC), pancreatic cancer, colorectal cancer, breast cancer, ovarian cancer, head and neck cancers, and lung cancer small cell (SCLC). In another embodiment, the tumors are lymphatic tumors. In one embodiment, the tumors are haematological tumors. In another embodiment, tumors arise from non-Hodgkin's lymphoma, leukemia, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B cell lymphoma, Burkitt's lymphoma, chronic myelocytic leukemia (CML), leukemia chronic lymphocytic (CLL) or hairy cell leukemia. In another embodiment, autoimmune disorders are rheumatoid diseases, arthritic diseases, or rheumatoid and arthritic diseases. In another embodiment, the disease or disorder is rheumatoid arthritis. In another embodiment, the degenerative disease is a neurodegenerative disease. In another embodiment, the neurodegenerative disease is multiple sclerosis.
    [026] In one embodiment, the second agent is a chemotherapeutic, radiotherapy or biological agent. In one embodiment, the second agent is selected from the group consisting of Duvelisibe, Ibrutinibe, Navitoclax and Venetoclax. In another embodiment, the second agent is an apoptotic agent. In one embodiment, the second apoptotic agent is selected from the group consisting of Bortezomibe, Azacitidine, Dasatinib and
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    8/64
    Gefitinib. In a particular embodiment, the pharmaceutical compositions disclosed in this report are administered to a patient via intravenous or subcutaneous administration. In other embodiments, the disclosed pharmaceutical compositions are administered to a patient by oral, parenteral, intramuscular, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelíaca, intracerebellar, intracerebroventricular, intracolonic, intracervical, intragastric, intrahepatic , intramiocardial, intraosteal, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, in bolus, vaginal, petal, buccal, subalual, sublingual.
    [027] In one embodiment, the TRAIL receptor agonist protein is administered as a single bolus. In another embodiment, the TRAIL receptor agonist protein can be administered in several divided doses. The TRAIL receptor agonist protein can be administered at about 0.1 to 100 mg / kg. In one embodiment, the TRAIL receptor agonist protein can be administered in a dosage selected from the group consisting of: about 0.1 to 0.5, 0.1 to 1, 0.1 to 10, 0 , 1 to 20, 0.1 to 50, 0.1 to 75, 1 to 10, 1 to 15, 1 to 7.5, 1.25 to 15, 1.25 to 7.5, 2.5 to 7 , 5, 2.5 to 15, 5 to 15, 5 to 7.5, 1 to 20, 1 to 50, 7 to 75, 1 to 100, 5 to 10, 5 to 15, 5 to 20, 5 to 25 , 5 to 50, 5 to 75, 10 to 20, 10 to 50, 10 to 75 and 10 to 100 mg / kg. In other embodiments, the TRAIL receptor agonist protein is present in pharmaceutical compositions at about 0.1 to 100 mg / ml. In one embodiment, the TRAIL receptor agonist protein is present in pharmaceutical compositions in an amount selected from the group consisting of: about 0.1 to 0.5, 0.1 to 1, 0.1 to 10 , 0.1 to 20, 0.1 to 50, 0.1 to 75, 1 to 10, 1 to 20, 1 to 50, 1 to 75, 1 to 100, 5 to 10, 5 to 15, 5 to 20 , 5 to 25, 5 to 50, 5 to 75, 10 to 20, 10 to 50, 10 to 75 or 10 to 100 mg / ml. In other embodiments, a
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    9/64 therapeutically effective amount of the TRAIL receptor agonist protein is administered to a subject. In another embodiment, a prophylactically effective amount of the TRAIL receptor agonist protein is administered to a subject.
    DESCRIPTION OF THE FIGURES
    Figure 1.
    [028] Domain structure of a single chain fusion polypeptide comprising three TRAIL domains. I., II., III. Soluble TRAIL domains.
    Figure 2.
    [029] Schematic photograph representing the general structure of TRAIL.
    Cell membrane, N-terminal located inside the cell, antiparallel β fold of the receptor binding domain (RBD),
    2. RBD interface and cell membrane,
    3. Protease cleavage site.
    Figure 3.
    [030] Schematic photograph representing the structure of the native TRAIL trimer. The cylindrical structures represent RBDs. N-terminals connect RBDs with the cell membrane.
    Figure 4.
    [031] Schematic photograph representing the structure of three soluble domains comprising the TRAIL receptor binding domain. I., II., III. Soluble TRAIL domains.
    Figure 5.
    [032] Trimerization of the soluble domains comprising the RBD of TRAIL, characterized by the fact that the N- and C-terminals of the three soluble domains form a surface.
    Figure 6.
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    10/64 [033] Schematic photograph depicting the single-chain TRAIL structure comprising all or part of the stem region illustrating the requirement for longer linkers to compensate for the distance to the N-terminus of the next soluble domain.
    Figure 7.
    [034] ScFv-TRAIL fusion protein known from the art.
    Figure 8.
    [035] Fc-TRAIL fusion protein known from the art.
    Figure 9A.
    [036] Single chain fusion polypeptide comprising an additional Fab antibody fragment.
    Figure 9B.
    [037] Single chain fusion polypeptide comprising an additional scFv antibody fragment.
    Figure 10.
    [038] Dimerization of two N-terminally fused scFc fusion polypeptides using disulfide bridges.
    Figure 11.
    [039] Dimerization of two C-terminally fused scFc fusion polypeptides using disulfide bridges.
    Figure 12.
    [040] Dimerization of single chain fusion polypeptides by means of a linker.
    Figure 13.
    [041] Single chain fusion polypeptide comprising an additional Fab antibody fragment further fused to a second fusion polypeptide or scFv fusion polypeptide.
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    11/64
    Figure 14.
    [042] Dimerization of two scFab fusion polypeptides using disulfide bridges.
    Figure 15.
    [043] N-terminally fused scFc fusion polypeptides still comprising an Fv and / or Fab antibody fragment.
    Figure 16.
    [044] C-terminally fused scFc fusion polypeptides still comprising an Fv and / or Fab antibody fragment.
    Figure 17A.
    [045] The exemplary TRAIL receptor agonist protein shown with the N-terminal signal peptide domain is shown in SEQ ID NO: 14. The mature protein (which does not include the N-terminal signal peptide domain) is shown in SEQ ID NO: 19.
    Figure 17B.
    [046] Schematic photograph depicting the overall structure and annotated sequence of an exemplary TRAIL receptor agonist protein.
    Figure 18.
    [047] Adjustment of the ELISA assay for the quantification of TRAIL receptor agonists containing an FC domain.
    Figure 19.
    [048] A TRAIL receptor agonist protein comprising two polypeptides having the amino acid sequence shown in SEQ ID NO: 19 induces cell death in human tumor cell lines in vitro. SKM-1, Colo205 or Jurkat cells were treated with increased concentrations of the TRAIL receptor agonist protein for 24 hours and cell viability assessed.
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    12/64
    Figures 20 (A to C).
    [049] A TRAIL receptor agonist protein comprising two polypeptides that have the amino acid sequence shown in SEQ ID NO: 19 synergizes with antitumor agents in vitro. SU-DHL-4 cells were incubated with increased concentrations of the TRAIL receptor agonist protein in the presence or absence of the indicated concentrations of venetoclax (Figure 20A) or navitoclax (Figure 20B) for 24 hours. Alternatively, (Figure 20C) NCI-H596 cells were treated with increased concentrations of the TRAIL receptor agonist protein in the presence or absence of the indicated concentrations of docetaxel (DTX) for 72 hours. Cell viability was assessed and synergy determined by the sum of Bliss.
    Figure 21.
    [050] Effect of the TRAIL receptor agonist protein comprising two polypeptides that present the amino acid sequence shown in SEQ ID NO: 19 on tumor growth in the Colo205 colorectal carcinoma xenograft model.
    Figure 22.
    [051] Effect of the TRAIL receptor agonist protein comprising two polypeptides that present the amino acid sequence shown in SEQ ID NO: 19 on tumor growth in the SKM-1 acute myeloid leukemia xenograft model.
    Figure 23.
    [052] Effect of the TRAIL receptor agonist protein comprising two polypeptides that have the amino acid sequence shown in SEQ ID NO: 19 on tumor growth in the H460LM non-small cell lung xenograft model.
    Figures 24 (A to G).
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    13/64 [053] Effect of the TRAIL receptor agonist protein comprising two polypeptides that have the amino acid sequence shown in SEQ ID NO: 19 on tumor growth in PDX models. Lozenges, treated with the TRAIL receptor agonist protein; Squares, untreated. Tumor volumes are shown for (A) CTG-0069, (B) CTG-0167, (C) CTG-0293, (D) CTG0785, (E) CTG-0714, (F) CTG-0136 and (G) CTG-0485.
    DETAILED DESCRIPTION OF THE INVENTION [054] In accordance with the present invention, it has been found that the fusion of a single-chain TRAIL receptor binding domain to an Fc domain results in a hexavalent TRAIL receptor agonist providing high biological activity combined with satisfactory stability . Consequently, a single chain fusion polypeptide comprising at least three soluble TRAIL domains connected by two peptide linkers and N-terminally and / or Cly terminally an antibody Fc fragment, is provided.
    [055] Preferably, the single chain fusion polypeptide is non-aggregating. The term "non-aggregator" refers to a monomer content of the preparation of> 50%, preferably> 70%, and, more preferably,> 90%. The ratio of the monomer content to the aggregate content can be determined by examining the amount of aggregate formation using size exclusion chromatography (SEC). Stability with respect to aggregation can be determined by SEC after defined periods of time, for example, from a few to several days, to weeks and months under different storage conditions, for example, at 4 ° C or 25 ° C . For the fusion protein, in order to be classified as substantially non-aggregating, it is preferred that the monomer content be as defined above after a period of time of several days, for example, 10 days, more preferably, after several weeks, for example, 2, 3 or 4 weeks, and, more preferably, after several months, for example, 2 or
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    14/64 months of storage at 4 ° C or 25 ° C.
    [056] The single-stranded fusion polypeptide may comprise additional domains that may be located at the N- and / or C-terminals thereof. Examples for additional fusion domains are, for example, an N-terminal signal peptide domain that can comprise a protease cleavage site or a C-terminal element that can comprise and / or connect to a recognition / purification domain. According to a preferred embodiment, the fusion polypeptide comprises a Strep tag at its C-terminus which is fused via a linker. An exemplary Strep tag including a short serine linker is shown in SEQ ID NO: 13.
    [057] The TRAIL receptor agonist protein of the present invention comprises three soluble domains derived from TRAIL. Preferably, such soluble domains are derived from a mammal, particularly human TRAIL, including allelic variants and / or derivatives thereof. Soluble domains comprise the extracellular portion of TRAIL, including the receptor binding domain without domains located on the membrane. Like other proteins in the TNF superfamily, TRAIL is anchored to the membrane through an N-terminal portion of 15 to 30 amino acids, the so-called stem region. The stem region contributes to trimerization and provides a certain distance to the cell membrane. However, the stem region is not part of the receptor binding domain (RBD).
    [058] Importantly, RBD is characterized by a particular location of its N- and C-terminal amino acids. Said amino acids are immediately adjacent and are located centrally to the axis of the trimer. The first N-terminal amino acids of the RBD form a beta antiparallel strip with the C-terminal amino acids of the RBD (Figs. 2 and 3).
    [059] Thus, the RBD antiparallel beta tape forms an interface with the
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    15/64 cell membrane, which is connected to, and anchored within the cell membrane by means of the amino acids in the stem region. It is highly preferred that the soluble TRAIL domains of the TRAIL receptor agonist protein comprise a TRAIL receptor binding domain devoid of any amino acids from the stem region (Figs. 4 and 5). Otherwise, a long link connecting the C-terminal of one of the soluble domains to the N-terminus of the next soluble domain would be required to compensate for the N-terminal stem region of the next soluble domain (Figure 6), which can result instability and / or formation of aggregates.
    [060] Another advantage of such soluble domains is that the N- and C-terminal amino acids of the RBD are not accessible for any anti-drug antibodies. Preferably, the single chain fusion polypeptide is capable of forming an ordered trimeric structure comprising at least one functional binding site for the respective TRAIL receptor.
    [061] The TRAIL receptor agonist protein comprises three functional TRAIL receptor binding sites, that is, amino acid sequences capable of forming a complex with a TRAIL receptor. Thus, the soluble domains are able to bind to the corresponding TRAIL receptor. In one embodiment, at least one of the soluble domains is capable of activating the receptor, whereby apoptotic and / or proliferative activity can be affected. In another embodiment, one or more soluble domains are selected as not being able to activate the receptor.
    [062] The soluble TRAIL domain can be derived from human TRAIL, as shown in SEQ ID NO: 1. Preferably, the soluble TRAIL domains are derived from human TRAIL, particularly starting from amino acids 120 to 122 and comprise, in particular, amino acids 120 to 281, 121 to 281 or 122 to 281 of SEQ ID NO: 1. Optionally, the amino acid Arg121 of SEQ ID NO: 1
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    16/64 can be replaced by an uncharged amino acid, for example, Ser or Gly.
    [063] Table 1: Human TRAIL Protein Sequence
    SEQ ID NO Sequence 1 MAMMEVQGGPSLGQTCVLIVIFTVLLQSLCVAVTYVYFTNELK QMQDKYSKSGIACFLKEDDSYWDPNDEESMNSPCWQVKWQ LRQLVRKMILRTSEETISTVQEKQQNISPLVRERGPQRVAAHI TGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLH LRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIY KYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKEND RIFVSVTNEHLIDMDHEASFFGAFLVG
    [064] As indicated above, soluble TRAIL domains can comprise wild type sequences, as indicated in SEQ ID NO: 1. It should be noted, however, that it is possible to introduce mutations in one or more between these soluble domains, for example , mutations that alter (for example, increase or decrease) the binding properties of soluble domains. In one embodiment, the soluble domains can be selected, which cannot bind to the corresponding cytokine receptor.
    [065] In another preferred embodiment of the invention, the soluble TRAIL domain (i) comprises a TRAIL mutant or a receptor binding domain thereof that binds to, and / or activates the TRAIL 1 receptor (TRAILR1) and / or TRAIL 2 receiver (TRAILR2). The binding and / or activity of the mutant can, for example, be determined by the assays described in van der Sloot et al. (PNAS, 2006, 103: 8634 - 8639), Kelley et al. (J. Biol. Chem., 2005, 280: 2205 to 2215) or MacFarlane et al. (Cancer Res., 2005, 65: 11265 to 11270).
    [066] The mutant can be generated by any technique and is known to the qualified person, for example, the techniques described in van der Sloot et al. (PNAS, 2006, 103: 8634 to 8639), Kelley et al. (J. Biol. Chem., 2005, 280: 2205 to 2215) or MacFarlane et al. (Cancer Res., 2005, 65: 11265 to 11270) and can comprise any type of structural mutations, for example, substitution, deletion,
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    17/64 duplication and / or insertion of an amino acid. A preferred embodiment is the generation of substitutions. The substitution can affect at least one TRAIL amino acid or a receptor binding domain thereof, as described in this report. In a preferred embodiment, the substitution can affect at least one among the TRAIL amino acids, for example, human TRAIL (for example, SEQ ID NO: 1). Preferred substitutions in this regard affect at least one of the following human TRAIL amino acids of SEQ ID NO: 1: R130, G160, Y189, R191, Q193, E195, N199, K201, Y213, T214, S215, H264, 1266, D267, D269. Preferred amino acid substitutions of human TRAIL of SEQ ID NO: 1 are at least one of the following substitutions: R130E, G160M, Y189A, Y189Q, R191K, Q193S, Q193R, E195R, N199V, N199R, K201R, Y213W, T214R, S215, H264R, I266L, D267Q, D269H, D269R or D269K.
    [067] Amino acid substitutions may affect the binding and / or activity of TRAIL, for example, human TRAIL, a, or in TRAILR1 or TRAILR2. Alternatively, amino acid substitutions can affect the binding and / or activity of TRAIL, for example, human TRAIL, a, or in TRAILR1 and TRAILR2. The binding and / or activity of TRAILR1 and / or TRAILR2 can be positively affected, that is, stronger, more selective or more specific binding and / or more activation of the receptor. Alternatively, TRAILR1 and / or TRAILR2 binding and / or activity may be negatively affected, i.e., weaker, less selective or less specific binding and / or less or no activation of the receptor.
    [068] Examples of TRAIL mutants with amino acid substitutions of the invention that affect the binding and / or activation of TRAILR1 and TRAILR2 can be found, for example, in Table 1 of MacFarlane et al. (as above) and can comprise a human TRAIL mutant with the following two amino acid substitutions of SEQ ID NO: 1 Y213W and S215D or with the following single amino acid substitution: Y189A.
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    18/64 [069] Examples of TRAIL mutants with amino acid substitutions of the invention that affect the binding and / or activation of TRAILR1 can be found, for example, in Table 1 of MacFarlane et al. (as above) and can comprise a human TRAIL mutant with the following four amino acid substitutions of SEQ ID NO: 1 N199V, K201R, Y213W and S215D or with the following five amino acid substitutions: Q193S, N199V, K201R, Y213W and S215D , or can be found in Table 2 by Kelley et al. (as above) and can comprise a human TRAIL mutant with the following six amino acid substitutions: Y213W, S215D, Y189A, Q193S, N199V and K201R, or with Y213W, S215D, Y189A, Q193S, N199R and K201R.
    [070] Examples of TRAIL mutants with amino acid substitutions of the invention that affect the binding and / or activation of TRAILR2 can be found, for example, in Table 1 of MacFarlane et al. (as above) or in Table 2 by Kelley et al. (as above) and can comprise a human TRAIL mutant with the following six amino acid substitutions of SEQ ID NO: 1: Y189Q, R191 K, Q193R, H264R, I266L and D267Q or can be found in Table 2 by van der Sloot et al. (as above) and can comprise a human TRAIL mutant with the following single amino acid substitution: D269H or with the following two amino acid substitutions: D269H and E195R or D269H and T214R.
    [071] Thus, a preferred embodiment is a TRAIL receptor agonist protein, as described in this report, in which at least one of the soluble domains comprises a mutant of TRAIL or a receptor binding domain thereof that binds a, and / or activates TRAILR1 and / or TRAILR2.
    [072] Other examples of TRAIL mutants, which show reduced TRAIL-induced receptor aggregation are H168 (S, T, Q), R170 (E, S, T, Q) and H177 (S, T).
    [073] A preferred embodiment of a protein agonist of
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    19/64 TRAIL receptor comprising a mutant of TRAIL or a receptor binding domain, as described in this report, is a TRAIL receptor agonist protein in which component (i) comprises at least one amino acid substitution, particularly as indicated below.
    [074] Such an amino acid substitution affects at least one of the following amino acid positions of human TRAIL (SEQ ID NO: 1): R130, G160, H168, R170, H177, Y189, R191, Q193, E195, N199, K201, Y213, T214, S215, H264, 1266, D267, D269.
    [075] Such an amino acid substitution is at least one of the following: R13OE, G16OM, H168 (S, T, Q), R170 (E, S, T, Q), H177 (SJ) 1 Y189A, Y189Q, R191K, Q193S, Q193R, E195R, N199V, N199R, K201R, Y213W, T214R, S215D, H264R, I266L, D267Q, D269H, D269R or D269K.
    [076] A preferred TRAIL-R2 selective domain comprises amino acid substitutions Y189Q, R191K, Q193R, H264R, I266L and D267Q.
    [077] A preferred TRAIL-R1 selective domain comprises amino acid substitutions Y189A, Q193S, N199V, K201R, Y213W and S215D.
    [078] The single chain fusion molecule of the present invention comprises three soluble TRAIL domains, that is, components (i), (iii) and (v). The stability of a single-chain TRAIL fusion polypeptide against aggregation is enhanced, if the second and / or the third soluble TRAIL domains are an N-terminally shortened domain that optionally comprises amino acid sequence mutations. Thus, preferably, the second and third soluble TRAIL domains are N-terminally shortened domains that optionally comprise amino acid sequence mutations in the N-terminal regions, preferably within the first five amino acids of the N-terminal of the soluble TRAIL domain. These mutations may include the replacement of charged amino acids, for example, acidic or basic, with neutral amino acids,
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    20/64 particularly, serine or glycine.
    [079] In contrast, the selection of the first soluble TRAIL domain is not as critical. In this report, a soluble domain that has a full-length Nterminal sequence can be used. It should also be noted, however, that the first soluble TRAIL domain can have an Nterminally shortened and optionally modified sequence.
    [080] In another preferred embodiment of the present invention, the soluble TRAIL domains (i), (iii) and (v) are soluble human TRAIL domains. The first soluble TRAIL domain (i) can be selected from native, shortened and / or modified sequences. Thus, the first soluble TRAIL domain (i) has an N-terminal sequence that can start between the amino acids GIu116 and Val122 of human TRAIL and where Arg121 can be replaced by a neutral amino acid, for example, by Ser or GIy. The second and third soluble TRAIL domains (iii) and (v) have a shortened N-terminal sequence that preferably starts between the human TRAIL amino acids Gln120 and Val122 and where Arg121 can be replaced by another amino acid, for example , Ser or Gly.
    [081] Preferably, the N-terminal sequence of the soluble TRAIL domains (iii) and (v) is selected from:
    (a) Arg121 - Val122 - Ala123 and (b) (Gly / Ser) 121.
    [082] The soluble TRAIL domain preferably ends with the human TRAIL amino acid Gly281. In certain embodiments, the TRAIL domain can comprise the internal mutations described above.
    [083] Components (ii) and (iv) of the TRAIL receptor agonist protein are elements of the peptide linker located between components (i) and (iii) or (iii) and (v), respectively. The flexible connector elements have a
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    21/64 length of 3 to 8 amino acids, particularly a length of 3, 4, 5, 6, 7 or 8 amino acids. The linker elements are preferably glycine / serine linkers, i.e., peptide linkers that consist substantially of the amino acids glycine and serine. In cases where the soluble cytokine domain ends with S or G (C-terminal), for example, human TRAIL, the linker starts after S or G. In cases where the soluble cytokine domain starts with S or G ( N-terminal), the linker ends before this S or G.
    [084] It should be noted that the connector (ii) and the connector (iv) do not have to be the same length. In order to decrease potential immunogenicity, it may be preferred to use shorter linkers. In addition, it has been found that shorter linkers lead to single chain molecules with a reduced tendency to form aggregates. While binders that are substantially longer than those disclosed in this report, they may exhibit unfavorable aggregation properties.
    [085] If desired, the linker can comprise an asparagine residue that can form a glycosylated Asn-Xaa-Ser site. In certain embodiments, one of the linkers, for example, linker (ii) or linker (iv), comprises a glycosylation site. In other embodiments, the linkers (iv) comprise glycosylation sites. In order to increase the solubility of sc TRAIL proteins and / or in order to reduce potential immunogenicity, it may be preferred that linker (ii) or linker (iv) or both comprise a glycosylation site.
    [086] Preferred linker sequences are selected from GSGSGSGS (SEQ ID NO: 3), GSGSGNGS (SEQ ID NO: 2), GGSGSGSG (SEQ ID NO: 4), GGSGSG (SEQ ID NO: 5), GGSG ( SEQ ID NO: 6), GGSGNGSG (SEQ ID NO: 7), GGNGSGSG (SEQ ID NO: 8), GGNGSG (SEQ ID NO: 9) and GSGS (SEQ ID NO: 23).
    [087] According to a more preferred embodiment, the sequences
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    22/64 of linker are GSGSGNGS, according to SEQ ID NO: 2. Examples of linker sequences are shown in Table 2.
    Table 2: Examples of Linker Strings
    SEQ ID NO Sequence 2 GSGSGNGS 3 GSGSGSGS 4 GGSGSGSG 5 GGSGSG 6 GGSG 7 GGSGNGSG 8 GGNGSGSG 9 GGNGSG 22 GSGSGS 23 GSGS 24 GSG
    [088] The TRAIL receptor agonist protein additionally comprises an antibody Fc fragment domain that may be located N-terminal to the first TRAIL (i) domain and / or C-terminal to the third TRAIL (v) domain. Preferably, the antibody Fc fragment domain comprises or consists of an amino acid sequence shown in SEQ ID NO: 10. Alternatively, the Fc fragment domain comprises or consists of an amino acid sequence shown in SEQ ID NO: 17. Examples of Fc fragment domains are shown in Table 3.
    Table 3: Examples of Fc Fragment Domains
    SEQ ID NO Sequence 10 PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRE EMTKN QVS LTC LVKG FYP DIAVEWESNGQP S and N NYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK 17 PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW
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    LNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSRE EMTKN QVS LTC LVKG FYP S DIAVEWESNGQP AND N NYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEKHH
    [089] The total number of glycosites and the individual position of carbohydrates in three dimensions impact on the in vivo stability of TRAIL receptor agonist proteins. In addition, carbohydrate recognition depends on the local density of terminal saccharides, the branch of the carbohydrate trunk and the relative position of the carbohydrate matter.
    [090] Depletion of carbohydrates from the CH2 domain is necessary in order to avoid cross-linking based on the Fc receptor in vivo and toxicity based on overgrouping the potential TRAIL receptor. In addition, partially degraded carbohydrates reduce the in vivo half-life of TRAIL receptor agonist proteins through the mechanisms triggered by lectin. By reducing the total number of glycosylation sites in the molecule, the resulting compound is less accessible to these mechanisms, increasing the half-life. Consequently, in one embodiment, the overall number of glycosites in the TRAIL receptor agonist proteins of the present invention has been reduced by the depletion of CH2 glycosides, resulting in TRAIL receptor agonist proteins comprising mutations equivalent to N297S (according to the numbering) creating aglicosl-CH2 domains.
    [091] CH2 glycosites present on the inner surface areas normally protect the subdomain from proteases during “open Fc conformation transits” in which the disulfide bonds between hinge chains are reduced and the bond between covalent chains is broken. This allows for the dissociation of CH2 and exposure of the internal surface area towards proteases.
    [092] TRAIL receptor agonist proteins comprising a mutation equivalent to N297S (according to the EU numbering system) creating
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    24/64 an aglycosl-CH2 are therefore probably less proteolytically stable than the equivalent structures with glycosylation of wild type CH2. This would impact the stability of the compound during USP / DSP / storage, where host cell proteases are present and have long-term access to the structure. Consequently, in certain embodiments, the TRAIL receptor agonist lacks CH2 glycosites, but comprises glycosites in the linker sequences of each polypeptide chain (e.g., GSGSGNGS, according to SEQ ID NO: 2). In certain exemplary embodiments, the TRAIL receptor agonist comprises two glycosites per polypeptide chain, for a total of four glycosites.
    [093] According to a preferred embodiment of the invention, the antibody Fc fragment domain is fused via a hinge linker element. The hinge connector element has a length of 10 to 30 amino acids, particularly a length of 15 to 25 amino acids, for example, 22 amino acids. The hinge connector element preferably comprises the hinge region sequence of an immunoglobulin, in this report referred to as "Ig hinge region". The term "Ig hinge region" means any polypeptide comprising an amino acid sequence that shares sequence identity or similarity with a portion of a naturally occurring Ig hinge region sequence that includes cysteine residues in which the disulfide bonds link the two immunoglobulin heavy chains.
    [094] Derivatives and analogues of the hinge region can be obtained by mutations. A derivative or analogue referred to in this report is a polypeptide comprising an amino acid sequence that shares sequence identity or similarity with the full-length wild-type sequence (or naturally occurring protein) except that it has one or more
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    25/64 differences in amino acid sequence attributable to a deletion, insertion and / or substitution. According to the present invention, however, the term "hinge linker" is not limited to those linkers comprising an Ig hinge region or a derivative thereof, but any linkers long enough to allow the domains linked by the linker element hinge reach biologically active confirmation.
    [095] The number of molecules with open Fc conformation in an individual TRAIL receptor agonist protein depends on the number of disulfide bonds between chains present in the hinge region. Consequently, in one embodiment, a third cysteine was introduced into the hinge region of the TRAIL receptor agonist proteins of the present invention, in order to improve the depletion effect of CH2 glycosites.
    [096] In addition, the TRAIL receptor agonist proteins of the invention additionally comprise mutation of the hinge lysine greater than a glycine to reduce proteolytic processing at this site.
    [097] A particularly preferred hinge linker element comprises or consists of the amino acid sequence shown in SEQ ID NO: 11 (Table 4).
    [098] The TRAIL receptor agonist protein may further comprise an N-terminal signal peptide domain, which allows processing, for example, extracellular secretion, into a suitable host cell. Preferably, the N-terminal signal peptide domain comprises a protease cleavage site, for example, a signal peptidase cleavage site and thus can be removed after or during expression to obtain the mature protein. A particularly preferred N-terminal signal peptide domain comprises the amino acid sequence shown in SEQ ID NO: 12 (Table 4).
    [099] In addition, the TRAIL receptor agonist protein may comprise
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    26/64 additionally a C-terminal element having a length, for example, from 1 to 50, preferably 10 to 30 amino acids, which can include or connect to a recognition / purification domain, for example, a FLAG domain, a Strep tag or Strep Il tag domain and / or a polyHis domain. According to a particularly preferred embodiment, the fusion polypeptide comprises a Strep tag fused to the C-terminus via a short serine linker, as shown in SEQ ID NO: 13 (Table 4).
    [0100] An exemplary hinge linker element, Nterminal signal peptide domain and short serine linker are shown in Table 4.
    Table 4: Exemplary domains and linkers
    SEQ ID NO Sequence 11 GPGSSSSSSSGSCDKTHTCPPC 12 METDTLLVFVLLVWVPAGNG 13 SSSSSSAWSHPQFEK 25 GPGSSSSSSGSCDKTHTCPPC
    [0101] According to a particularly preferred embodiment of the invention, the fusion polypeptide comprises three soluble TRAIL domains fused by peptide linker elements of SEQ ID NO: 2. The first soluble TRAIL domain (i) consists of amino acids 120 to 281 of human TRAIL, according to SEQ ID NO: 1, and the soluble TRAIL domains (iii) and (v) consist of amino acids 121 to 281 of human TRAIL, according to SEQ ID NO: 1. Additionally, the polypeptide The fusion domain comprises an antibody Fc fragment domain, according to SEQ ID NO: 10, which is fused C-terminally to the soluble TRAIL domain (v) via a hinge ligand, according to SEQ ID NO: 11. The inventors surprisingly found that this particular fusion polypeptide provides improved biological activity and is particularly stable. The amino acid sequence of an embodiment
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    An exemplary example of a TRAIL receptor agonist protein of the invention is shown in SEQ ID NO: 19.
    [0102] In addition, the fusion polypeptide can comprise an N-terminal signal peptide domain for example, according to SEQ ID NO: 12. A specific example of a TRAIL receptor agonist protein of the invention is shown in SEQ ID NO: 14.
    [0103] According to another preferred embodiment, the fusion polypeptide may additionally comprise a Strep Cterminal tag which is fused to the polypeptide of the invention by means of a short serine linker, as shown in SEQ ID NO: 13. According to this aspect of the invention, the Fc fragment preferably consists of the amino acid sequence shown in SEQ ID NO: 10 or 17. In addition, the Fc fragment can consist of a shorter Fc fragment, for example, including amino acids 1 to 217 of SEQ ID NO: 10. Particularly preferred examples of fusion polypeptides comprising a Strep C-terminal tag are shown in SEQ ID NOs: 15 and 18.
    [0104] The exemplary TRAIL receptor agonist proteins shown in SEQ ID NOs: 14, 15 and 18 comprise an N-terminal signal peptide domain. The signal peptide domain includes amino acids 1 to 20. In each case, the mature protein starts with amino acid 21. The exemplary mature TRAIL receptor agonist proteins of the present invention are shown in SEQ ID NO: 19, 20, 21, 26 , 27, 28, 29 and 30. Exemplary TRAIL receptor agonist proteins described above are shown in Table 5.
    [0105] The TRAIL receptor agonist shown in SEQ ID NO: 19 shows a reduced total number of glycosylation sites (the N297S mutation in the CH2 region providing an aglycosylated CH2 domain), an increased number of disulfide bonds between chains in the hinge region and a lysine mutation
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    28/64 of the upper hinge on a glycine. These changes provide a decrease in potential degradation and overgrouping of the TRAIL receptor (along with concomitant toxicity), while increasing the molecule's half-life. In some embodiments, the N-terminal glutamine is modified into pyroglutamate (Liu et al. 2011, J. Biol. Chem. 286: 11211 to 11217).
    Table 5: Exemplary TRAIL receptor agonist proteins
    SEQ ID NO Sequence 14 METDTLLVFVLLVWVPAGNGQRVAAHITGTRGRSNTLSSPNS KNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYI YSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSAR NSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDH EASFFGAFLVGGSGSGNGSRVAAHITGTRGRSNTLSSPNSK NEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIY SQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARN SCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHE ASFFGAFLVGGSGSGNGSRVAAHITGTRGRSNTLSSPNSKN EKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYS QTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNS CWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEA SFFGAFLVGGPGSSSSSSSGSCDKTHTCPPCPAPELLGGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 15 METDTLLVFVLLVWVPAGNGQRVAAHITGTRGRSNTLSSPNS KNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYI YSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSAR NSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDH EASFFGAFLVGGSGSGNGSRVAAHITGTRGRSNTLSSPNSK NEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIY SQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARN SCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHE ASFFGAFLVGGSGSGNGSRVAAHITGTRGRSNTLSSPNSKN EKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYS QTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNS CWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEA SFFGAFLVGGPGSSSSSSSGSCDKTHTCPPCPAPELLGGPS
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    VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDG VEVHNAKTKPREEQYSSTYRVVSVLTVLHQDWLNGKEYKCK VSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSS SSSSAWSHPQFEK 18 METDTLLVFVLLVWVPAGNGQRVAAHITGTRGRSNTLSSPNS KNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYI YSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSAR NSCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDH EASFFGAFLVGGSGSGNGSRVAAHITGTRGRSNTLSSPNSK NEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIY SQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARN SCWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHE ASFFGAFLVGGSGSGNGSRVAAHITGTRGRSNTLSSPNSKN EKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGFYYIYS QTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNS CWSKDAEYGLYSIYQGGIFELKENDRIFVSVTNEHLIDMDHEA SFFGAFLVGGPGSSSSSSSGSCDKTHTCPPCPAPPVAGPSV FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKGLPSSIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
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    19 QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGH SFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDK QMVQYIYKYTSYPDPILLM KSARNSCWSKDAEYGLYSIYQGGI FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGNGS RVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHS FLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQ MVQYIYKYTSYP DPILLM KSARNSCWS KDAEYG LYSIYQGGIF ELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGNGSR VAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSF LSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQM VQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFE LKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGPGSSSSSSS GSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 20 QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGH SFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDK QMVQYIYKYTSYPDP IIIm KSARNSCWSKDAEYGLYS IYQGGI FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGNGS RVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHS FLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQ MVQYIYKYTSYP DPILLM KSARNSCWS KDAEYG LYSIYQGGIF ELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGNGSR VAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSF LSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQM VQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFE LKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGPGSSSSSSS GSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGSSSSSSAWSHPQFEK
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    21 QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGH SFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDK QMVQYIYKYTSYPDPILLM KSARNSCWSKDAEYGLYSIYQGGI FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGNGS RVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHS FLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQ MVQYIYKYTSYP DPILLM KSARNSCWS KDAEYG LYSIYQGGIF ELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGNGSR VAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSF LSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQM VQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFE LKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGPGSSSSSSS GSCDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK 26 QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGH SFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDK QMVQYIYKYTSYPDP IIIm KSARNSCWSKDAEYGLYS IYQGGI FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGNGS RVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHS FLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQ MVQYIYKYTSYP DPILLM KSARNSCWS KDAEYG LYSIYQGGIF ELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGNGSR VAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSF LSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQM VQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFE LKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGPGSSSSSSG SDKTHTCPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS VLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH EALHNHYTQKSLSLSPGSSSSSSAWSHPQFEK
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    27 QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGH SFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDK QMVQYIYKYTSYPDPILLM KSARNSCWSKDAEYGLYSIYQGGI FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGNGS RVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHS FLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQ MVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIF ELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSRVAAHI TGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLH LRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIY KYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKEND RIFVSVTNEHLIDMDHEASFFGAFLVGGPGSSSSSSSGSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGSSSSSSAWSHPQFEK 28 QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGH SFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDK QMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGI FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGSRV AAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFL SNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMV QYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFEL KENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGSRVAAHI TGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLH LRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIY KYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKEND RIFVSVTNEHLIDMDHEASFFGAFLVGGPGSSSSSSSGSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGSSSSSSAWSHPQFEK
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    29 QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGH SFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDK QMVQYIYKYTSYPDPILLM KSARNSCWSKDAEYGLYSIYQGGI FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGNGS RVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHS FLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQ MVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIF ELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSRVAAHI TGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLH LRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIY KYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKEND RIFVSVTNEHLIDMDHEASFFGAFLVGGPGSSSSSSSGSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK 30 QRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGH SFLSNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDK QMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGI FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGSRV AAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFL SNLHLRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMV QYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFEL KENDRIFVSVTNEHLIDMDHEASFFGAFLVGGSGSGSRVAAHI TGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLH LRNGELVIHEKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIY KYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELKEND RIFVSVTNEHLIDMDHEASFFGAFLVGGPGSSSSSSSGSCDK THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYSSTYRVVSVL TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV YTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK
    [0106] Another aspect of the present invention relates to a nucleic acid molecule that encodes a TRAIL receptor agonist protein, as described in this report. The nucleic acid molecule can be a molecule of
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    DNA, for example, a double-stranded or single-stranded DNA molecule or an RNA molecule. The nucleic acid molecule can encode the TRAIL receptor agonist protein or a precursor thereof, for example, a pro- or pre-form of the TRAIL receptor agonist protein that can comprise a signal sequence or other portions of heterologous amino acids for secretion or purification which are preferably located at the N- and / or C-terminal of the TRAIL receptor agonist protein. The heterologous amino acid moieties can be linked to the first and / or the second domain via a protease cleavage site, for example, a Factor X3 protease cleavage site, thrombin or IgA. A specific example of a nucleic acid sequence of the invention is shown in Table 6 as SEQ ID NO: 16. This nucleic acid molecule encodes the fusion polypeptide of SEQ ID NO: 14.
    Table 6: Nucleic Acid Sequence of TRAIL Receptor Agonist Protein
    SEQ ID NO Sequence 16 gatatcggtaccgccaccatggaaaccgacaccctgctggtgttcgtgctgctcgtgtg ggtgccagccggcaatggacagagagtggccgctcatatcaccggcacccggggc agatctaacaccctgtccagccccaactccaagaacgagaaggccctgggccgga agatcaactcctgggagtcctccagatccggccactcctttctgtccaacctgcacctga gaaacggcgagctggtcatccacgagaagggcttctactacatctactcccagaccta cttcaggtttcaggaagagatcaaagagaacacaaagaacgacaagcagatggtg cagtatatctacaagtacacctcctaccccgaccccatcctgctgatgaagtccgcccg gaactcctgctggtccaaggatgctgagtacggcctgtacagcatctaccagggcggc atcttcgagctgaaagagaacgaccggatcttcgtgtccgtgaccaacgagcacctga tcgacatggaccacgaggccagctttttcggcgcctttctcgtgggcggatccggaagc ggaaacggcagtagagtggctgcccacattaccggaaccagaggccggtccaaca ccctgagcagccctaacagcaaaaatgagaaagctctcgggcgcaagatcaacag ctgggaatctagcagaagcggccacagctttctgagcaatctgcatctgcggaacggc gaactcgtgattcatgagaaggggttttattatatctatagccagacatactttcgattcca ggaggaaatcaaggaaaacaccaaaaatgataaacagatggtccagtacatttata agtataccagctaccctgatcctatcctcctcatgaagtctgccagaaactcttgttggag the caaggacgccgagtatggactgtactctatctatcagggggggatctttgaactcaaag aaacgatcgcatctttgtcagcgtcaccaatgagcatctcattgatatggatcatgaag ctagtttcttcggggcattcctcgtgggaggctccggctctggcaacggatctagagtcg ccgcacacatcacagggaccagaggcagaagcaataccctgtcctccccaaatagt aaaaacgaaaaggcactcggccgcaaaattaattcctgggagagcagcagatccg ggcacagttttctgtctaatctccatctgaggaatggggagctggtgattcacgaaaaag
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    gattttactacatttacagtcagacttactttcgttttcaggaagagattaaggaaaatacc aaaaacgacaagcagatggtccagtacatctataaatacacctcttatcctgacccaat tctgctcatgaagagtgcccgcaacagctgctggtctaaagacgccgaatacgggctg tattccatttaccaggggggaatttttgagctgaaggaaaatgatcggatttttgtctctgtc acaaacgaacacctcatcgatatggatcacgaagcctctttctttggcgccttcctggtc ggaggccctggctcgagttccagctcctcttctggctcctgcgacaagacccacacctg tcccccttgtcctgcccctgaactgctgggcggaccttccgtgttcctgttccccccaaag cccaaggacaccctgatgatctcccggacccccgaagtgacctgcgtggtggtggat gtgtctcacgaggaccctgaagtgaagttcaattggtacgtggacggcgtggaagtgc acaacgccaagaccaagcccagagaggaacagtactcctccacctaccgggtggt gtctgtgctgaccgtgctgcaccaggactggctgaacggcaaagagtacaagtgcaa ggtgtccaacaaggccctgcctgcccccatcgaaaagaccatctccaaggccaagg gccagccccgggaaccccaggtgtacacactgccccctagccgggaagagatgac caagaaccaggtgtccctgacctgcctggtcaagggcttttacccctccgacattgccgt ggaatgggagtccaacggccagcctgagaacaactacaagaccaccccccctgtg ctggactccgacggctcattcttcctgtactccaagctgacagtggacaagtcccggtg gcagcagggcaacgtgttctcctgctccgtgatgcacgaggccctgcaca accactac acccagaagtccctgtccctgagccccggcaaatgatagaagcttgatatc
    [0107] The nucleic acid molecule can be operably linked to an expression control sequence, for example, an expression control sequence that allows expression of the nucleic acid molecule in a desired host cell. The nucleic acid molecule can be located in a vector, for example, a plasmid, a bacteriophage, a viral vector, a chromosomal integration vector, etc. Examples of expression control sequences and suitable vectors are described, for example, by Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press, and Ausubel et al. (1989), Current Protocols in Molecular Biology, John Wiley & Sons or more recent editions.
    [0108] Various expression vector / host cell systems can be used to express the nucleic acid sequences encoding the TRAIL receptor agonist proteins of the present invention. Suitable host cells include, but are not limited to prokaryotic cells, such as bacteria, for example, E.coli, eukaryotic host cells, such as yeast cells, insect cells, plant cells or animal cells, preferably mammals and, more preferably, human cells. In addition
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    The invention relates to a non-human organism transformed or transfected with a nucleic acid molecule described above. Such transgenic organisms can be generated by known methods of genetic transfer, including homologous recombination.
    [0109] Another aspect of the present invention relates to a pharmaceutical or diagnostic composition comprising as the active agent at least one TRAIL receptor agonist protein, a nucleic acid encoding the respective one, therefore, or a transformed or transfected cell, all as described in this report.
    [0110] The term “disease or disorder associated with TRAIL”, as used in this report, is any disease or disorder that can be ameliorated by the addition of a TRAIL receptor agonist. At least one TRAIL receptor agonist protein, therefore nucleic acid encoding the respective, or transformed or transfected cell, all as described in this report, can be used in therapy, for example, in the prophylaxis and / or treatment of disorders caused by, associated with and / or accompanied by TRAIL dysfunction, particularly, proliferative disorders, such as tumors, for example, solid or lymphatic tumors; infectious diseases; inflammatory diseases; metabolic diseases; autoimmune disorders, for example, rheumatoid and / or arthritic diseases; degenerative diseases, for example, neurodegenerative diseases, such as multiple sclerosis; diseases associated with apoptosis or transplant rejections.
    [0111] The term “TRAIL dysfunction”, as used in this report, should be understood as any TRAIL function or expression that deviates from the normal TRAIL function or expression, for example, the overexpression of the TRAIL gene or protein, expression reduced or abolished TRAIL gene or protein compared to the normal physiological expression level of TRAIL,
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    37/64 increased TRAIL, reduced or canceled TRAIL activity, increased TRAIL binding to any binding pairs, for example, to a receptor, particularly a TRAIL receptor or another cytokine molecule, reduced or canceled binding to any pair binding, for example, to a receptor, particularly a TRAIL receptor or another cytokine molecule, compared to normal physiological activity or TRAIL binding.
    [0112] In various embodiments, a method is provided to diagnose and / or treat a human being suffering from a disorder that can be diagnosed and / or treated by targeting TRAIL receptors comprising administering an agonist protein to humans of the TRAIL receptor disclosed in this report, such that the effect on the activity of the target, or targets, in humans is agonistic, one or more symptoms are relieved and / or treatment is obtained. The TRAIL receptor agonist proteins provided in this report can be used to diagnose and / or treat humans suffering from primary and metastatic cancers, including carcinomas of the breast, colon, rectum, lung (eg, small cell lung cancer “SCLC ”And non-small cell lung cancer“ NSCLC ”), oropharyngeal, hypopharyngeal, esophagus, stomach, pancreas, liver, gallbladder and bile duct, small intestine, urinary tract (including kidney, bladder and urothelium), female genital tract ( including cervix, uterus and ovaries, as well as choriocarcinoma and gestational trophoblastic disease), male genital tract (including prostate, seminal vesicles, germ cell testes and tumors), endocrine glands (including thyroid, adrenal and pituitary glands), and skin, as well as hemangiomas, melanomas, sarcomas (including those arising from bone and soft tissue, as well as sarcoma of Kaposi), tumors of the brain, nerves, eyes and meninges (including astrocytomas, gliomas, glioblastomas, retinoblastomas, neuromas, neuroblastomas, Schwannomas and meningiomas), tumors arising from hematopoietic malignancies, leukemia
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    38/64 acute, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), B cell lymphoma, Burkitt's lymphoma, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hair cell leukemia, Hodgkin's lymphoma and non-Hodgkin, DLBCL, follicular lymphomas, hematopoietic malignancies, Kaposi's sarcoma, malignant lymphoma, malignant histiocytosis, malignant melanoma, multiple myeloma, paraneoplastic syndrome / malignancy hypercalcemia or solid tumors.
    [0113] A pharmaceutical composition comprising a TRAIL receptor agonist protein disclosed in this report and a pharmaceutically acceptable carrier is provided. In some embodiments, the pharmaceutical composition comprises at least one additional therapeutic agent for treating a disorder. For example, the additional agent can be a therapeutic agent, a chemotherapeutic agent; an imaging agent, a cytotoxic agent, an angiogenesis inhibitor, a kinase inhibitor (including, but not limited to a KDR and TIE-2 inhibitor), a co-stimulus molecule modulator or an immunological checkpoint inhibitor (including, but not limited to anti-B7.1, antiB7.2, anti-B7.3, anti-B7.4, anti-CD28, anti-B7RP1, CTLA4-Ig, anti-CTLA-4, anti-PD-1, anti-PD-L1, anti-PD-L2, anti-ICOS, anti-LAG-3, anti-Tim3, anti-VISTA, anti-HVEM, anti-BTLA, LIGHT fusion protein, anti-CD137, anti-CD137L , anti-OX40, antiOX40L, anti-CD70, anti-CD27, anti-GAL9, anti-A2AR, anti-KIR, anti-IDO-1, antiCD20), a dendritic cell modulator / antigen presenting cell (including, but not limited to) not limited to anti-CD40, anti-CD40 L, anti-DC-SIGN, anti-Dectin-1, anti-CD301, anti-CD303, anti-CD123, anti-CD207, anti-DNGR1, antiCD205, anti-DCIR , anti-CD206, anti-ILT7), a modulator for Toll-like receptors (including, but not limited to anti-TLR-1, anti-TLR-2, anti-TLR-3 , anti-TLR-4, anti-TLR-4, anti-TLR-5, anti-TLR-6, anti-TLR-7, anti-TLR-8, anti-TLR-9), an adhesion molecule blocker (including, but not limited to, an anti-LFA-1 antibody, an anti-E / L selectin antibody, a small molecule inhibitor), a
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    39/64 anti-cytokine antibody or functional fragment thereof (including, but not limited to an anti-IL-18 antibody, anti-TNF or anti-IL-6 / cytokine receptor), bispecific redirected T cells or cytotoxicity of NK cells (including, but not limited to a BiTE®), a chimeric T cell receptor (CAR-T) based therapy, a T cell receptor based therapy (TCR), a therapeutic cancer vaccine, methotrexate, cyclosporine, rapamycin, FK506, a detectable tag or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroidal anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anesthetic , a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteroid, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressant, a growth hormone, a hormone replacement drug, a pro radiopharmaceutical product, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or analog, a cytokine or a cytokine antagonist.
    [0114] In one embodiment, a method for treating cancer or for preventing or inhibiting metastasis from the tumors described in this report, the TRAIL receptor agonist protein can be used alone or in combination with one or more additional agents, for example, a chemotherapy product, radiation therapy or biological agent. In some embodiments, the agent can include the following: 13-cis-Retinoic acid; 2-CdA; 2-Chlorodeoxyadenosine; 5Azacitidine; 5-Fluorouracil; 5-FU; 6-Mercaptopurine; 6-MP; 6-TG; 6-Thioguanine; Abraxan; Accutane®; Actinomycin-D; Adriamycin®; Adrucil®; Afinitor®; Agrylin®; AlaCort®; Aldesleukin; Alentuzumab; ALIMTA; Alitretinoin; Alkaban-AQ®; Alkeran®; All-transretinoic acid; Alpha Interferon; Altretamine; Amethopterin; Amifostine; Aminoglutetimide; Anagrelide; Anandron ® ; Anastrozole; Arabinosilocytosine; Ara-C Aranesp®; Aredia®; Arimidex®; Aromasin®; Arranon®; Arsenic Trioxide; Arzerra ™;
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    Asparaginase; ATRA; Avastin®; Azacitidine; BCG; BCNU; Bendamustine; Bevacizumab; Bexarotene; BEXXAR®; Bicalutamide; BiCNU; Blenoxane®; Bleomycin; Bortezomib; Busulfan; Busulfex®; C225; Leukovorin Calcium; Campath®; Camptosar®; Camptothecin-11; Capecitabine Carac ™; Carboplatin; Carmustine; Carmustine Wafer; Casodex®; CC-5013; CCI-779; CCNU; CDDP; CeeNU; Cerubidine®; Cetuximab; Chlorambucil; Cisplatin; Citrovorum factor; Cladribine; Cortisone; Cosmegen®; CPT-11; Cyclophosphamide; Cytadren®; Cytarabine; Liposomal cytarabine; Cytosar-U®; Cytoxan®; Dacarbazine; Dacogen; Dactinomycin; Darbepoeitin Alpha; Dasatinib; Daunomycin; Daunorubicin; Daunorubicin Hydrochloride; Liposomal Daunorubicin; DaunoXome ® ; Decadron; Decitabine; Delta-Cortef ® ; Deltasone ® ; Denileucine; Diftitox; DepoCyt ™; Dexamethasone; Dexamethasone acetate; Sodium Dexamethasone Phosphate; Dexasone; Dexrazoxane; DHAD; DIC; Diodex; Docetaxel; Doxil®; Doxorubicin; Liposomal Doxorubicin; Droxia ™; DTIC; DTIC-Dome ® ; Duralone ® ; Duvelisibe; Efudex ® ; Eligard ™; Ellence ™; Eloxatin ™; Elspar ® ; Emcyt ® ; Epirubicin; Epoietin Alpha; Erbitux; Erlotinib; Erwinia L-asparaginase; Stramustine; Ethyol Etopophos®; Etoposid; Etoposide Phosphate; Eulexin®; Everolimo; Evista®; Exemestane; Fareston ® ; Faslodex ® ; Femara ® ; Filgrastim; Floxuridine; Fludara ® ; Fludarabine; Fluoroplex®; Fluorouracil; Fluorouracil (cream); Fluoxymesterone; Flutamide; Folinic Acid; FUDR ® ; Fulvestrante; Gefitinib; Gemcitabine; Gentuzumab ozogamycin; Genzar; Gleevec ™; Gliadel® wafer; GM-CSF; Goserelin; Granulocyte Colony Stimulating Factor (G-CSF); Macrophage Granulocyte Colony Stimulating Factor (G-MCSF); Halotestin ® ; Herceptin ® ; Hexadrol; Hexalen ® ; Hexamethylmelamine; HMM; Hycamtin ® ; Hydrea ® ; Hydrocort Acetate ® ; Hydrocortisone; Sodium Hydrocortisone Phosphate; Sodium Hydrocortisone Succinate; Hydrocortone Phosphate; Hydroxyurea; Ibrutinib; Ibritumomab; Ibritumomab Tiuxetano; Idamycin®; Idarubicin Ifex®; Interferon-alpha; Interferon-alpha-2b (PEG Conjugate);
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    Ifosfamide; Interleukin-11 (IL-11); Interleukin-2 (IL-2); Imatinib mesylate; Imidazole Carboxamide; Intron A®; ipilimumab, Iressa®; Irinotecan; Isotretinoin; Ixabepilone; Ixempra ™; KADCYCLA®; Kidrolase (t) Lanacort®; Lapatinib; Lasparaginase; CSF; Lenalidomide; Letrozole; Leucovorin; Leucerano; Leukine ™; Leuprolide; Leurocristine; Leustatin ™; Lirilumab; Liposomal Ara-C; Pred® Liquid; Lomustine; L-PAM; L-Sarcolysin; Lupron®; Lupron Depot®; Matulane®; Maxidex; Mecloretamina; Mecloretamine Hydrochloride; Medralone®; Medrol®; Megace®; Megestrol; Megestrol acetate; MEK inhibitors; Melfalano; Mercaptopurine; Mesna; Mesnex ™; Methotrexate; Sodium Methotrexate; Methylprednisolone; Meticorten®; Mitomycin; Mitomycin-C; Mitoxantrone M-Prednisol®; TCM; MTX; Mustargen®; Mustina; Mutamycin®; Myleran®; Mylocel ™; Mylotarg®; Navitoclax; Navelbine®; Nelarabine; Neosar®; Neulasta ™; Neumega®; Neupogen®; Nexavar®; Nilandron®; Nilotinib; Nilutamide; Nipent ® ; Nitrogen Mustard Novaldex ® ; Nivolumab; Novantrone®; Nplate; Octreotide; Octreotide acetate; Ofatumumab; Oncospar®; Oncovin®; Ontak®; Onxal ™; Oprelvecin; Orapred®; Orasone®; Oxaliplatin; Paclitaxel; bound to Paclitaxel Protein; Pamidronata; Panitumumab; Panretin®; Paraplatin®; Pazopanib; Pediapred®; PEG Interferon; Pegaspargase; Pegfilgrastim; PEG-INTRON ™; PEG-L-asparaginase; PEMETREXED; Pembrolizumab; Pentostatin; Pertuzumab; Phenylalanine mustard; Pidilizumab; Platinol®; Platinum-AQ®; Prednisolone; Prednisone; Prelone®; Procarbazine; PROCRIT®; Proleukin®; Prolifeprospan 20 with Carmustine Implant; Purinethol®; BRAF inhibitors; Raloxifene; Revlimid®; Rheumatrex®; Rituxan®; Rituximab; Roferon-A®; Romiplostim; Rubex ® ; Rubidomycin hydrochloride; Sandostatin ® ; Sandostatin LAR ® ; Sargramostim; Solu-Cortef®; Solu-Medrol®; Sorafenib; SPRYCEL ™; STI-571; STIVAGRA ™, Streptozocin; SU11248; Sunitinib; Sutent®; Tamoxifen Tarceva®; Targretin ® ; Tasigna ® ; Taxol ® ; Taxotere ® ; Temodar ® ; Temozolomide Tensirolimus; Teniposide; TESPA; Thalidomide; Thalomid ® ; TheraCys ® ; Thioguanine; Thioguanine
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    Tabloid®; Thiophosfoamide; Thioplex®; Thiotepa; TICE®; Toposar®; Topotecan; Toremifene; Torisel®; Tositumomab; Trastuzumab; Treanda®; Tremelimumab; Tretinoin; Trexall ™; Trisenox®; TSPA; TYKERB®; Urelumab; VCR; Vectibix ™; Velban®; Velcade®; Venetoclax; VePesid®; Vesanoid®; Viadur ™; Vidaza®; Vinblastine; Vinblastine Sulfate; Vincasar Pfs ® ; Vincristine; Vinorelbine; Vinorelbine tartrate; VLB; VM-26; Vorinostate; Votriente; VP-16; Vumon®; Xeloda®; Zanosar®; Zevalin ™; Zinecard®; Zoladex®; Zoledronic acid; Zolinza; or Zometa®, and / or any other agent not specifically listed in this report that targets similar routes.
    [0115] When two or more substances or principles are to be used as part of a combined treatment regimen, they can be administered through the same route of administration or through different routes of administration, essentially at the same time or at different times (for example, essentially simultaneously, consecutively, or, according to an alternate regime). When substances or principles are to be administered simultaneously through the same route of administration, they can be administered as different pharmaceutical formulations or compositions or as part of a combined pharmaceutical formulation or composition, as will be apparent to a skilled person.
    [0116] In addition, when two or more active substances or ingredients are to be used as part of a combined treatment regimen, each of the substances or principles can be administered in the same amount and according to the same regimen, as used when compound or principle is used alone, and such combined use may or may not lead to a synergistic effect. However, when the combined use of two or more active substances or ingredients leads to a synergistic effect, it may also be possible to reduce the amount of one, more than one or all of the substances or principles that will be
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    43/64 administered, while still obtaining the desired therapeutic action. For example, this can be useful to prevent, limit or reduce any unwanted side effects that are associated with the use of one or more between substances or principles when they are used in their usual amounts, while still obtaining the desired pharmaceutical or therapeutic effect. .
    [0117] The effectiveness of the treatment regimen used, according to the invention, can be determined and / or followed in any way known to you for the disease or disorder involved, as will be evident to the physician. The physician will also be able, where appropriate and on a case-by-case basis, to modify a particular treatment regimen in order to obtain the desired therapeutic effect, to avoid, limit or reduce unwanted side effects and / or to achieve an appropriate balance between obtaining the desired therapeutic effect, on the one hand, and avoiding, limiting or reducing unwanted side effects, on the other hand.
    [0118] In general, the treatment regimen will be followed until the desired therapeutic effect is achieved and / or as long as the desired therapeutic effect is maintained. Again, this can be determined by the doctor.
    [0119] In various embodiments, pharmaceutical compositions comprising one or more TRAIL receptor agonist proteins, alone or in combination with prophylactic agents, therapeutic agents and / or pharmaceutically acceptable carriers are provided in this report. In various embodiments, non-limiting examples of the uses of the pharmaceutical compositions disclosed in this report include diagnosis, detection and / or monitoring of a disorder, prevention, treatment, administration and / or amelioration of a disorder or one or more symptoms of it and / or in research. The formulation of pharmaceutical compositions, alone or in combination with prophylactic agents, therapeutic agents and / or pharmaceutically acceptable carriers, is known to a skilled person in the art (U.S. Patent Publication No. 20090311253 A1).
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    44/64 [0120] In various embodiments, a pharmaceutical formulation may comprise one or more amino acids, one or more polysaccharides and / or polysorbates and a TRAIL receptor agonist protein present in a concentration between about 0.1 and 100 mg / ml, including end points (for example, 0.1 to 10, 1 to 10, 0.01 to 50, 1 to 50, 1 to 100, 10 to 100, 25 to 100, 25 to 50, or 50 to 100 mg / ml), where the formulation is at a pH between about 5.0 and 7.0, including end points (for example, a pH of about 5.0 to 6.0, 5.5 to 6 , 0, 5.0 to 6.5, 5.5 to 6.5 or 6.0 to 7.0). In one embodiment, at least one amino acid in the formulation is histidine and is present in a concentration of about 10 to 20 mM, 10 to 15 mM, 15 to 20 mM or about 15 mM. In one embodiment, at least one polysaccharide in the formulation is sucrose and is present in a concentration of about 0 to 8.0% w / v (w / v). In one embodiment, the polysorbate in the formulation is polysorbate 80 and is in a concentration of about 0 to 0.06% w / v. In one embodiment, at least one amino acid in the formulation is arginine and is present in a concentration of about 0 to 1.5% w / v (for example, 0.5 to 1.5, 1.0 to 1, 5 or 0.5 to 1.0 w / v). In one embodiment, the TRAIL receptor agonist protein is present in the formulation at a concentration of about 0.1 to 100 mg / ml, (for example, about 1 to 100 mg / ml, or about 1 to 15 mg / ml, or about 1 to 7.5 mg / ml, or about 2.5 to 7.5 mg / ml, or about 5 to 7.5 mg / ml, or about 25 to 100 mg / ml, or about 20 to 60 mg / ml, or about 25 to 50 mg / ml, or about 25 mg / ml, or about 50 mg / ml, or about 0.1 to 60 mg / ml, or about 0.1 to 25 mg / ml, or about 1.0 to 60 mg / ml, or about 0.5 to 60 mg / ml, or about 0.1 to 2.0 mg / ml ml, or about 0.5 to 2.0 mg / ml, or about 1 to 5 mg / ml, or about 1 to 7.5 mg / ml, or about 1 to 15 mg / ml, or about 0.5 mg / ml, or about 1.0 mg / m).
    [0121] As used in this report, the phrase “effective amount” means an amount of TRAIL agonist protein that results in an improvement
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    45/64 detectable (for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65% , 70%, 75% or more from the reference value) on one or more parameters associated with a TRAIL dysfunction or with a TRAIL-associated disease or disorder.
    [0122] In various embodiments, the pharmaceutical formulation is an aqueous formulation, a lyophilized formulation or a lyophilized and rehydrated formulation. In one embodiment, the hydrant solution is dextrose and / or saline (for example, dextrose in a concentration of about 5% w / v / or the saline in a concentration of about 0.9% w / v ). In one embodiment, the pharmaceutical formulation comprises about 15 mM histidine, about 0.03% (w / v) polysorbate 80, about 4% (w / v) sucrose and about 0.1 to 25 mg / ml of the TRAIL receptor agonist protein or about 1 to 15 mg / ml of the TRAIL receptor agonist protein and is at a pH of about 6. In one embodiment, the formulation further comprises at least one additional agent .
    [0123] In various embodiments, a formulation is used containing about 25 mg / ml of TRAIL receptor agonist protein, about 15 mM histidine, 0.03% polysorbate 80 (weight / volume, w / v) , 4.0% sucrose (w / v) and a pH of about 6.0. In some embodiments, the formulation does not comprise arginine. In some embodiments, the formulation unexpectedly exhibits improved freeze-thaw stability, liquid formulation stability and / or lyophilized formulation stability compared to other formulations comprising other components or concentrations.
    [0124] Methods for administering a therapeutic agent provided in this report include, but are not limited to, oral administration, parenteral administration (eg, intradermal, intramuscular, intraperitoneal, intravenous and subcutaneous), epidural administration, intratumoral administration, administration
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    46/64 mucosal (eg, intranasal and oral routes) and pulmonary administration (eg, aerosolized compounds administered with an inhaler or nebulizer). The formulation of pharmaceutical compositions for particular routes of administration and techniques and materials needed for the various administration methods are available and known to a skilled person in the art (U.S. Patent Publication No. 20090311253 A1).
    [0125] In various embodiments, dosage regimens can be adjusted to provide an ideal desired response (for example, a therapeutic or prophylactic response). For example, a single cake can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased, as indicated by the requirements of the therapeutic situation. In some embodiments, parenteral compositions are formulated in unit dosage form for ease of administration and uniformity of dosage. The term "dosage unit form" refers to physically distinct units suitable as unitary dosages for the mammals to be treated; each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
    [0126] An exemplary, non-limiting range for a therapeutic or prophylactically effective amount of a TRAIL receptor agonist protein provided in this report is about 0.1 to 100 mg / kg, (for example, about 0.1 to 0 , 5, 0.1 to 1, 0.1 to 10, 0.1 to 20, 0.1 to 50, 0.1 to 75, 1 to 10, 1 to 15, 1 to 7.5, 1.25 to 15, 1.25 to 7.5, 2.5 to 7.5, 2.5 to 15, 5 to 15, 5 to 7, 5.1 to 20, 1 to 50, 7 to 75, 1 to 100 , 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 50, 5 to 75, 10 to 20, 10 to 50, 10 to 75 or 10 to 100 mg / kg, or any concentration between this range ). In some embodiments, the TRAIL receptor agonist protein is present in a pharmaceutical composition in a therapeutically effective concentration, for example, a
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    47/64 concentration of about 0.1 to 100 mg / ml (for example, about 0.1 to 0.5, 0.1 to 1, 0.1 to 10, 0.1 to 20, 0.1 to 50, 0.1 to 75, 1 to 10, 1 to 20, 1 to 50, 1 to 75, 1 to 100, 5 to 10, 5 to 15, 5 to 20, 5 to 25, 5 to 50, 5 to 75, 10 to 20, 10 to 50, 10 to 75 or 10 to 100 mg / ml, or any concentration within this range). Note that dosage values may vary with the type and / or severity of the condition that will be alleviated. It should also be understood that for any particular subject, specific dosage regimens can be adjusted over time, according to the individual need and / or the professional judgment of the person who administers or supervises the administration of the compositions and that the ranges of Dosages presented in this report are exemplary only and are not intended to limit the scope or practice of the claimed composition.
    EXAMPLES
    1. Manufacture of a TRAIL receptor agonist protein (sc TRAIL by weight)
    1.1. Polypeptide structure
    A) Met1 - Gly20 amino acids [0127] Ig-cap signal peptide, signal peptidase cleavage site considered after the GIy 20 amino acid.
    B) Amino acids Gln21 - Gly182 [0128] First soluble cytokine domain of the human TRAIL ligand (TRAIL, amino acid 120 to 281 of SEQ ID NO: 1).
    C) Amino acids GIy 183 - Ser 190 [0129] First element of the peptide linker of SEQ ID NO: 2.
    D) Arg191 - Gly351 amino acids [0130] Second soluble cytokine domain of the human TRAIL ligand (TRAIL, amino acids 121 to 281 of SEQ ID NO: 1).
    E) Amino acids GIy352 - Ser359.
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    48/64 [0131] Second peptide linker element of SEQ ID NO: 2.
    F) Arg360 - Gly520 amino acids [0132] Third soluble cytokine domain of the human TRAIL ligand (TRAIL, amino acids 121 to 281 of SEQ ID NO: 1).
    G) Amino acids Gly521 - Cys542 [0133] Hinge linker element of SEQ ID NO: 11.
    H) Pro543 - Lys760 Amino Acids [0134] Fc antibody fragment domain of SEQ ID NO: 10.
    [0135] The above TRAIL receptor agonist protein is shown in SEQ ID NO: 14.
    [0136] The indicated linkers can be replaced with other preferred linkers, for example, as shown in SEQ ID NOs: 3 to 9.
    [0137] It should be noted that the first and second peptide linkers need not be identical.
    [0138] The signal peptide sequence (a) can be replaced by any other suitable sequence, for example, mammalian signal peptide sequence.
    1.2. Gene cassette encoding the polypeptide [0139] The synthetic gene can be optimized in view of its use of codon for expression in suitable host cells, for example, insect cells or mammalian cells. A preferred nucleic acid sequence is shown in SEQ ID NO: 16.
    2. Expression and Purification [0140] Cloning, expression and purification of fusion polypeptides [0141] The aforementioned fusion proteins were recombinantly expressed in two different eukaryotic host cells:
    [0142] For the initial analysis of receptor agonist fusion proteins
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    TRAIL previously mentioned, Hek293T cells cultured in DMEM + GlutaMAX (GibCo) supplemented with 10% FBS, Penicillin 100 units / ml and Streptomycin 100 [mu] g / ml were transiently transfected with a plasmid containing an expression cassette for a polypeptide of fusion and an appropriate selection marker, for example, a functional expression cassette comprising a gene resistant to blasticidin, puromycin or hygromycin. In such cases, where a plurality of polypeptide chains are required to obtain the final product, the expression cassettes have been combined into one plasmid or positioned on different plasmids during transfection. The cell culture supernatant containing recombinant fusion polypeptide was collected three days post-transfection and purified by centrifugation at 300 x g, followed by filtration through a 0.22 pm sterile filter.
    [0143] For the larger scale expression of the TRAIL receptor agonist fusion proteins that will be used in vivo, synthetic DNA cassettes encoding the previously mentioned proteins have been inserted into eukaryotic expression vectors comprising appropriate selection markers (for example, a functional expression cassette comprising a gene resistant to blasticidin, puromycin or hygromycin) and suitable genetic elements to enhance the number of transcriptionally active insertion sites within the host cell genome. The expression vectors verified by the sequence were introduced by electroporation into Chinese adapted Hamster Ovary cells in suspension (CHO-S, Invitrogen). The appropriate selection pressure was applied three days post-transfection to the transfected cells. The surviving cells carrying the resistant genes derived from the vector were recovered by subsequent cultivation under selection pressure. After the stable growth of the selected cell clusters in a chemically defined medium (PowerCHO2CD, Lonza) at 37 ° C and 7% CO2 atmosphere in an orbital shaker incubator
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    50/64 (100 rpm, 50 mm stirring pitch), the individual supernatants were analyzed by ELISA assays detecting the proteins and the previously mentioned cell clusters with the highest specific productivity were expanded in shake flasks prior to protein production ( orbital shaker, 100 rpm, shaking pitch 50 mm).
    [0144] For laboratory-scale protein production, individual cell clusters were cultured for 7 to 12 days in a chemically defined medium (PowerCHO2-CD, Lonza) at 37 ° C and 7% CO2 atmosphere in a Wave 20 / bioreactor 50 EHT (GE-Healthcare). The basal medium was PowerCHO2-CD supplemented with 4 mM Glutamax. The Wave culture started with a viable cell concentration of 0.3 to 0.4 x 10 and 6 cells / ml and the following settings (for a five or ten liter bag): shaking frequency 18 rpm, shaking angle 7 °, gas flow 0.2 to 0.3 L / min, 7% CO2, 36.5 ° C. During Wave conduction, the cell culture was fed twice with PowerFeed A (Lonza), usually on day 2 (20% feeding) and day 5 (30% feeding). After the second feeding, the stirring frequency was increased to 22 rpm, as well as the stirring angle at 8 °.
    [0145] The bioreactor was usually collected between day 7 to day 12 when cell viability dropped below 80%. First, the culture supernatant was purified using a manual depth filtration system (Millipore Millistak Pod, MC0HC 0.054 m 2 ). For Strep-labeled proteins, Avidin was added at a final concentration of 0.5 mg / L. Finally, the culture supernatant containing the TRAIL receptor agonist fusion protein was filtered and sterilized using a bottle top filter (0.22 pm, PES, Corning) and stored at 2 to 8 ° C until further processing.
    [0146] For affinity purification, Streptactin Sepharose was packed in a column (1 ml gel bed), equilibrated with 15 ml W buffer
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    51/64 (100 mM Tris-HCI, 150 mM NaCI, pH 8.0) or PBS pH 7.4 and the cell culture supernatant was applied to the column at a flow rate of 4 ml / min. Subsequently, the column was washed with 15 ml W buffer and the bound polypeptide was eluted gradually by the addition of 7 x 1 ml E buffer (100 mM Tris HCI, 150 mM NaCI, 2.5 mM Destiobiotin, pH 8.0 ). Alternatively, PBS pH 7.4 containing 2.5 mM Destiobiotin can be used for this step.
    [0147] Alternatively to the Streptactin Sepharose-based method, affinity purification was performed using an immobilized Protein-A column as an affinity binder and an Akta chromatography system (GEHealthcare). A solid-phase material with high affinity for the FC domain of the fusion protein was chosen: MABSelect Sure TM (GE Healthcare). Briefly, the purified cell culture supernatant was loaded onto a HiTrap MabSelectSure column (CV = 5 ml) equilibrated in wash buffer-1 (20 mM Pi, 95 mM NaCl, pH 7.2) not exceeding a load of fusion protein 10 mg per ml of column bed. The column was washed with ten column volumes (10 CV) of equilibration buffer previously mentioned, followed by four column volumes (4 CV) of wash buffer-2 (20 mM Pi, 95 mM NaCl, pH 8.0) to deplete host cell protein and host cell DNA. The column was then eluted with elution buffer (20 mM Pi, 95 mM NaCl, pH 3.5) and the eluate was collected in up to ten fractions with each fraction having a volume equal to the column bed volume (5 ml). Each fraction was neutralized with an equal volume of wash buffer-2 mentioned above. The linear velocity was adjusted to 150 cm / h and kept constant during the aforementioned affinity chromatography method.
    [0148] The amount of protein in the eluate fractions was quantified and the peak fractions were concentrated by ultrafiltration and further purified by size exclusion chromatography (SEC).
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    52/64 [0149] SEC was performed on Superdex 200 10/300 GL or HiLoad 26/60 columns using an Akta chromatography system (GE-Healthcare). The columns were equilibrated with phosphate buffered saline and the concentrated affinity purified polypeptide was loaded onto the SEC column with the sample volume not exceeding 2% (v / v) of the column volume. In the case of Superdex200 10/300 GL columns (GE Healthcare), a flow rate of 0.5 ml per minute was applied. In the case of HiLoad 26/60 Superdex200 columns, a flow rate of 2.5 ml per minute was applied. The elution profile of the polypeptide was monitored by absorbance at 280 nm.
    [0150] For the determination of the apparent molecular weight of the purified fusion polypeptide under native conditions, a Superdex 200 column was loaded with standard proteins of known molecular weight. Based on the elution volume of the standard proteins, a calibration curve was plotted and the apparent molecular weight of the purified fusion polypeptide was determined. The FC domain comprising TRAIL receptor agonist fusion proteins typically eluted from Supoerdex200 columns with an apparent molecular weight for the homodimer of approx. 160 to 180 kDa.
    3. Apoptosis Assay [0151] A cell assay with a Jurkat A3 permanent T cell line was used to determine the apoptosis-inducing activity of TRAIL receptor agonist fusion proteins. Jurkat cells were grown in flasks with RPMI 1640 + GlutaMAX (GibCo) medium supplemented with 10% FBS, Penicillin 100 units / ml and Streptomycin 100 pg / ml. Prior to the assay, 100,000 cells were seeded per well in a 96-well microtiter plate. The addition of different concentrations of fusion peptides to the wells was followed by a 3 hour incubation at 37 ° C. The cells were lysed by adding lysis buffer (250 mM HEPES, 50 mM MgCl 2, 10 mM EGTA, 5% Triton-X-100,
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    100 mM DTT, 10 mM AEBSF, pH 7.5) and the plates were placed on ice for 30 minutes to 2 hours. Apoptosis is paralleled by increased caspase activity, for example, Caspase-3. Consequently, the cleavage of the specific Caspase substrate Ac-DEVD-AFC (Biomol) was used to determine the extent of apoptosis. In fact, Caspase activity correlates with the percentage of apoptotic cells morphologically determined after pigmentation of the cells with propidium iodide and Hoechst-33342. For the assay of caspase activity, 20 µl of cell lysate was transferred to a black 96-well microtiter plate. After adding 80 pl of buffer containing 50 mM HEPES, 1% Sucrose, 0.1% CHAPS, 50 pM Ac-DEVD-AFC and 25 mM DTT, pH 7.5, the plate was transferred to a microtiter plate Tecan Infinite 500 and the increase in fluorescence intensity was monitored (excitation wavelength 400 nm, emission wavelength 505 nm).
    3.1. Cell death assay [0152] To determine cell death in HT1080 fibrosarcoma cells, 15,000 cells were plated in 96-well plates overnight in RPM1 1640 + GlutaMAX (GibCo) medium supplemented with 10% FBS (Biochrom). The cells were matched with cycloheximide (Sigma) at a final concentration of 2.5 g / ml. Cell death was quantified by pigmentation with KV buffer (0.5% violet crystal, 20% methanol). After pigmentation, the wells were washed with water and air dried. The dye was eluted with methanol and the optical density at 595 nm was measured with an ELISA reader.
    4. Stability / Aggregation Test
    4.1. Principle of aggregation analysis (Definition for soluble protein) [0153] The content of monomers (defined quarterly assembly of the TRAIL receptor binding modules) and aggregates is determined by analytical SEC, as
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    54/64 described in Example 2. For this particular purpose, the analysis is performed in buffers containing physiological salt concentrations at physiological pH (for example, 0.9% NaCI, pH 7.4; PBS pH 7.4). A typical aggregation analysis is performed on a Superdex200 column (GE Healthcare). This column separates proteins in the range between 10 to 800 kDa.
    [0154] For the determination of the apparent molecular weight of the purified fusion polypeptide under native conditions, a Superdex 200 column is loaded with standard proteins of known molecular weight. Based on the elution volume of the standard proteins, a calibration curve is plotted and the apparent molecular weight of the purified fusion polypeptide is calculated based on the elution volume.
    [0155] SEC analysis of non-aggregated soluble proteins, for example, trimeric TRAIL, typically shows a distinct single protein peak at a defined elution volume. This elution volume corresponds to the apparent native molecular weight of the particular protein and approximately conforms to the theoretical molecular weight calculated on the basis of the primary amino acid sequence.
    [0156] If protein aggregation occurs, SEC analysis shows additional protein peaks with lower retention volumes. For TRAIL, the aggregation of soluble proteins occurs in a characteristic manner. Proteins tend to form oligomers of the "trimers", forming nonamers (3 x 3) and 27mers (3 x 9). These oligomers serve as aggregation seeds and a high oligomer content potentially leads to protein aggregation. Large molecular weight oligomers and aggregates elute in the empty volume of the Superdex200 column and cannot be analyzed by SEC with respect to their native molecular weight.
    [0157] Due to induction of (complete) aggregation, purified preparations of TRAIL-SF fusion proteins should preferably contain only
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    55/64 defined quarterly proteins and only a very low amount of oligomerized protein. The degree of aggregation / oligomerization of a particular TRAIL-SF protein preparation is determined on the basis of SEC analysis by calculating the peak areas of the OD280 diagram for the defined quarterly fraction and oligomer / aggregate, respectively. Based on the total peak area, the percentage of the trimester protein defined is calculated, as follows:
    (% trimer content = [eak area trimer] / [total peak area] x 100) [0158] The definition for the soluble protein used in this text describes a protein preparation of the purified TRAIL protein in a buffer of physiological saline concentrations at the physiological pH which contains a defined soluble protein content (trimeric assembly of TRAIL domains) of> 90% within a typical protein concentration range of 0.2 to 10.0 mg / ml.
    5. Determination of half-life [0159] Molecules A to D consist of two polypeptides covalently linked by disulfide bonds between chains. The number of glycosites and cysteines on the hinge (resulting in disulfide bonds between chains between proteins) were tested in order to determine the effect that altering these characteristics has on the half-life of these compounds.
    [0160] Female NMRI mice were treated with 1.2 mg / kg bw and / or 4 mg / kg bw of the specified compounds as a single intravenous bolus injection. Whole blood was collected before application (pre-dose) and up to 168 hours after administration of the test item. The serum was prepared and the samples were stored at -80 ° C until the serum concentrations were determined. Pharmacokinetic parameters were calculated using the mean serum concentrations and the PK Solutions Version 2.0 pharmacokinetic evaluation program for the analysis of non-compartmental pharmacokinetic data (Summit Research Services, Montrose, CO). PK Solutions is an application based on Excel
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    56/64 that computes the pharmacokinetic parameters from the concentration-time data obtained from the analysis, for example, of biological samples following intravenous or extravascular routes of administration. PK Solutions calculates the results without assuming any specific compartmental model.
    [0161] Quantification of test items in serum was performed with an ELISA assay detecting the individual TRAIL receptor agonists shown in Table 7 independent of a Strep tag being part of the molecules. The general outline is shown in Figure 18. The results are summarized in Table 7.
    [0162] Molecule A (consisting of two polypeptides of SEQ ID NO: 26) has two hinge cysteines (forming two disulfide bonds between chains) and an N residue at position 297 of the Fc region (according to the EU index), resulting in glycosylation of wild type CH2. Molecule A also has glycosites at positions 168 and 337. Molecule B (consisting of two polypeptides of SEQ ID NO: 19) has three hinge cysteines (forming three disulfide bonds between chains) (at positions 513, 519, and 522) and an N297S mutation at position 297 of the Fc region (according to the EU index), resulting in aglycosylation of the CH2 domain. Molecule B also has glycosites at positions 168 and 337. Molecule C (consisting of two polypeptides of SEQ ID NO: 27) has three hinge cysteines (forming three disulfide bonds between chains) and an N297S mutation at position 297 of the Fc region (according to the EU index), resulting in aglycosylation of the CH2 domain. In addition, there is a glycoside at position 168 (linker 1), but not at position 337 (linker 2). The D molecule (consisting of two polypeptides of SEQ ID NO: 28) has three hinge cysteines (forming three disulfide bonds between chains) and an N297S mutation at position 297 of the Fc region (according to the EU index), resulting in aglycosylation of the CH2 domain. In addition, the glycosites in linker 1
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    57/64 and linker 2 (positions 168 and 337, respectively) were depleted in Molecule D.
    [0163] The in vivo stability (determined by the half-life of the compound) of Molecule B (glycosylated linkers, depleted CH2 glycosides and the addition of a third hinge cysteine) was enhanced compared to Molecule A. In addition, depletion of all glycosites from the compound (Molecule D) resulted in reduced in vivo stability and low productivity during transient expression. Molecule C (first glycosylated linker, second aglycosylated linker, depleted CH2 glycosides) demonstrated intermediate in vivo stability compared to Molecules B and D (see results in Table 7).
    Table 7: Results of the Compound Half-Life Test in NMRI Mice _______________________________________________________________
    Molecule Number of glycosylation sites Number of hinge cysteines Half lifeTerminal 4 mg / kg i.v. (hour) Terminal half-life 1.2 mg / kg i.v. (hour) THE 6 2 23.1 17.7 B 4 3 33.94 28.28 Ç 2 3 21.03 - D 0 3 8.81 -
    [0164] These experimental results demonstrate that the combination of linker glycosylation (in linkers 1 and 2) with a third disulfide link between chains (through the addition of a hinge cysteine) and the deglycosylation of the CH2 domain in the Fc region results in greater in vivo stability in the molecules of the present invention.
    6. In vitro demonstration of effectiveness
    6.1. TRAIL receptor agonist protein of SEQ ID NO: 19 inhibits the survival of hematological tumor cells and human solid in vitro [0165] Tumor cells were seeded in 10,000 cells / well in 96-well plates in the recommended medium containing 10% FBS and treated with a TRAIL receptor agonist protein consisting of two polypeptides that
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    58/64 show the amino acid sequence shown in SEQ ID NO: 19 for 24 hours at 37 ° C in a humidified 5% CO2 atmosphere. Cell viability was subsequently assessed using CellTiter-Glo® reagent, as described by the manufacturer's instructions (Promega; Madison, WI). IC50 values were determined by nonlinear regression analysis of normalized concentration response data in untreated control cells. Examples of the resulting concentration response curves for Colo205, Jurkat and SKM-1 cells that demonstrate a loss in cell viability in response to treatment with TRAIL receptor agonist protein consisting of two polypeptides that have the amino acid sequence shown in SEQ ID NO: 19 are shown in Figure 19. Table 8 shows the results of hematological tumor cell lines (A; (n = 40; Non-Hodgkin's Lymphoma, NHL; Acute Myeloid Lymphoma, AML; Acute Lymphoblastic Leukemia, ALL) and solid (B; (n = 44; Non-Small Cell Lung Carcinoma, NSCLC; Pancreatic; Colorectal Cancer, CRC; Breast Cancer, BrCa; Ovary, Fibrosarcoma; Head and Neck, H&N; Small Cell Lung Cancer, SCLC) treated with a TRAIL receptor agonist protein consisting of two polypeptides that have the amino acid sequence shown in SEQ ID NO: 19 for 24 hours and the viability assessed by CellTiter-Glo®. Resulting 50s for the effects mediated by the TRAIL receptor agonist protein consisting of two polypeptides that present the amino acid sequence shown in SEQ ID NO: 19 on the tumor cell viability are presented.
    Table 8
    Potency of the TRAIL receptor agonist protein of SEQ ID NO: 19 in human tumor cancer cell lines in vitro
    Tumor cell line
    Tumor Type
    SEQ ID NO: 19
    IC50 (ng / ml)
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    THE SU-DHL-8 NHL 1.36 NUDHL-1 NHL 6.50 OCI-Ly8 NHL 7.49 ULA NHL 8.44 OCI-Ly2 NHL 18.98 OCI-LY19 NHL 26.34 WSU-NHL NHL 31.60 OCI-Ly7 NHL 63.76 SU-DHL-5 NHL 82.07 OCI-Ly18 NHL 196.20 OCI-Ly1 NHL 416.95 SU-DHL-16 NHL 545.55 SU-DHL-2 NHL 1000.00 WSU-DLCL2 NHL 1000.00 Toledo NHL 1000.00 OCI-LY3 NHL 1000.00 RL NHL 1000.00 SU-DHL-4 NHL 1000.00 U2932 NHL 1000.00 HT NHL 1000.00 RC-K8 NHL 1000.00 SKM-1 AML 0.95 PL-21 AML 10.67 EOL-1 AML 18.31 HL-60 AML 76.62 OCI-AML2 AML 124.32 UKE-1 AML 205.35 MV4 - 11 AML 312.55 SET-2 AML 384.80 MOLM-13 AML 722.10 OCI-AML5 AML 1032.60 Kasumi-1 AML 1000.00 KG-1 AML 1000.00 OCI-AML3 AML 1000.00 SHI-1 AML 1000.00 SKNO-1 AML 1000.00 TF-1 AML 1000.00 THP-1 AML 1000.00 HEL AML 1000.00 Jurkat ALL 3.08 B
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    NCI-H847 NSCLC 14.53 NCI-H647 NSCLC 24.75 NCI-H2444 NSCLC 27.75 NCI-H2170 NSCLC 30.16 NCI-H460 NSCLC 36.85 NCI-H838 NSCLC 44.48 NCI-H1792 NSCLC 61.09 NCI-H2347 NSCLC 81.06 NCI-H1373 NSCLC 125.15 NCI-H522 NSCLC 259.87 NCI-H2110 NSCLC 314.20 NCI-H596 NSCLC 397.80 HCC4006 NSCLC 407.24 NCI-H2122 NSCLC 480.55 NCI-H1299 NSCLC 716.00 NCI-H1975 NSCLC 741.50 HCC827 NSCLC 2824.50 NCI-H727 NSCLC 3178.00 NCI-H1944 NSCLC 4068.75 NCI-H1299 NSCLC 4214.87 Calu-6 NSCLC 4757.00 NCI-H1693 NSCLC 5000.00 HCC2935 NSCLC 5000.00 A549 NSCLC 5000.00 NCI-H1395 NSCLC 5000.00 NCI-H2172 NSCLC 5000.00 Calu-1 NSCLC 5000.00 NCI-H441 NSCLC 5000.00 NCI-H23 NSCLC 5000.00 NCI-H661 NSCLC 5000.00 NCI-H1650- NSCLC > 3 GFPBxPC3 Pancreatic 16.00 Capan-1 Pancreatic 393.00 MIA PaCa-2 Pancreatic 158.00 PANC-1 Pancreatic > 1000 SW48 CRC 6.10 Colo205 CRC 1.30 SW480 CRC 132.00 HCT 116- CRC 337.00 GFP HCC38 BrCa 3.00
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    HCC1569 BrCa 219.00 MCF7 BrCa > 3000 MDA-MB-231 BRCa 235.00 HeyA8-GFP Ovary 141.00 Fadu-GFP H&N > 3 HT-1080 Fibrosarcoma 377.00 NCI-H211 SCLC 72.58
    6.2. TRAIL receptor agonist protein of SEQ ID NO: 19 synergizes with antitumor agents to induce tumor cell death [0166] Tumor cells were seeded in 10,000 cells / well in 96-well plates in the recommended medium containing 10% FBS and co-treated with a TRAIL receptor agonist protein consisting of two polypeptides that have the amino acid sequence shown in SEQ ID NO: 19 and venetoclax (ABT-199), navitoclax (ABT-263) or docetaxel (DTX) for 24 h at 37 ° C in a humidified 5% CO2 atmosphere. Cell viability was subsequently assessed using CellTiter-Glo® reagent, as described by the manufacturer's instructions. The Bliss independence model (Wong et al., 2012; Mol. Cancer Ther. 11: 1026 to 1035; Bernebaum, 1981 Adv. Cancer Res. 35: 269 to 335; Borisy et al., 2003 Proc. Natl. Acad. Sci. USA 100: 7977 to 7982) was used to evaluate the combination activity, with negative integers indicating antagonism, a value of zero indicating additive activity and positive integers indicating synergy. Bliss scores were calculated for each combination in the dose matrix and totaled to provide a “Bliss sum” value. An example of synergistic tumor cell death induced by co-treatment of human tumor cells with a TRAIL receptor agonist protein consisting of two polypeptides that have the amino acid sequence shown in SEQ ID NO: 19 and venetoclax, navitoclax or DTX, with the sum Associated bliss is shown in Figures 20 (A to C). The Bliss sums determined for these combinations in a number of tumor cell lines are shown in Table 9.
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    Table 9
    Evaluation of Bliss cell death synergy by the TRAIL receptor agonist protein of SEQ ID NO: 19 in combination with DTX in NSCLC (a) cell lines and venetoclax or navitoclax in NHL & AML (B) cell lines in vitro.
    LineageCell phoneTumor Sum Bliss(SEQ IDNO: 19 +DTX) LineageCell phoneTumor Sum Bliss (SEQ ID NO:19 +venitoclax) Sum Bliss (SEQ ID NO:19 + navitoclax) LG0552 748.2 WSU-DLCL2 1292 560 NCI-H522 549.1 SU-DHL-4 898 617 NCI-H647 452 OCI-AML3 831.9 456.4 NCI-H727 429.4 OCI-AML5 777.8 174.5 NCI-H1373 387.1 U2932 736.2 636.1 NCI-H596 261 PL-21 600.8 244.9 HCC2935 224.2 ULA 343.8 79.4 NCI-H2347 154 OCI-Ly18 309.8 8.3 NCI-H2444 135 MV4; 11 301.1 351 A549 118.1 RL 286.1 446.7 NCI-H23 70.6 MOLM-13 270.6 264.8 NCI-H847 64.2 SKM-1 222.9 88.1 HCC4006 15.75 OCI-Ly1 218.8 69.1 NCI-H2170 -97.1 SU-DHL-16 217.5 142.5 LG0567 -105.2 OCI-AMl2 160.2 145.5 HCC2935 -183 OCI-Ly8 154.9 177 HCC827 -292.8 THP-1 152.7 43.1 NCI-H661 -344.5 OCI-Ly3 146.5 -242.2 NCI-H441 -362 OCI-Ly2 145 127.2 NCI-H1395 -512 OCI-Ly19 114.9 37.3 NCI-H1944 -565 SKNO-1 104.7 -138.9 NCI-H1693 -584 UKE-1 80.5 28.9 Calu-6 -628.7 WSU-NHK 79.8 84 LG0481 -803 EOL-1 69.7 -6.3 NCI-H2172 -1404 SU-DHL-2 53.5 -31.8 Toledo 51.4 -68.2 HEL 21.5 -92.4 NuDHL-1 -28.4 18.7 TF-1 -100.6 -50.2 RC-K8 -131 -68 HT -173 12.1 HL-60 -176 -112.7
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    SHI-1
    SU-DHL-8
    SU-DHL-5
    SET-2
    KG-1
    Kasumi-1
    -208.4
    -210.6
    -233.8
    -248.4 -260
    -356.4
    -122.6
    -37 -280.6
    71.2
    -20.3 -241.2
    7. Treatment with the TRAIL receptor agonist protein of SEQ ID NO: 19 inhibits tumor growth in vivo [0167] The effect of a TRAIL receptor agonist protein consisting of two polypeptides that have the amino acid sequence shown in SEQ ID NO: 19 on tumor growth was evaluated in Colo205 (colorectal) subcutaneous xenograft, SKM-1 (acute myeloid leukemia) and H460LM (non-small cell lung) tumors implanted in female SCID mice (Charles Rivers Laboratories; Wilmington, MA ). Briefly, human cancer cells were subcutaneously inoculated on the right rear flank of female SCID mice on day 0 of the study. The administration of the TRAIL receptor agonist protein of SEQ ID NO: 19 (0.3, 1 or 3 mkd dosed IV, QDx5 or IP, Q2Dx5, as indicated) was initiated at the size matching time. The tumor volume was measured during the experiment until the average tumor volume in each group reached a point of> 2000 mm 3 for Colo205 and SKM-1 or> 2500 mm 3 for H460LM. The results are shown in Figures 21 to 23. The administration of a TRAIL receptor agonist protein consisting of two polypeptides that have the amino acid sequence shown in SEQ ID NO: 19 significantly induced the inhibition of tumor growth in xenograft tumor models Colo205, SKM-1 and H460LM.
    [0168] The effect of a TRAIL receptor agonist protein consisting of two polypeptides that have the amino acid sequence shown in SEQ ID NO: 19 on tumor growth was also evaluated in xenograft models derived from patient CTG-0069 (colorectal) , CTG-0167 (NSCLC), CTG-0293 (pancreatic), CTG-0714 (sarcoma), CTG-0136 (esophageal), CTG-485
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    64/64 (gastric) and CTG-0785 (Ewing's sarcoma) implanted in female NSG mice (Champions Oncology; Hackensack, NJ). Briefly, the tumor fragments were subcutaneously propagated on the right rear flank of the female NSG mice on day 0 of the study. The administration of a TRAIL receptor agonist protein consisting of two polypeptides that have the amino acid sequence shown in SEQ ID NO: 19 (3 mkd dosed IP, Q2Dx5) was initiated at the size matching time. The tumor volume was measured during the experiment until the average tumor volume in each group reached a point of> 2000 mm 3 or 60 days. The results are shown in Figures 24 (A to G). The administration of a TRAIL receptor agonist protein consisting of two polypeptides that have the amino acid sequence shown in SEQ ID NO: 19 significantly induced tumor growth inhibition in PDX models CTG-0069 (colorectal), CTG-0167 (NSCLC) , CTG-0293 (pancreatic), CTG-0714 (sarcoma), CTG-0136 (esophageal), CTG-485 (gastric) and CTG-0785 (Ewing's sarcoma).
    权利要求:
    Claims (11)
    [1]
    1. TRAIL receptor agonist protein CHARACTERIZED by the fact that it comprises a polypeptide having the amino acid sequence shown in SEQ ID NO: 19.
    [2]
    2. TRAIL receptor agonist protein CHARACTERIZED by the fact that it comprises a dimer of two polypeptides having the amino acid sequence shown in SEQ ID NO: 19.
    [3]
    3. TRAIL receptor agonist protein according to claim 2, CHARACTERIZED by the fact that the two polypeptides are covalently linked through three disulfide bonds between chains formed between the cysteine residues 513, 519 and 522 of each polypeptide.
    [4]
    4. TRAIL receptor agonist protein according to claim 1, CHARACTERIZED by the fact that one or more of the asparagine residues at positions 168 and 337 of the polypeptide are N-glycosylated.
    [5]
    5. TRAIL receptor agonist protein according to claim 1, CHARACTERIZED by the fact that the asparagine residues at positions 168 and 337 of the polypeptide are both N-glycosylated.
    [6]
    6. TRAIL receptor agonist protein, according to claim 1, CHARACTERIZED by the fact that the polypeptide is still post-translationally modified.
    [7]
    7. TRAIL receptor agonist protein according to claim 6, CHARACTERIZED by the fact that the post-translational modification comprises modification of the N-terminal glutamine for pyroglutamate.
    [8]
    8. TRAIL receptor agonist protein according to claim 3, CHARACTERIZED by the fact that one or more of the asparagine residues at positions 168 and 337 of the polypeptides are N-glycosylated.
    [9]
    9. TRAIL receptor agonist protein according to claim 8,
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    CHARACTERIZED by the fact that the asparagine residues at positions 168 and 337 of the polypeptides are both N-glycosylated.
    [10]
    10. TRAIL receptor agonist protein according to claim 8, CHARACTERIZED by the fact that one or more of the polypeptides is still post-institutionally modified.
    [11]
    11. TRAIL receptor agonist protein according to claim 10, CHARACTERIZED by the fact that the post-translational modification comprises modification of the N-terminal glutamine in pyroglutamate.
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    法律状态:
    2018-07-31| B65X| Notification of requirement for priority examination of patent application|
    2018-08-21| B65Y| Grant of priority examination of the patent application (request complies with dec. 132/06 of 20061117)|
    2019-04-24| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|
    2019-08-20| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |
    2019-12-03| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|
    2020-03-10| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2020-03-31| 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 23/04/2015, OBSERVADAS AS CONDICOES LEGAIS. |
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    US201461983152P| true| 2014-04-23|2014-04-23|
    US61/983,152|2014-04-23|
    PCT/US2015/027270|WO2015164588A1|2014-04-23|2015-04-23|Single-chain trail-receptor agonist proteins|
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