Anti-B7H3 antibodies for the treatment of cancer

Abstract

The present invention relates to humanized antibodies and antigen-binding fragments. More specifically, the present invention relates to humanized antibodies and antigen-binding fragments capable of binding to antigens, which preferably contain at least 50% amino acid identity to the human germline versions of the antibodies or antigen-binding fragments. The present invention further relates to antibodies with minimal potential for immunogenicity for treating B7-H3(+) solid tumors by intraperitoneal or systemic administration. The present invention further relates to fully humanized antibodies against B7-H3 that have a high human content, strong binding to B7-H3, high stability, high purity, and high expression titers.

Description

[Technical Field]

[0001] The present specification contains a Sequence Listing in computer readable format, which has been submitted with this application and which forms part of this disclosure and is incorporated herein in its entirety.

[0002] The present invention relates to humanized antibodies and antigen-binding fragments. More specifically, the present invention relates to humanized antibodies and antigen-binding fragments capable of binding to antigens, wherein the antibodies or antigen-binding fragments preferably contain at least 50%, more preferably at least 75%, amino acids identical to the human germline amino acids of the antibodies or antigen-binding fragments. The present invention further relates to antibodies with minimal potential for immunogenicity for treating B7-H3(+) solid tumors by intraperitoneal or systemic administration. The present invention further relates to fully humanized antibodies against B7-H3 that have high human content, strong binding to B7-H3, high stability, high purity, and high expression titers. [Background technology]

[0003] Technology background Human B7-H3 (also known as CD276) is a member of the B7 / CD28 immunoglobulin superfamily. B7-H3 has been identified as a type I transmembrane protein, with two isoforms (2Ig B7H3) containing one extracellular V-like Ig domain and one extracellular C-like Ig domain according to Chapoval et al. [1], and two sets of V-like and C-like Ig domains (4Ig B7H3) according to Steinberger et al. [2]. According to Steinberger et al., B7-H3 was initially identified as a costimulatory molecule capable of inducing IFN-γ. Later, B7-H3 was shown to inhibit T cell proliferation, correlating with reduced IFN-γ production [2].

[0004] International Patent Application WO2016033225 discloses humanized and / or affinity-matured versions of the 8H9 antibody. According to WO2016033225, four humanized 8H9 (hu8H9) IgG1 variants have been produced. The variant with the most human-like content did not bind to B7-H3 as strongly as the other three variants, but none of them exhibited optimal binding capacity. Six mutations were introduced into one of the hu8H9 antibodies to create hu8H9 H3L3, and 12 additional humanization mutations were introduced into hu8H9 H3L3 to create hu8H94.1. Six affinity maturation mutations were incorporated into the hu8H9 H3L3 sequence to create the hu8H9 3.1 scFv and IgG1 variants. Five affinity maturation mutations were incorporated into hu8H9 H3L3 to create the hu8H9 5.1 scFv and IgG1 variants.

[0005] Summary of the Invention According to Loos et al. and Modak et al., B7-H3 is widely expressed in various human solid tumors, including pediatric solid tumors such as brain tumors and sarcomas. [3, 4] However, its expression in normal human tissues is limited. Furthermore, B7-H3 expression in solid tumors has been found to correlate with poor patient survival, increased risk of clinical cancer recurrence, and cancer-specific death in a number of cancers, including prostate cancer, pancreatic cancer, gastric cancer, ovarian cancer, osteosarcoma, neuroblastoma, and glioblastoma. [5-11] B7-H3 is an ideal target for immunotherapy. [12-14] The anti-B7-H3 murine monoclonal antibody 8H9 has been successfully used as radioimmunotherapy in clinical trials by compartmentalized intrathecal delivery to treat patients with recurrent metastatic central nervous system neuroblastoma

[15] and by single intraperitoneal administration to patients with desmoplastic small cell tumors and other solid tumors involving the peritoneum. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] International Publication No. 2016 / 033225

Non-licensed literature

[0007] [Non-licensed document 1] Chapoval, AI, et al., B7-H3: A costimulatory molecule for T cell activation and IFN-γ production. Nature Immunology, 2001. 2×3): p. 269-274. [Non-licensed document 2] Steinberger, P., et al., Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains. The Journal of Immunology, 2004. 172×4): p. 2352-2359. [Non-licensed document 3] Loos, M., et al., B7-H3 and Its Role in Antitumor Immunity. Clinical and Developmental Immunology, 2010. 2010: p. 1-7.

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[0008] All cited references are incorporated by reference. Summary of the Invention [Means for solving the problem]

[0009] As one of the leading causes of cancer-related deaths worldwide, metastatic gastric cancer has a low reported median survival rate of 1 year

[16] . The 5-year survival rate for women with metastatic epithelial ovarian cancer remains at 30% [Siegel et al. 2020, American Cancer Society, 2020]. To minimize the potential immunogenicity of anti-B7-H3 antibodies administered intraperitoneally or systemically to treat these and other B7-H3+ solid tumors, we present here a fully humanized antibody, Hu8H9, directed against B7-H3, with high human content, strong binding to B7-H3, high stability, high purity, and high expression titer. Previous attempts to humanize the murine mAb 8H9, which has low human germline content in the variable domains (73% human germline content in the V-kappa domain and 76.5% human germline content in the VH domain), required three affinity maturation mutations in the CDR regions to restore the affinity of murine 8H9

[17] .

[0010] For B7-H3-targeted delivery of radiopharmaceuticals and to eliminate antibody interactions with effector cells or complement, we engineered the mutations "N298A and K323A" and "L235A, L236A, and K323A" (numbered according to Kabat: N297A and K322A; L234A, L235A, and K322A) in the Fc of IgG to eliminate ADCC (antibody-dependent cellular cytotoxicity), ADCP (antibody-dependent cellular phagocytosis), and complement activation. The modified Fc is silent in terms of effect, i.e., a null Fc.

[0011] Based on the sequences of the leading candidates, we also describe a novel class of SADA (self-assembly and disassembly) antibodies that bind to both human B7-H3 and DOTA (dodecanetetraacetic acid, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, a metal chelator) for pretargeted radioimmunotherapy. The SADA antibodies include an ScFv against B7-H3 and an ScFv against DOTA (based on the humanized C825 antibody from patent WO2016130539A2, all of which are incorporated by reference in their entirety). This type of construct may be tetramerized and monomerized in vivo for faster clearance without the use of clearing agents.

[0012] In certain embodiments, antibodies or antigen-binding fragments thereof that bind to self-assembly degradation (SADA) polypeptides are disclosed in International Patent Application Publication No. WO2018204873 (all of which are incorporated by reference in their entirety).

[0013] In a specific embodiment, the tetramerization domain is identical to SEQ ID NO:139.

[0014] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an engineered protein with high affinity for a DOTA chelate and is disclosed in U.S. Pat. No. US8648176 or International Patent Application Publication No. WO2010099536, all of which are incorporated by reference in their entirety.

[0015] In one aspect, the present invention relates to a humanized antibody or antigen-binding fragment thereof capable of binding to the B7H3 antigen, which comprises a CDR region having an overall identity of at least 90% to a CDR sequence selected from SEQ ID NOs: 25 to 30 or 64 to 99, and a FR region having an overall identity of at least 70%, preferably at least 75%, to a FR sequence selected from SEQ ID NOs: 40 to 63 or 108 to 131.

[0016] The CDR region refers to the complementarity-determining region (CDR), while the FR region refers to the framework region. The framework region is a part of the variable region (Fab) of an antibody. The variable region consists of seven amino acid regions, four of which are framework regions and three of which are hypervariable regions. The framework region acts as a scaffold for the complementarity-determining region (CDR), also called the hypervariable region of the Fab.

[0017] The challenge is to humanize mouse antibodies so as to maintain their affinity for the antigen while introducing a degree of humanization that avoids adverse reactions such as side effects. A further challenge is to obtain antibodies that exhibit good stability and have the potential to be used as drugs in terms of efficacy and safety.

[0018] In certain embodiments, the stability of antibodies or antibody fragments can be improved using techniques known in the art, such as the introduction of additional disulfide bonds and the replacement of oxidation-labile residues. Disulfide bonds can be formed between the thiol groups of cysteine ​​residues. The introduction of additional disulfide bonds can be achieved by examining the structure of the antibody or antibody fragment, identifying residues in the framework regions within a suitable distance for disulfide bonding, and substituting cysteine ​​residues capable of forming disulfide bonds with the amino acids at those positions. Preferred examples of suitable positions for introducing cysteine ​​residues to form additional disulfide bonds are positions 3 in LFR4 and 9 in HFR2. Without being bound by theory, it is surprising that altering the LFR region can maintain affinity and still enable a high degree of humanization of the antibody while keeping the sequences of the CDR regions close to or identical to those of a non-humanized mouse antibody.

[0019] LFRs may be defined as light chain framework regions, and HFRs may be defined as heavy chain framework regions.

[0020] According to another aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a region having at least 90% identity overall, preferably at least 95% identity, such as at least 96% identity, for example at least 97% identity, to SEQ ID NOs: 28-30, 67-69, 73-75, 79-81, 85-87, 91-93; for example at least 98% identity, for example at least 99% identity or preferably 100% identity; a region having the sequence of one of SEQ ID NOs: 25-27, 64-66, 70-72, 76-78, 82-84, 88-90, 94-99, or a region having a sequence which differs from one of SEQ ID NOs: 25-27, 64-66, 70-72, 76-78, 82-84, 88-90, 94-99 at one, two or three positions, the difference being selected from a substitution, deletion or insertion; a region having at least 75% identity overall to a sequence according to SEQ ID NOs: 40-43, 48-51, 56-59 or 108-119, and A region having at least 75% identity overall to a sequence according to SEQ ID NOs: 44 to 47, 52 to 55, 60 to 63, or 120 to 131 The present invention relates to a humanized antibody or antigen-binding fragment thereof capable of binding to the B7H3 antigen, comprising:

[0021] According to another aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: A region having at least 90%, more preferably 95%, identity overall to SEQ ID NOs: 28-30, 67-69, 73-75, 79-81, 85-87, 91-93, and / or A region having at least 75% identity overall to SEQ ID NOs: 40 to 43, 48 to 51, 56 to 59, or 108 to 119 The present invention relates to a humanized antibody or antigen-binding fragment thereof comprising:

[0022] In another aspect, the present invention relates to a humanized antibody or antigen-binding fragment thereof comprising a region having at least 80%, more preferably 85%, preferably 90%, more preferably 95% identity overall to SEQ ID NOs: 40-43, 48-51, 56-59, or 108-119.

[0023] According to another aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: A region having at least 90%, more preferably 95%, identity overall to SEQ ID NOs: 25-27, 64-66, 70-72, 76-78, 82-84, 88-90, 94-99, and / or A region having at least 75% identity overall to SEQ ID NOs: 44 to 47, 52 to 55, 60 to 63, or 120 to 131 The present invention relates to a humanized antibody or antigen-binding fragment thereof comprising:

[0024] In another aspect, the present invention relates to a humanized antibody or antigen-binding fragment thereof, comprising a region having at least 80%, more preferably 85%, preferably 90%, more preferably 95% identity overall to SEQ ID NOs: 44-47, 52-55, 60-63, or 120-131.

[0025] In another aspect, the present invention relates to a humanized antibody or antigen-binding fragment thereof, comprising a region selected from SEQ ID NOs: 25-30 and 64-99.

[0026] According to another aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: a region selected from SEQ ID NOs: 28 to 30, 67 to 69, 73 to 75, 79 to 81, 85 to 87, and 91 to 93; a region selected from SEQ ID NOs: 25 to 27, 64 to 66, 70 to 72, 76 to 78, 82 to 84, 88 to 90, and 94 to 99; A region selected from SEQ ID NOs: 40 to 43, 48 to 51, 56 to 59, or 108 to 119; and A region selected from SEQ ID NOs: 44 to 47, 52 to 55, 60 to 63, or 120 to 131 The present invention relates to a humanized antibody or antigen-binding fragment thereof comprising:

[0027] According to another aspect, the present invention relates to a humanized antibody or antigen-binding fragment thereof comprising Fc, Fc2, or Null-Fc.

[0028] According to another aspect, the present invention relates to a humanized antibody or antigen-binding fragment thereof capable of binding to the B7H3 antigen, wherein the antibody or antigen-binding fragment comprises a sequence having at least 70% identity to a sequence selected from among any of SEQ ID NOs: 36, 37, 38, and 39.

[0029] According to another aspect, the present invention relates to an antibody or antigen-binding fragment thereof capable of binding to an antigen, wherein the antibody or antigen-binding fragment comprises a sequence according to SEQ ID NO: 15, 16, 17 or 132.

[0030] According to another aspect, the present invention relates to a self-assembly and disassembly (SADA) polypeptide, which binds to an antibody or antigen-binding fragment according to the present invention.

[0031] According to another aspect, the present invention provides a method for producing a medicament for the treatment of a pulmonary arthritis, comprising: A self-assembly and disassembly (SADA) polypeptide according to the present invention, and Antibodies or antigen-binding fragments according to the present invention The present invention relates to a polypeptide conjugate comprising:

[0032] According to another aspect, the present invention relates to a polypeptide conjugate comprising a self-assembly and disassembly (SADA) polypeptide, which conjugate further comprises a bispecific antibody according to the present invention, wherein the first antigen is B7H3 and the second antigen is DOTA.

[0033] According to another aspect, the present invention relates to a polypeptide conjugate comprising a self-assembly and disassembly (SADA) polypeptide and at least a first binding domain that binds to a first target and is covalently linked to the SADA polypeptide.

[0034] According to another aspect, the present invention relates to an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment according to the invention.

[0035] According to another aspect, the present invention relates to an isolated nucleic acid molecule comprising a sequence according to SEQ ID NO: 18, 19, 20, 21 or 22.

[0036] According to another aspect, the present invention relates to a recombinant vector comprising an isolated nucleic acid molecule according to the present invention.

[0037] According to another aspect, the present invention relates to a host cell comprising a recombinant vector according to the invention.

[0038] According to another aspect, the present invention relates to a method for the production of an antibody or antigen-binding fragment thereof according to the invention, comprising culturing a host cell according to the invention in a culture medium under conditions that allow expression of the antibody or fragment, and separating the antibody or fragment from the culture medium.

[0039] According to another aspect, the present invention relates to a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment according to the invention.

[0040] According to another aspect, the present invention relates to a CAR-T cell expressing a CAR.

[0041] According to another aspect, the present invention relates to a population of CAR-T cells.

[0042] According to another aspect, the present invention relates to a composition comprising a population of CAR-T cells.

[0043] According to another aspect, the present invention relates to CAR-NK cells expressing a CAR.

[0044] According to another aspect, the present invention relates to a population of CAR-NK cells.

[0045] According to another aspect, the present invention relates to a composition comprising a population of CAR-NK cells.

[0046] According to another aspect, the present invention relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment according to the invention.

[0047] According to another aspect, the present invention relates to a T cell comprising an antibody or antigen-binding fragment according to the invention.

[0048] According to another aspect, the present invention relates to a method of treating, preventing, alleviating, and / or diagnosing a symptom of a condition in a subject, the condition being characterized by expression of the B7H3 antigen, comprising the step of intraperitoneally administering an antibody, antigen-binding fragment, bispecific antibody, trispecific antibody, polypeptide conjugate, composition, and / or CAR.

[0049] According to another aspect, the present invention relates to a method for imaging intraperitoneal tumors, which imaging comprises the use of an antibody or antigen-binding fragment thereof, wherein the tumor is characterized by expression of the B7H3 antigen, and wherein the imaging method is preceded by a step of intraperitoneal administration of the antibody or antigen-binding fragment thereof.

[0050] According to another aspect, the present invention relates to the use of a composition according to the invention in the manufacture of a medicament for the treatment of cancer for use in a method according to the invention.

[0051] According to another aspect, the present invention relates to the use of an antigen-binding fragment antibody according to the invention in the manufacture of a medicament for the treatment of cancer and / or for use in a method according to the invention.

[0052] According to another aspect, the present invention relates to the in vitro use of an antibody or antigen-binding fragment thereof according to the invention.

[0053] According to another aspect, the present invention relates to a method for treating, preventing, alleviating, and / or diagnosing the symptoms of a condition in a subject, the condition being characterized by expression of the B7H3 antigen, comprising the step of intraperitoneally administering an antibody or antigen-binding fragment according to the present invention.

[0054] According to another aspect, the present invention relates to a method for imaging intraperitoneal tumors, which imaging comprises the use of an antibody or antigen-binding fragment thereof according to the present invention, wherein the medical tumor is characterized by expression of the B7H3 antigen, and wherein the imaging method is preceded by a step of intraperitoneal administration of an antibody or antigen-binding fragment thereof according to the present invention. [Brief explanation of the drawings]

[0055] [Figure 1] SEC-HPLC results for chimeric IgG are shown. [Figure 2] SEC-HPLC results for L2H3 are shown. [Figure 3] SEC-HPLC results for L2H4 are shown. [Figure 4] SEC-HPLC results for L2H5 are shown. [Figure 5] Schematic design of SADA: The tetramerization domain was linked to the ScFv against DOTA via a linker, and the ScFv against DOTA was linked to the ScFv against B7-H3 via a second linker. [Figure 6] 1 shows the binding of SADA to ovarian cancer and glioblastoma cell lines. DETAILED DESCRIPTION OF THE INVENTION

[0056] Disclosure Details According to one embodiment, the present invention provides a method for manufacturing a semiconductor device comprising: a CDR region having at least 90% identity overall to a CDR sequence selected from SEQ ID NOs: 25 to 30 or 64 to 99; and An FR region having at least 70%, preferably at least 75%, identity overall to an FR sequence selected from SEQ ID NOs: 40 to 63 or 108 to 131. The present invention relates to a humanized antibody or antigen-binding fragment thereof capable of binding to a B7H3 antigen, comprising:

[0057] The CDR region refers to the complementarity-determining region (CDR), while the FR region refers to the framework region. The framework region is a part of the variable region (Fab) of an antibody. The variable region consists of seven amino acid regions, four of which are framework regions and three of which are hypervariable regions. The framework region acts as a scaffold for the complementarity-determining region (CDR), also called the hypervariable region of the Fab.

[0058] The challenge is to humanize mouse antibodies so as to maintain their affinity for the antigen while introducing a degree of humanization that avoids adverse reactions such as side effects. A further challenge is to obtain antibodies that exhibit good stability and have the potential to be used as drugs in terms of efficacy and safety.

[0059] Without being bound by theory, it appears surprising that altering the LFR region can maintain affinity by keeping the CDR regions closer to the non-humanized murine antibody, yet still allow for a high degree of humanization of the antibody.

[0060] According to one embodiment, the present invention provides a method for manufacturing a semiconductor device comprising: A region having at least 90% identity overall to SEQ ID NOs: 28 to 30, 67 to 69, 73 to 75, 79 to 81, 85 to 87, and 91 to 93; A region having at least 90% identity overall to SEQ ID NOs: 25 to 27, 64 to 66, 70 to 72, 76 to 78, 82 to 84, 88 to 90, and 94 to 99; a region having at least 75% identity overall to a sequence according to SEQ ID NOs: 40-43, 48-51, 56-59 or 108-119, and A region having at least 75% identity overall to a sequence according to SEQ ID NOs: 44 to 47, 52 to 55, 60 to 63, or 120 to 131 The present invention relates to a humanized antibody or antigen-binding fragment thereof capable of binding to the B7H3 antigen, comprising:

[0061] According to one embodiment, the present invention provides a method for manufacturing a semiconductor device comprising: A region having at least 90%, more preferably 95%, identity overall to SEQ ID NOs: 28-30, 67-69, 73-75, 79-81, 85-87, 91-93, and / or A region having at least 75% identity overall to SEQ ID NOs: 40 to 43, 48 to 51, 56 to 59, or 108 to 119 The present invention relates to a humanized antibody or antigen-binding fragment thereof comprising:

[0062] According to one embodiment, the present invention relates to a humanized antibody or antigen-binding fragment thereof comprising a region having at least 80%, more preferably 85%, preferably 90%, more preferably 95% identity overall to SEQ ID NOs: 40-43, 48-51, 56-59, or 108-119.

[0063] According to one embodiment, the present invention provides a method for manufacturing a semiconductor device comprising: A region having at least 90%, more preferably 95%, identity overall to SEQ ID NOs: 25-27, 64-66, 70-72, 76-78, 82-84, 88-90, 94-99, and / or A region having at least 75% identity overall to SEQ ID NOs: 44 to 47, 52 to 55, 60 to 63, or 120 to 131 The present invention relates to a humanized antibody or antigen-binding fragment thereof comprising:

[0064] According to one embodiment, the present invention provides a method for manufacturing a semiconductor device comprising: A region having at least 80%, more preferably 85%, preferably 90%, more preferably 95% identity overall to SEQ ID NOs: 44 to 47, 52 to 55, 60 to 63, or 120 to 131 The present invention relates to a humanized antibody or antigen-binding fragment thereof comprising:

[0065] According to one embodiment, the present invention relates to a humanized antibody or antigen-binding fragment thereof, comprising a region selected from among SEQ ID NOs: 25-30 and 64-99.

[0066] According to one embodiment, the present invention provides a method for manufacturing a semiconductor device comprising: a region selected from SEQ ID NOs: 28 to 30, 67 to 69, 73 to 75, 79 to 81, 85 to 87, and 91 to 93; a region selected from SEQ ID NOs: 25 to 27, 64 to 66, 70 to 72, 76 to 78, 82 to 84, 88 to 90, and 94 to 99; A region selected from SEQ ID NOs: 40 to 43, 48 to 51, 56 to 59, or 108 to 119, and A region selected from SEQ ID NOs: 44 to 47, 52 to 55, 60 to 63, or 120 to 131 The present invention relates to a humanized antibody or antigen-binding fragment thereof comprising:

[0067] According to one embodiment, the present invention relates to a humanized antibody or antigen-binding fragment thereof comprising Fc, Fc2 or Null-Fc.

[0068] According to one embodiment, the present invention relates to a humanized antibody or antigen-binding fragment thereof capable of binding to the B7H3 antigen, wherein the antibody or antigen-binding fragment comprises a sequence having at least 70% identity to a sequence selected from among any of SEQ ID NOs: 36, 37, 38, and 39.

[0069] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof according to the invention, wherein the antibody or antigen-binding fragment comprises a sequence having at least 75% identity to a sequence selected from among any of SEQ ID NOs: 36, 37, 38 or 39.

[0070] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment according to the present invention, which comprises at least one sequence selected from a heavy chain variable region CDR1 according to SEQ ID NOs: 25, 64, 70, 76, 82, 88, 94 and 97, a heavy chain variable region CDR2 according to SEQ ID NOs: 26, 65, 71, 77, 83, 89, 95 and 98, a heavy chain variable region CDR3 according to SEQ ID NOs: 27, 66, 72, 78, 84, 90, 96 and 99, a light chain variable region CDR1 according to SEQ ID NOs: 28, 67, 73, 79, 85 and 91, a light chain variable region CDR2 according to SEQ ID NOs: 29, 68, 74, 80, 86 and 92, and a light chain variable region CDR3 according to SEQ ID NOs: 30, 69, 75, 81, 87 and 93.

[0071] According to one embodiment, the invention relates to an antibody or antigen-binding fragment according to the invention, which antibody comprises a heavy chain sequence according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8 or 9 and / or a light chain sequence according to SEQ ID NO: 10, 11, 12, 13 or 14.

[0072] According to one embodiment, the invention relates to an antibody or antigen-binding fragment, wherein the antibody has a sequence identity at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, or 9. % sequence identity to the sequence set forth in SEQ ID NO: 10, 11, 12, 13 or 14 and / or a light chain sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequence set forth in SEQ ID NO: 10, 11, 12, 13 or 14.

[0073] According to one embodiment, the invention relates to an antibody or antigen-binding fragment, which comprises at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 77.5%, at least 80%, at least 82%, at least 84%, at least 86%, at least 88%, or at least 90% amino acids identical to human germline amino acids of the antibody or antigen-binding fragment.

[0074] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment, which binds to an epitope, and which epitope is an epitope of B7H3.

[0075] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment, wherein the antibody of the antigen-binding fragment binds to a sequence according to SEQ ID NO:33.

[0076] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment, which binds to an antigen, and which antigen comprises a sequence selected from among SEQ ID NOs: 31 and 32.

[0077] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment, the antigen of which is presented on a cancer cell.

[0078] According to one embodiment, the invention relates to an antibody or antigen-binding fragment according to the invention, wherein said cancer cells originate from a metastasis.

[0079] According to one embodiment, the invention relates to an antibody or antigen-binding fragment according to the invention, wherein the cancer cells and / or metastases are prostate cancer, desmoplastic small cell tumor, ovarian cancer, gastric cancer, pancreatic cancer, liver cancer, renal cancer, breast cancer, non-small cell lung cancer, melanoma, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, Ewing's sarcoma, Wilms' sarcoma, neuroblastoma, ganglioneuroblastoma, medulloblastoma, high-grade glioma, diffuse intrinsic pontine glioma, embryonal tumor with multilayered rosettes, or a B7H3-expressing cancer.

[0080] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment comprising an Fc region that does not interact with an Fc gamma receptor.

[0081] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment further comprising an Fc region, which Fc region is not reactive or shows only little reactivity.

[0082] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment, which comprises a null Fc.

[0083] According to one embodiment, the invention relates to an antibody or antigen-binding fragment thereof, which has an immunogenicity of less than 50%, less than 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, or less than about 10%.

[0084] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, wherein said agent or antibody is a murine antibody or antigen-binding fragment thereof.

[0085] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, wherein said agent or antibody is a chimeric antibody or antigen-binding fragment thereof.

[0086] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, wherein said agent or antibody is a humanized antibody or antigen-binding fragment thereof.

[0087] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment is radiolabeled with a radioisotope.

[0088] According to one embodiment, the invention relates to an antibody or antigen-binding fragment thereof, wherein said radioisotope is selected from among a PET label and / or a SPECT label.

[0089] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, wherein said PET label comprises: 124 I, 225 Ac and 89 Zr is selected from the group consisting of

[0090] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, wherein said SPECT label comprises: 131 I, 177 Lu, 99 mTc, 64 Cu and 89 Zr is selected from the group consisting of

[0091] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment is conjugated to a chelator compound.

[0092] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, wherein said chelator compound is conjugated to a radioisotope.

[0093] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, wherein the radioisotope is: 124I, 131 I and 177 Lu or 99 mTc, 64 Cu and 89 Zr is selected from the group consisting of

[0094] According to one embodiment, the invention relates to an antibody or antigen-binding fragment thereof, wherein said chelator compound is selected from among DOTA, DTPA, NOTA and DFO.

[0095] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, wherein said DOTA is a variant of DOTA, such as benzyl-DOTA.

[0096] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, wherein said DTPA is a variant of DTPA, such as CHX-A″-DTPA.

[0097] According to one embodiment, the invention relates to an antibody or antigen-binding fragment, wherein said radioisotope is an alpha, beta or positron emitting radionuclide.

[0098] According to one embodiment, the invention relates to an antibody or antigen-binding fragment comprising a structure selected from among IgG, IgG1, IgG2, IgG3, and IgG4.

[0099] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment comprising a structure selected from among IgG, IgM, IgA, IgD, and IgE.

[0100] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof capable of binding to an antigen, wherein the antibody or antigen-binding fragment comprises a sequence according to SEQ ID NO: 15, 16, 17 or 132.

[0101] According to one embodiment, the present invention relates to a self-assembly and disassembly (SADA) polypeptide, which binds to an antibody or antigen-binding fragment according to the present invention.

[0102] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment thereof, which antibody or antigen-binding fragment thereof is a bispecific and / or trispecific binding antibody.

[0103] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment, wherein said bispecific and / or trispecific binding antibody comprises a first antibody or antigen-binding fragment thereof according to the invention for binding to a first antigen, and a second antibody or antigen-binding fragment thereof for binding to a second antigen.

[0104] According to one embodiment, the invention relates to an antibody or antigen-binding fragment, wherein said second antibody or antigen-binding fragment thereof binds to DOTA and / or DTPA.

[0105] DOTA (dodecanetetraacetic acid) is also called 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and has the formula (CH2CH2NCH2CO2H)4.

[0106] DTPA (diethylenetriaminepentaacetic acid) is also known by the IUPAC name 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid. DTPA has the molecular formula C 14 H 23 N3O 10 It has.

[0107] According to one embodiment, the present invention relates to an antibody or antigen-binding fragment, which binds to a self-assembly degradation (SADA) polypeptide.

[0108] In certain embodiments, the antibody of the antigen-binding fragment binds to the self-assembly disassembly (SADA) polypeptide disclosed in International Patent Application Publication No. WO2018204873, all of which are incorporated by reference in their entirety.

[0109] In one embodiment, the present invention relates to an antibody or antigen-binding fragment, wherein the self-assembly and disassembly (SADA) polypeptide has an amino acid sequence that exhibits at least 75% identity to the amino acid sequence of a human homomultimerizing polypeptide and is characterized by one or more multimerization dissociation constants (KD).

[0110] According to one embodiment, the present invention provides a method for manufacturing a semiconductor device comprising: A self-assembly and disassembly (SADA) polypeptide according to the present invention, and Antibodies or antigen-binding fragments according to the present invention The present invention relates to a polypeptide conjugate comprising:

[0111] According to one embodiment, the present invention relates to a polypeptide conjugate comprising a self-assembly and degradation (SADA) polypeptide, and the conjugate further comprises a bispecific antibody according to the present invention, wherein the first antigen is B7H3 and the second antigen is DOTA.

[0112] According to one embodiment, the present invention relates to a polypeptide conjugate comprising a self-assembly and disassembly (SADA) polypeptide and at least a first binding domain that binds to a first target and is covalently linked to the SADA polypeptide.

[0113] In one embodiment, the present invention relates to a polypeptide conjugate, wherein the self-assembly and disassembly (SADA) polypeptide has an amino acid sequence that exhibits at least 75% identity to the amino acid sequence of a human homomultimerizing polypeptide and is characterized by one or more multimerization dissociation constants (KD); and the conjugate is constructed and organized to have a first multimerization state and one or more higher multimerization states, wherein: the first multimerization state is less than about 70 kDa in size; and at least one of the higher multimerization states is a homotetramer or higher homomultimer greater than 150 kDa in size; the higher homomultimerized conjugates are stable in aqueous solution when present at concentrations higher than the SADA polypeptide KD; and transition from the higher multimerization state(s) to the first multimerization state under physiological conditions when the concentration of the conjugate is lower than the SADA polypeptide KD.

[0114] According to one embodiment, the present invention relates to a polypeptide conjugate, which comprises a chelator.

[0115] According to one embodiment, the present invention relates to a conjugate, wherein the chelator comprises a metal ion.

[0116] According to one embodiment, the present invention relates to a conjugate, wherein said metal ion is a radionuclide.

[0117] According to one embodiment, the present invention relates to an isolated nucleic acid molecule encoding an antibody or antigen-binding fragment of the present invention.

[0118] According to one embodiment, the present invention relates to an isolated nucleic acid molecule comprising a sequence according to SEQ ID NO: 18, 19, 20, 21 or 22.

[0119] According to one embodiment, the present invention relates to a recombinant vector comprising the isolated nucleic acid molecule of the present invention.

[0120] According to one embodiment, the present invention relates to a host cell comprising a recombinant vector according to the invention.

[0121] According to one embodiment, the present invention relates to a method for the production of an antibody or antigen-binding fragment thereof according to the invention, comprising culturing a host cell according to the invention in a culture medium under conditions allowing expression of the antibody or fragment, and separating the antibody or fragment from the culture medium.

[0122] According to one embodiment, the present invention relates to a chimeric antigen receptor (CAR) comprising an antibody or antigen-binding fragment according to the present invention.

[0123] According to one embodiment, the present invention relates to a CAR-T cell expressing a CAR.

[0124] According to one embodiment, the present invention relates to a population of CAR-T cells.

[0125] According to one embodiment, the present invention relates to a composition comprising a population of CAR-T cells.

[0126] According to one embodiment, the present invention relates to CAR-NK cells expressing a CAR.

[0127] According to one embodiment, the present invention relates to a population of CAR-NK cells.

[0128] According to one embodiment, the present invention relates to a composition comprising a population of CAR-NK cells.

[0129] According to one embodiment, the present invention relates to a pharmaceutical composition comprising an antibody or antigen-binding fragment according to the invention.

[0130] According to one embodiment, the invention relates to a T cell comprising an antibody or antigen-binding fragment according to the invention.

[0131] According to one embodiment, the present invention relates to a method of treating, preventing, alleviating, and / or diagnosing a symptom of a condition in a subject, the condition being characterized by expression of the B7H3 antigen, comprising the step of intraperitoneally administering an antibody, antigen-binding fragment, bispecific antibody, trispecific antibody, polypeptide conjugate, composition, and / or CAR.

[0132] According to one embodiment, the present invention relates to a method for imaging intraperitoneal tumors, which imaging comprises the use of an antibody or antigen-binding fragment thereof, wherein the tumor is characterized by expression of the B7H3 antigen, and the imaging method is preceded by a step of intraperitoneal administration of the antibody or antigen-binding fragment thereof.

[0133] According to one embodiment, the present invention relates to a method, wherein said antibody, antigen-binding fragment, bispecific antibody, trispecific antibody, polypeptide conjugate, composition and / or CAR is an antibody, antigen-binding fragment, bispecific antibody, trispecific antibody, polypeptide conjugate, composition and / or CAR according to the present invention.

[0134] According to one embodiment, the present invention relates to a method, wherein the antibody or antigen-binding fragment thereof comprises at least one sequence selected from a heavy chain variable region CDR1 according to SEQ ID NO: 25, a heavy chain variable region CDR2 according to SEQ ID NO: 26, a heavy chain variable region CDR3 according to SEQ ID NO: 27, a light chain variable region CDR1 according to SEQ ID NO: 28, a light chain variable region CDR2 according to SEQ ID NO: 29, and a light chain variable region CDR3 according to SEQ ID NO: 30.

[0135] According to one embodiment, the invention relates to a method, wherein said antibody comprises a heavy chain sequence according to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 23 and / or a light chain sequence according to SEQ ID NO: 10, 11, 12, 13, 14 or 24.

[0136] According to one embodiment, the invention provides an antibody having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity to the sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, or 23. The present invention relates to a method comprising administering to a mammalian subject the invention comprising administering to a mammalian subject a heavy chain sequence and / or a light chain sequence having at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98% or about 99% sequence identity to the sequence set forth in SEQ ID NO: 10, 11, 12, 13, 14 or 24.

[0137] According to one embodiment, the present invention relates to the use of a composition according to the invention in the manufacture of a medicament for the treatment of cancer for use in a method according to the invention.

[0138] According to one embodiment, the present invention relates to the use of an antigen-binding fragment antibody according to the invention for the manufacture of a medicament for the treatment of cancer and / or for use in a method according to the invention.

[0139] According to one embodiment, the present invention relates to the in vitro use of an antibody or antigen-binding fragment thereof according to the invention.

[0140] According to one embodiment, the present invention relates to a method wherein said condition is cancer.

[0141] According to one embodiment, the present invention relates to a method, wherein said cancer and / or said tumor is a metastasis.

[0142] According to one embodiment, the present invention relates to a method, wherein the cancer, tumor and / or metastasis is prostate cancer, desmoplastic small cell tumor, ovarian cancer, gastric cancer, pancreatic cancer, liver cancer, renal cancer, breast cancer, non-small cell lung cancer, melanoma, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, Ewing's sarcoma, Wilms' sarcoma, neuroblastoma, ganglioneuroblastoma, medulloblastoma, high-grade glioma, childhood diffuse intrinsic pontine glioma, embryonal tumor with multilayered rosettes, or a B7H3-expressing cancer.

[0143] According to one embodiment, the present invention relates to a method of treating, preventing, alleviating the symptoms of, and / or diagnosing a condition in a subject, wherein the condition is characterized by expression of the B7H3 antigen, comprising intraperitoneal administration of an antibody or antigen-binding fragment according to the present invention.

[0144] According to one embodiment, the present invention relates to a method for imaging intraperitoneal tumors, which imaging comprises the use of an antibody or antigen-binding fragment thereof according to the present invention, wherein the medical tumor is characterized by expression of the B7H3 antigen, and the imaging method is preceded by a step of intraperitoneal administration of an antibody or antigen-binding fragment thereof according to the present invention.

[0145] In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

[0146] Affinity: As known in the art, "affinity" is a measure of the strength with which a particular ligand (e.g., an antibody) binds to its partner (e.g., an epitope). Affinity can be measured in a variety of ways.

[0147] Antibody: The term "antibody" is an art-recognized term and is intended to include molecules or active fragments of molecules that bind to a known antigen. Examples of active fragments of molecules that bind to a known antigen include Fab and F(ab')2 fragments. These active fragments can be obtained from the antibodies of the present invention by a number of techniques. For example, purified monoclonal antibodies can be cleaved with an enzyme such as pepsin and subjected to HPLC gel filtration. Appropriate fractions containing Fab fragments can then be collected and concentrated, such as by membrane filtration. The term "antibody" also includes bispecific and chimeric antibodies, as well as other available formats.

[0148] Antibody fragment: An antibody fragment is a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, or sFv. Regardless of structure, an antibody fragment binds to the same antigen recognized by the intact antibody. For example, the 3F8 monoclonal antibody fragment binds to the epitope recognized by 3F8. The term "antibody fragment" also includes any synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex. For example, antibody fragments include isolated fragments consisting of the variable region (e.g., an "Fv" fragment consisting of the heavy and light chain variable regions), recombinant single-chain polypeptide molecules in which the light and heavy chain variable regions are linked by a peptide linker ("scFv protein"), and minimal recognition units consisting of amino acid residues mimicking the hypervariable region.

[0149] Bispecific antibodies: Bispecific antibodies are antibodies that can simultaneously bind to two targets of different structures. Bispecific antibodies (bsAb) and bispecific antibody fragments (bsFab) have at least one arm, specifically an arm that binds to an antigen, e.g., GD2, and at least one other arm, specifically an arm that binds to another antigen, e.g., a targetable conjugate carrying a therapeutic or diagnostic agent. Various bispecific fusion proteins can be generated using molecular engineering. In one form, the bispecific fusion protein is bivalent, e.g., consisting of an scFv with one binding site for one antigen and a Fab fragment with one binding site for a second antigen. In another form, the bispecific fusion protein is tetravalent, e.g., consisting of an IgG with two binding sites for one antigen and two identical scFvs for a second antigen.

[0150] Chimeric antibody: A chimeric antibody is a recombinant protein that contains the variable domains, including the complementarity determining regions (CDRs), of an antibody from one species, for example, a rodent antibody, while the constant domains of the antibody molecule are derived from the constant domains of a human antibody. The constant domains of a chimeric antibody may also be derived from the constant domains of another species, for example, a cat or dog.

[0151] Effective amount: As used herein, the term "effective amount" refers to the amount of a given compound, conjugate, or composition necessary or sufficient to realize a desired biological effect. An effective amount of a given compound, conjugate, or composition according to the methods of the invention is the amount that achieves this selected result, and such an amount can be determined as a matter of routine by one of ordinary skill in the art without necessitating undue experimentation.

[0152] Humanized antibody: A humanized antibody is a recombinant protein in which the CDRs of an antibody from one species, such as a rodent antibody, have been transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains. The constant domains of the antibody molecule are derived from the constant domains of a human antibody.

[0153] Human antibodies may also be obtained from transgenic mice that have been "engineered" to produce specific human antibodies in response to antigenic challenge. In this technique, elements of human heavy and light chain loci are introduced into strains of mice derived from embryonic stem cell lines containing targeted disruptions of the endogenous heavy and light chain loci. The transgenic mice are capable of synthesizing human antibodies specific to human antigens, and these mice can be used to produce human antibody-secreting hybridomas.

[0154] Immunogenicity: Immunogenicity can be defined as the propensity of a therapeutic protein product to generate an immune response against self and related proteins or to induce immunologically related adverse clinical events (FDA). Immunogenicity can refer to the ability of a molecule or substance to elicit an immune response. Unwanted immunogenicity can be an immune response by an organism against the therapeutic antigen. The incidence of anti-therapeutic antibody responses in patients is described as a % of the patient population.

[0155] Immunogenicity can be tested by ELISA for HAMA (human anti-mouse antibodies), in which serum samples are analyzed through an established ELISA assay using mouse antibodies as the capture antigen.

[0156] Prevent: As used herein, the terms "prevent," "preventing," and "prevention" refer to the prevention of the recurrence or onset of one or more symptoms of a disorder in a subject as a result of the administration of a prophylactic or therapeutic agent.

[0157] Radioisotopes: Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include, but are not limited to: 211 At, 14 C. 51 Cr, 57 Co, 58 Co, 67 Cu, 152 EU, 67 Ga, 3 H, 111 In, 59 Fe, 212Pb, 177 Lu, 32 P, 223 Ra, 224 Ra, 186 Re, 188 Re, 75 Se, 35 S, 99m Tc, 227 Th, 89 Zr, 90 Y, 123 I, 124 I, 125 I, 131 I, 94m Tc, 64 Cu, 68 Ga, 66 Ga, 76 Br, 86 Y, 82 Rb, 110m In, 13 N, 11 C. 18 F and alpha-emitting particles. Non-limiting examples of alpha-emitting particles include: 209 Bi, 211 Bi, 212 Bi, 213 Bi, 210 Po, 211 Po, 212 Po, 214 Po, 215 Po, 216 Po, 218 Po, 211 At, 215 At, 217 At, 218 At, 218 Rn, 219 Rn, 220 Rn, 222 Rn, 226 Rn, 221 Fr, 223 Ra, 224 Ra, 226 Ra, 225 Ac, 227 Ac, 227 Th, 228 Th, 229 Th, 230 Th, 232 Th, 231 Pa, 233 U, 234U, 235 U, 236 U, 238 U, 237 Np, 238 Pu, 239 Pu, 240 Pu, 244 Pu, 241 Am, 244 Cm, 245 Cm, 248 Cm, 249 Cf, and 252 Cf is an example.

[0158] Sequence alignment: Sequence alignment simply refers to any method of placing two sequences one above the other. It is a method of aligning DNA, RNA, or protein sequences to identify regions of similarity between the sequences. Different alignment algorithms exist, and they usually have a scoring function that assigns a numerical score to every alignment indicating how good the alignment is, and they try to find the best alignment according to the scoring function.

[0159] Sequence identity: The term "sequence identity" is used herein as a measure of relatedness between two amino acid or nucleic acid sequences. To calculate the sequence identity between two sequences, the sequences are aligned and the longest overlap is identified. Sequence identity is calculated as the percentage of identical bases at corresponding positions in the overlap, over the length of the overlap.

[0160] A variety of computer algorithms are available to those skilled in the art for creating sequence alignments and calculating sequence identity. As used herein, sequence alignment refers to pairwise alignment. Several algorithms accomplish this, including the sequence alignment program FASTA, which uses the Smith-Waterman algorithm.

[0161] As used herein, sequence alignment may refer to the following algorithms and parameters: Algorithm: FASTA (3.8 Nov 2011) [Optimized] Parameters: BL50 matrix(15:-5), open / ext:-10 / -2 ktup:2, E-join:1(1), E-opt:0.2(1), width:16

[0162] Subject: "Subject" or "individual" or "animal" or "patient" or "mammal" means any subject for whom diagnosis, prognosis, or treatment is desired, particularly a mammalian subject. Mammalian subjects include humans and other primates, domestic animals, farm animals, and zoo animals, sport animals, or pet animals, such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cows, cows, etc.

[0163] Treatment: As used herein, the terms "treatment," "treat," "treated," or "treating" refer to prevention and / or treatment, particularly prevention and / or treatment with the goal of preventing or slowing (alleviating) an undesirable physiological change or disorder (such as the progression of multiple sclerosis). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, whether detectable or undetectable, reduction in the extent of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, palliation or remission of the disease state, and remission (either partial or total). "Treatment" can also mean longer survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder, as well as those susceptible to the condition or disorder or those in whom the condition or disorder is to be prevented.

[0164] The accompanying figures and examples are provided to illustrate rather than limit the invention. It will be apparent to those skilled in the art that the aspects, embodiments, claims and optional items of the invention may be combined.

[0165] Unless otherwise stated, all percentages are weight / weight. Unless otherwise stated, all measurements are made under standard conditions (ambient temperature and pressure). Unless otherwise stated, test conditions are in accordance with European Pharmacopoeia 8.0. [Example]

[0166] Example 1: Design of a humanized anti-B7-H3 antibody The murine 8H9 antibody, comprising a heavy chain sequence according to SEQ ID NO:23 and a light chain sequence according to SEQ ID NO:24, was humanized by grafting IMGT CDR residues onto a human IgG1 framework using the human germline sequences IGKV6-21*02 (SEQ ID NO:37) for the kappa variable domain and IGHV1-8*01 (SEQ ID NO:38) for the variable heavy domain. Selected back mutations were then inserted based on rational design and molecular modeling of the crystal structure of the murine 8H9 antibody Fab fragment (Protein Data Bank structure 5CMA: anti-B7H3 monoclonal antibody ch8H9 Fab fragment), comprising a heavy chain sequence according to SEQ ID NO:34 and a light chain sequence according to SEQ ID NO:35, to generate six humanized VH candidates and four humanized VL candidates, each with a human germline content of greater than 85%. Computer modeling was performed using Biovia Discovery Studio software (Dassault Systemes). Each of the humanized sequences was rationally engineered to retain the affinity of murine 8H9.

[0167] Example 2: Generation and characterization of humanized anti-B7-H3 antibodies Chimeric and humanized antibodies were generated using a CDR-grafting method with highly homologous human germline sequences. Six different humanized VH and four humanized VL sequences were combined to generate 24 different humanized IgG1 antibodies. The antibodies were expressed in HEK293 cells and purified using Protein A resin. The antibodies were buffer-exchanged into PBS and quantified by OD280, and titers were calculated. Sample purity and aggregation state were determined by analytical size-exclusion chromatography (SEC-HPLC), which separates molecules based on molecular mass and hydrodynamic volume. The area percentage of each peak was calculated based on the total area of ​​the peak. Peak symmetry was calculated to determine fronting peaks (less than 1) or tailing peaks (greater than 1). A 300A pore size column was used for detection at 280 nm, and PBS was used as the running buffer. Surface plasmon resonance studies to evaluate binding affinity were performed on a Carterra LSA instrument. Anti-human Fc antibody "lawn" was prepared by amine coupling on an HC30M chip. The chip was activated with equal volumes of 100 mM MES pH 5.5, 100 mM S-NHS, and 400 mM EDC. Anti-human IgG Fc antibody was immobilized. The chip was inactivated with 1 M ethanolamine pH 8.5. The antibody was diluted and printed onto the anti-human Fc lawn. An eight-point series of 4Ig human B7-H3 was prepared. For kinetic studies, association was observed for 5 minutes, and dissociation was observed for 15 minutes.

[0168] Expression titer (mg of purified protein per L of culture), purity (% monomer peak by analytical SEC-HPLC) and SPR (ka, kd and K D The characterization data, including affinity measurements by ), are summarized in Table 1 and Figures 1-14.

[0169] [Table 1] TIFF0007759348000002.tif62161

[0170] The IMGT / DomainGapAlign (imgt.org) tool was used to calculate human germline content for the heavy chain H1-H6 and light chain L1-L4 variable regions (Table 2).

[0171] [Table 2]

[0172] Three constructs, L2H3, L2H4, and L2H5, were selected as lead candidates based on expression titer, % purity, binding affinity relative to chimeric IgG, and human germline content. L2H5 had the highest human content (both VH5 and VL2) and purity (Figures 1-4) of the three, all of which were higher than the parent chimeric antibody. L2H5 also had nearly identical affinity for chimeric IgG (9.4 nM and 10 nM, respectively). D ). Therefore, L2H5 was selected for further investigation, including the generation of an anti-B7H3 x anti-DOTA bispecific SADA (self-assembly and degradation) antibody construct.

[0173] Example 3: Production and characterization of anti-B7-H3 SADA for radioimmunotherapy Three anti-B7H3 x anti-DOTA bispecific SADA constructs (3BH-1, 3BH-2, and 3BH-3) were designed as shown in Figure 5. 3BH-1 consists of an anti-B7H3 scFv derived from the murine 8H9 antibody, which contains the heavy chain sequence according to SEQ ID NO:23 and the light chain sequence according to SEQ ID NO:24 in a VH-VL orientation. 3BH-2 consists of an anti-B7H3 scFv derived from the L2H5 sequence in a VH-VL orientation. 3BH-3 has a disulfide bond in the L2H5 scFv for improved stability (Nh44-VL100 according to Kabat numbering). All three constructs contain the same anti-DOTA scFv (humanized C825) with VH-VL disulfide stabilization.

[0174] Example 4: SADA binding to B7H3(+) cell lines via flow cytometry Two cancer cell lines were separately incubated with SADA protein using a titration series for 30 minutes at 4°C. After a washing step, the cells were incubated with a fluorophore-conjugated secondary antibody (anti-His) for 30 minutes at 4°C. After washing again, the cells were analyzed using a flow cytometer. 3BH-1 and 3BH-3 showed concentration-dependent binding to the B7-H3-positive cell lines, glioblastoma cell line U-87MG and ovarian adenocarcinoma cell line SK-OV-3 (Figure 6).

[0175] Example 5: 131 In vivo antitumor effect of I-labeled anti-B7-H3 antibody To evaluate the antitumor effect of the humanized anti-B7H3 antibody, luciferase-transfected human ovarian cancer SKOV-3 cells or gastric cancer NCI-N87 cells were injected intraperitoneally into nude mice. In the planned protocol, iodine-131 ( 131 I) Labeled YB8-L2H5 will be administered as a single injection at various doses and tumor burden will be assessed weekly using bioluminescence imaging.

[0176] Further related sequences are provided below.

[0177] SEQ ID NO: 1: Chimeric heavy chain (parent) QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIFPGDGSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTATWFAYWGQGT LVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 2: YB8 H1 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQGLEWIGWIFPGDGSTQYNEKFQGRVTLTTNTSISTAYMELSSLRSEDTAVYYCARQTTATWFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 3: YB8 H2 QVQLQQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQGLEWIGWIFPGDGSTQYNEKFKGRATLTRNTSISTAYMELSSLRSEDTAVYYCARQTTATWFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 4: YB8 H3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQGLEWMGWIFPGDGSTQYNEKFKGRVTLTRNTSISTAYMELSSLRSEDTAVYFCARQTTATWFAYWGQGTLVTSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK sequence number 5:YB8 H4 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQGLEWIGWIFPGDGSTQYNEKFQGRVTMTTNTSISTAYMELSSLRSEDTAVYFCARQTTATWFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 6: YB8 H5 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQGLEWMGWIFPGDGSTQYNEKFQGRVTMTTNTSISTAYMELSSLRSEDTAVYYCARQTTATWFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 7: YB8 H5 N297A and K322A QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQGLEWMGWIFPGDGSTQYNEKFQGRVTMTTNTSISTAYMELSSLRSEDTAVYYCARQTTATWFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 8: YB8_H5 L234A, L235A and K322A QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQGLEWMGWIFPGDGSTQYNEKFQGRVTMTTNTSISTAYMELSSLRSEDTAVYYCARQTTATWFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPPAPEAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 9: YB8 H6 QVQLQQSGAEVKKPGASVKLSCKASGYTFTNYDINWVRQATGQGLEWIGWIFPGDGSTQYAQKFQGRATLTTNTSISTAYMELSSLRSEDTAVYFCARQTTATWFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 10: Chimeric light chain (parent) DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 11: YB8 L1 EIVLTQSPDFQSVTPKEKVTLTCRASQSISDYLHWYQQKPDQSPKLLIKYASQSISGVPSRFSGSGSGSDFTLTINSLEAEDAATYYCQNGHSFPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 12: YB8 L2 EIVMTQSPDFQSVTPKEKVTITCRASQSISDYLHWYQQKPDQSPKLLIKYASQSISGVPSRFSGSGSGSDFTLTINSLEAEDAATYYCQNGHSFPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 13: YB8 L3 EIVMTQSPDFQSVTPKEKVTLTCRASQSISDYLHWYQQKPDQSPKLLIKYASQSISGVPSRFSGSGSGSDFTLTINSLEAEDAGVYYCQNGHSFPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 14: YB8 L4 EIVMTQSPDFQSVTPKEKVTLTCRASQSISDYLHWYQQKPDQSPKLLIKYASQSISGIPSRFSGSGSGTDFTLTINSVEAEDAGVYYCQNGHSFPLTFGQGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 15:3BH-1 QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIFPGDGSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTATWFAYWGQGTLVTVSAGGGGSGGGGSGGGGSGGGGSGGGGGSGGGGSD IVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELKGGGGSGGGGSGGGGSGGGGSHVQLVESGGGLVQPGGSLRLSCA ASGFSLTDYGVHWVRQAPGKGLEWLGVIWSGGGTAYNTALISRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGSYPYNYFDAWGCGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCG SSTGAVTASNYANWVQQKPGQCPRGLIGGHNNRPPGVPARFSGSLLGGKAALTLLGAQPEDEAEYYCALWYSDHWVIGGGTKLTVLGTPLGDTTHTSGKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSGGAPHHHHHH SEQ ID NO: 16:3BH-2 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQGLEWWMGWIFPGDGSTQYNEKFQGRVTMTTNTSISTAYMELSSLRSEDTAVYYCARQTTATWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGSGGGGSE IVMTQSPDFQSVTPKEKVTITCRASQSISDYLHWYQQKPDQSPKLLIKYASQSISGVPSRFSGSGSGSDFTLTINSLEAEDAATYYCQNGHSFPLTFGQGTKLEIKGGGGSGGGGSGGGGSGGGGSHVQLVESGGGLVQPGGSLRLSCA ASGFSLTDYGVHWVRQAPGKGLEWLGVIWSGGGTAYNTALISRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGSYPYNYFDAWGCGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCG SSTGAVTASNYANWVQQKPGQCPRGLIGGHNNRPPGVPARFSGSLLGGKAALTLLGAQPEDEAEYYCALWYSDHWVIGGGTKLTVLGTPLGDTTHTSGKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSGGAPHHHHHH SEQ ID NO: 17:3BH-3 QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQCLEWMGWIFPGDGSTQYNEKFQGRVTMTTNTSISTAYMELSSLRSEDTAVYYCARQTTATWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGGSGGGGSE IVMTQSPDFQSVTPKEKVTITCRASQSISDYLHWYQQKPDQSPKLLIKYASQSISGVPSRFSGSGSGSDFTLTINSLEAEDAATYYCQNGHSFPLTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSHVQLVESGGGLVQPGGSLRLSCA ASGFSLTDYGVHWVRQAPGKGLEWLGVIWSGGGTAYNTALISRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGSYPYNYFDAWGCGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVTLTCG SSTGAVTASNYANWVQQKPGQCPRGLIGGHNNRPPGVPARFSGSLLGGKAALTLLGAQPEDEAEYYCALWYSDHWVIGGGTKLTVLGTPLGDTTHTSGKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSGGAPHHHHHH SEQ ID NO: 18: YB8 L2H5 heavy chain Accession number 19: YB8 L2H5 light chain GAGATCGTGATGACCCAGTCTCCTGACTTCCAGAGCGTGACCCCTAAAGAGAAAGTCACCATCACCTGTCGGGCCAGCCAGTCCATCTCTGACTACCTGCACTGGTATCAGCAGAAGCCCGATCAGTCCCCTAAGCTGCTGATTAAGTACGCCAGCCAGAGCATCTCCGGCGTGCCATCCAGATTTTCTGGCTCCGGCTCTGGCTCTGACTTCACCCTGACCATCAATTCCCTGGAAGCCGAGGATGCCGCCACCTACTACTGTCAGAATGGCCACAGCTTCCCTCTGACCTTTGGCCAGGGCACCAAGCTGGAAATCAAGAGAACCGTGGCCGCTCCTTCCGTGTTCATCTTCCCACCATCTGACGAGCAGCTGAAGTCTGGCACCGCTTCTGTCGTGTGCCTGCTGAACAACTTCTACCCTCGGGAAGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAGTCCGGCAACTCCCAAGAGTCTGTGACCGAGCAGGACTCCAAGGACTCTACCTACAGCCTGTCCTCCACACTGACCCTGTCTAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAAGTGACCCACCAGGGACTGTCTAGCCCCGTGACCAAGTCTTTCAACCGGGGCGAGTGTTGA Accession number 20: YB8 L2H5 N297A K322A (null Fc) heavy chain SEQ ID NO: 21: YB8 L2H5 L234A L235A K322A (null Fc) heavy chain SEQ ID NO: 22:3BH-3 SEQ ID NO: 23: Mouse 8H9 heavy chain QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIFPGDGSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTATWFAYWGQ GTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCG CKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTI SKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK SEQ ID NO: 24: Mouse 8H9 light chain DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLELK RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC SEQ ID NO: 25: 8H9 heavy chain CDR-1 NYDIN SEQ ID NO: 26: 8H9 heavy chain CDR-2 WIFPGDGSTQYNEKFKG SEQ ID NO: 27: 8H9 heavy chain CDR-3 QTTATWFAY SEQ ID NO: 28: 8H9 light chain CDR-1 RASQSISDYLH SEQ ID NO: 29: 8H9 light chain CDR-2 YASQSIS SEQ ID NO: 30: 8H9 light chain CDR-3 QNGHSFPLT SEQ ID NO: 31:4Ig-B7H3 MLRRRGPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAA VSLQVAAPYSKPSMTLENKDLRPGDTVTITCSSYQGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNPVLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGTDATLR CSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGRDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPGDTVTITCSSYRGYPEAEVFWQDGQ GVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKEDGQEIA SEQ ID NO: 32:2Ig-B7H3 MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPEDPVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPG DTTVITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDSKEDGQEIA SEQ ID NO: 33: B7H3 epitope IRFD SEQ ID NO: 34: Antibody ch8H9 Fab heavy chain QVQLQQSGAELVKPGASVKLSCKASGYTFTNYDINWVRQRPEQGLEWIGWIFPGDGSTQYNEKFKGKATLTTDTSSSTAYMQLSRLTSEDSAVYFCARQTTATWFAYWGQ GTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKS SEQ ID NO: 35: Antibody ch8H9 Fab light chain DIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYYCQNGHSFPLTFGAGTKLEL KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGE SEQ ID NO: 36: IGKV3D-11 EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGPGTDFTLTISSLEPEDFAVYYCQQRSNWH SEQ ID NO: 37: IGKV6-21 EIVLTQSPDFQSVTPKEKVTITCRASQSIGSSLHWYQQKPDQSPKLLIKYASQSFSGVPSRFSGSGSGTDFTLTINSLEAEDAATYYCHQSSSLP SEQ ID NO: 38: IGHV1-8 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYDINWVRQATGQGLEWMGWMNPNSGNTGYAQKFQGRVTMTRNTSISTAYMELSSLRSEDTAVYYCAR SEQ ID NO: 39: IGHV1-46 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYMHWVRQAPGQGLEWMGIINPSGGSTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCAR SEQ ID NO: 40: LFR1 VL L2H5 EIVMTQSPDFQSVTPKEKVTITC SEQ ID NO: 41: LFR2 VL L2H5 WYQQKPDQSPKLLIK SEQ ID NO: 42: LFR3 VL L2H5 GVPSRFSGSGSGSSDFTLTINSLEAEDAATYYC SEQ ID NO: 43: LFR4 VL L2H5 FGQGTKLEIK SEQ ID NO: 44: HFR1 VH L2H5 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SEQ ID NO: 45: HFR2 VH L2H5 WVRQATGQGLEWMG SEQ ID NO: 46: HFR3 VH L2H5 RVTMTTNTSISTAYMELSSLRSEDTAVYYCAR SEQ ID NO: 47: HFR4 VH L2H5 WGQGTLVTVSS SEQ ID NO: 48: LFR1 VL L2H3 EIVMTQSPDFQSVTPKEKVTITC SEQ ID NO: 49: LFR2 VL L2H3 WYQQKPDQSPKLLIK SEQ ID NO: 50: LFR3 VL L2H3 GVPSRFSGSGSGSSDFTLTINSLEAEDAATYYC SEQ ID NO: 51: LFR4 VL L2H3 FGQGTKLEIK SEQ ID NO: 52: HFR1 VH L2H3 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SEQ ID NO: 53: HFR2 VH L2H3 WVRQATGQGLEWMG SEQ ID NO: 54: HFR3 VH L2H3 RVTLTRNTSISTAYMELSSLRSEDTAVYFCAR SEQ ID NO: 55: HFR4 VH L2H3 WGQGTLVTVSS SEQ ID NO: 56: LFR1 VL L2H4 EIVMTQSPDFQSVTPKEKVTITC SEQ ID NO: 57: LFR2 VL L2H4 WYQQKPDQSPKLLIK SEQ ID NO: 58: LFR3 VL L2H4 GVPSRFSGSGSGSSDFTLTINSLEAEDAATYYC SEQ ID NO: 59: LFR4 VL L2H4 FGQGTKLEIK SEQ ID NO: 60: HFR1 VH L2H4 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SEQ ID NO: 61: HFR2 VH L2H4 WVRQATGQGLEWIG SEQ ID NO: 62: HFR3 VH L2H4 RVTMTTNTSISTAYMELSSLRSEDTAVYFCAR SEQ ID NO: 63: HFR4 VH L2H4 WGQGTLVTVSS SEQ ID NO: 64: IMGT CDR mouse 8H9 heavy chain CDR1 GYTFTNYD SEQ ID NO: 65: IMGT CDR mouse 8H9 heavy chain CDR2 IFPGDGST SEQ ID NO: 66: IMGT CDR mouse 8H9 heavy chain CDR3 ARQTTATWFAY SEQ ID NO: 67: IMGT CDR mouse 8H9 light chain CDR1 QSISDY SEQ ID NO: 68: IMGT CDR mouse 8H9 light chain CDR2 YA SEQ ID NO: 69: IMGT CDR mouse 8H9 light chain CDR3 QNGHSFPLT SEQ ID NO: 70: IMGT CDR H1 heavy chain CDR1 GYTFTNYD SEQ ID NO: 71: IMGT CDR H1 heavy chain CDR2 IFPGDGST SEQ ID NO: 72: IMGT CDR H1 heavy chain CDR3 ARQTTATWFAY SEQ ID NO: 73: IMGT CDR L1 light chain CDR1 QSISDY SEQ ID NO: 74: IMGT CDR L1 light chain CDR2 YA SEQ ID NO: 75: IMGT CDR L1 light chain CDR3 QNGHSFPLT SEQ ID NO: 76: IMGT CDR H2 heavy chain CDR1 GYTFTNYD SEQ ID NO: 77: IMGT CDR H2 heavy chain CDR2 IFPGDGST SEQ ID NO: 78: IMGT CDR H2 heavy chain CDR3 ARQTTATWFAY SEQ ID NO: 79: IMGT CDR L2 light chain CDR1 QSISDY SEQ ID NO: 80: IMGT CDR L2 light chain CDR2 YA SEQ ID NO: 81: IMGT CDR L2 light chain CDR3 QNGHSFPLT SEQ ID NO: 82: IMGT CDR H3 heavy chain CDR1 GYTFTNYD SEQ ID NO: 83: IMGT CDR H3 heavy chain CDR2 IFPGDGST SEQ ID NO: 84: IMGT CDR H3 heavy chain CDR3 ARQTTATWFAY SEQ ID NO: 85: IMGT CDR L3 light chain CDR1 QSISDY SEQ ID NO: 86: IMGT CDR L3 light chain CDR2 YA SEQ ID NO: 87: IMGT CDR L3 light chain CDR3 QNGHSFPLT SEQ ID NO: 88: IMGT CDR H4 heavy chain CDR1 GYTFTNYD SEQ ID NO: 89: IMGT CDR H4 heavy chain CDR2 IFPGDGST SEQ ID NO: 90: IMGT CDR H4 heavy chain CDR3 ARQTTATWFAY SEQ ID NO: 91: IMGT CDR L4 light chain CDR1 QSISDY SEQ ID NO: 92: IMGT CDR L4 light chain CDR2 YA SEQ ID NO: 93: IMGT CDR L4 light chain CDR3 QNGHSFPLT SEQ ID NO: 94: IMGT CDR H5 heavy chain CDR1 GYTFTNYD SEQ ID NO: 95: IMGT CDR H5 heavy chain CDR2 IFPGDGST SEQ ID NO: 96: IMGT CDR H5 heavy chain CDR3 ARQTTATWFAY SEQ ID NO: 97: IMGT CDR H6 heavy chain CDR1 GYTFTNYD SEQ ID NO: 98: IMGT CDR H6 heavy chain CDR2 IFPGDGST SEQ ID NO: 99: IMGT CDR H6 heavy chain CDR3 ARQTTATWFAY SEQ ID NO: 100: m8H9 LFR1 DIVMTQSPATLSVTPGDRVSLSC SEQ ID NO: 101: m8H9 LFR2 WYQQKSHESPRLLIK SEQ ID NO: 102: m8H9 LFR3 GIPSRFSGSGSGSDFTLSINSVEPEDVGVYYC SEQ ID NO: 103: m8H9 LFR4 FGAGTKLELK SEQ ID NO: 104: m8H9 HFR1 QVQLQQSGAELVKPGASVKLSCKASGYTFT SEQ ID NO: 105: m8H9 HFR2 WVRQRPEQGLEWIG SEQ ID NO: 106: m8H9 HFR3 KATLTTDTSSSTAYMQLSRLTSEDSAVYFCAR SEQ ID NO: 107: m8H9 HFR4 WGQGTLVTVSA SEQ ID NO: 108: L1 LFR1 EIVLTQSPDFQSVTPKEKVTLTC SEQ ID NO: 109: L1 LFR2 WYQQKPDQSPKLLIK SEQ ID NO: 110: L1 LFR3 GVPSRFSGSGSGSSDFTLTINSLEAEDAATYYC SEQ ID NO: 111: L1 LFR4 FGQGTKLEIK SEQ ID NO: 112: L3 LFR1 EIVMTQSPDFQSVTPKEKVTLTC SEQ ID NO: 113: L3 LFR2 WYQQKPDQSPKLLIK SEQ ID NO: 114: L3 LFR3 GVPSRFSGSGSGSSDFTLTINSLEAEDAGVYYC SEQ ID NO: 115: L3 LFR4 FGQGTKLEIK SEQ ID NO: 116: L4 LFR1 EIVMTQSPDFQSVTPKEKVTLTC SEQ ID NO: 117: L4 LFR2 WYQQKPDQSPKLLIK SEQ ID NO: 118: L4 LFR3 GIPSRFSGSGSGTDFTLTINSVEAEDAGVYYC SEQ ID NO: 119: L4 LFR4 FGQGTKLEIK SEQ ID NO: 120: H1 HFR1 QVQLVQSGAEVKKPGASVKVSCKASGYTFT SEQ ID NO: 121: H1 HFR2 WVRQATGQGLEWIG SEQ ID NO: 122: H1 HFR3 RVTLTTNTSISTAYMELSSLRSEDTAVYYCAR SEQ ID NO: 123: H1 HFR4 WGQGTLVTVSS SEQ ID NO: 124: H2 HFR1 QVQLQQSGAEVKKPGASVKVSCKASGYTFT SEQ ID NO: 125: H2 HFR2 WVRQATGQGLEWIG SEQ ID NO: 126: H2 HFR3 RATLTRNTSISTAYMELSSLRSEDTAVYYCAR SEQ ID NO: 127: H2 HFR4 WGQGTLVTVSS SEQ ID NO: 128: H6 HFR1 QVQLQQSGAEVKKPGASVKLSCKASGYTFT SEQ ID NO: 129: H6 HFR2 WVRQATGQGLEWIG SEQ ID NO: 130: H6 HFR3 RATLTTNTSISTAYMELSSLRSEDTAVYFCAR SEQ ID NO: 131: H6 HFR4 WGQGTLVTVSS SEQ ID NO: 132: 3BH-3 without His tag QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQCLEWMGWIFPGDGSTQYNEKFQGRVTMTTNTSISTAYMELSSLRSEDTAVYYCARQTTATWFAYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGSGGGG SEIVMTQSPDFQSVTPKEKVTITCRASQSISDYLHWYQQKPDQSPKLLIKYASQSISGVPSRFSGSGSGSDFTLTINSLEAEDAATYYCQNGHSFPLTFGCGTKLEIKGGGGSGGGGSGGGGSGGGGSHVQLVESGGGLVQPGGSLRL SCAASGFSLTDYGVHWVRQAPGKGLEWLGVIWSGGGTAYNTALISRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARRGSYPYNYFDAWGCGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSQAVVTQEPSLTVSPGGTVT LTCGSSTGAVTASNYANWVQQKPGQCPRGLIGGHNNRPPGVPARFSGSLLGGKAALTLLGAQPEDEAEYYCALWYSDHWVIGGGTKLTVLGTPLGDTTHTSGKPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEPGGSGGAP SEQ ID NO: 133: LFR4 VL L2H5 substitute FGCGTKLEIK SEQ ID NO: 134: HFR2 VH L2H5 substitute WVRQATGQCLEWMG SEQ ID NO: 135: YB8 H5 substitute QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQCLEWMGWIFPGDGSTQYNEKFQGRVTMTTNTSISTAYMELSSLRSEDTAVYYCARQTTATWFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 136: YB8 H5 N297A and K322A substitution QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQCLEWMGWIFPGDGSTQYNEKFQGRVTMTTNTSISTAYMELSSLRSEDTAVYYCARQTTATWFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 137: YB8_H5 L234A, L235A and K322A substitutions QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYDINWVRQATGQCLEWMGWIFPGDGSTQYNEKFQGRVTMTTNTSISTAYMELSSLRSEDTAVYYCARQTTATWFAYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKT HTCPCPPAPEAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO: 138: YB8 L2 substitute EIVMTQSPDFQSVTPKEKVTITCRASQSISDYLHWYQQKPDQSPKLLIKYASQSISGVPSRFSGSGSGSDFTLTINSLEAEDAATYYCQNGHSFPLTFGCGTKLEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO: 139: Tetramer domain, SADA polypeptide KPLDGEYFTLQIRGRERFEMFRELNEALELKDAQAGKEP

[0178] References: 1. Chapoval, A.I., et al., B7-H3: A costimulatory molecule for T cell activation and IFN-γ production. Nature Immunology, 2001. 2×3): p. 269-274. 2. Steinberger, P., et al., Molecular Characterization of Human 4Ig-B7-H3, a Member of the B7 Family with Four Ig-Like Domains. The Journal of Immunology, 2004. 172×4): p. 2352-2359. 3. Loos, M., et al., B7-H3 and Its Role in Antitumor Immunity. Clinical and Developmental Immunology, 2010. 2010: p. 1-7. 4. Modak, S., et al., Monoclonal antibody 8H9 targets a novel cell surface antigen expressed by a wide spectrum of human solid tumors. Cancer Res, 2001. 61×10): p. 4048-54. 5. Roth, T.J., et al., B7-H3 ligand expression by prostate cancer: a novel marker of prognosis and potential target for therapy. Cancer Res, 2007. 67×16): p. 7893-900. 6. Zang, X., et al., B7-H3 and B7x are highly expressed in human prostate cancer and associated with disease spread and poor outcome. Proc Natl Acad Sci U S A, 2007. 104×49): p. 19458-63. 7. Crispen, P.L., et al., Tumor cell and tumor vasculature expression of B7-H3 predict survival in clear cell renal cell carcinoma. Clin Cancer Res, 2008. 14×16): p. 5150-7. 8. Zang, X., et al., Tumor associated endothelial expression of B7-H3 predicts survival in ovarian carcinomas. Modern Pathology, 2010. 23×8): p. 1104-1112. 9. Lemke, D., et al., Costimulatory Protein 4IgB7H3 Drives the Malignant Phenotype of Glioblastoma by Mediating Immune Escape and Invasiveness. Clinical Cancer Research, 2012. 18×1): p. 105-117. 10. Wang, L., et al., B7-H3 is overexpressed in patients suffering osteosarcoma and associated with tumor aggressiveness and metastasis. PLoS One, 2013. 8×8): p. e70689. 11. Gregorio, A., et al., Small round blue cell tumours: diagnostic and prognostic usefulness of the expression of B7-H3 surface molecule. Histopathology, 2008. 53×1): p. 73-80. 12. Du, H., et al., Antitumor Responses in the Absence of Toxicity in Solid Tumors by Targeting B7-H3 via Chimeric Antigen Receptor T Cells. Cancer Cell, 2019. 35×2): p. 221-237 e8. 13. Majzner, R.G., et al., CAR T Cells Targeting B7-H3, a Pan-Cancer Antigen, Demonstrate Potent Preclinical Activity Against Pediatric Solid Tumors and Brain Tumors. Clin Cancer Res, 2019. 25×8): p. 2560-2574. 14. Castellanos, J.R., et al., B7-H3 role in the immune landscape of cancer. American Journal of Clinical and Experimental Immunology, 2017. 6×4): p. 66-75. 15. Kramer, K., et al., Compartmental intrathecal radioimmunotherapy: results for treatment for metastatic CNS neuroblastoma. J Neurooncol, 2010. 97×3): p. 409-18. 16. Van Cutsem, E., et al., Gastric cancer. Lancet, 2016. 388×10060): p. 2654-2664. 17. Ahmed, M., et al., Humanized Affinity-matured Monoclonal Antibody 8H9 Has Potent Antitumor Activity and Binds to FG Loop of Tumor Antigen B7-H3. Journal of Biological Chemistry, 2015. 290×50): p. 30018-30029. 18. Siegel, RL. et al. Cancer statistics, 2020. CA: A Cancer Journal for Clinicians, Vol 70, Issue 1, Jan / Feb 2020, p. 7-30 19. American Cancer Society website, survival rates https: / / www.cancer.org / cancer / ovarian-cancer / detection-diagnosis-staging / survival-rates.html Accessed May 26, 2020

Claims

1. A humanized antibody or antigen-binding fragment thereof capable of binding to a B7H3 antigen, comprising heavy chain CDR regions of SEQ ID NOs: 64-66 and light chain CDR regions of SEQ ID NOs: 67-69, and light chain FR regions of SEQ ID NOs: 40-43 and heavy chain FR regions of SEQ ID NOs: 44-47.

2. The antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody or antigen-binding fragment thereof is a bispecific and / or trispecific binding antibody.

3. A polypeptide conjugate comprising a self-assembly disassembly (SADA) polypeptide having the sequence of SEQ ID NO: 139 bound to the antibody or antigen-binding fragment of claim 1 or 2.

4. The polypeptide conjugate of claim 3 , wherein the antibody or antigen-binding fragment thereof is a bispecific and / or trispecific binding antibody.

5. 5. The polypeptide conjugate of claim 4, wherein the bispecific and / or trispecific binding antibody comprises a first antibody or antigen-binding fragment thereof of claim 1 and a second antibody or antigen-binding fragment thereof capable of binding to DOTA.

6. 6. The polypeptide conjugate of claim 5, comprising a sequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO:

132.

7. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment of claim 1 or 2 or the polypeptide conjugate of any one of claims 3 to 6.

8. A recombinant vector comprising the isolated nucleic acid molecule of claim 7.

9. A host cell comprising the recombinant vector described in claim 8.

10. A pharmaceutical composition comprising the antibody or antigen-binding fragment of claim 1 or 2 or the polypeptide conjugate of any one of claims 3 to 6.

11. 11. The composition of claim 10 for use in the treatment, prevention, mitigation, and / or diagnosis of cancer.

12. 12. The composition of claim 11, wherein the cancer is prostate cancer, desmoplastic small cell tumor, ovarian cancer, gastric cancer, pancreatic cancer, liver cancer, renal cancer, breast cancer, non-small cell lung cancer, melanoma, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, Ewing's sarcoma, Wilms' sarcoma, neuroblastoma, ganglioneuroblastoma, medulloblastoma, high-grade glioma, pediatric brainstem glioma (diffuse intrinsic pontine glioma), embryonal tumor with multilayered rosettes, or a B7H3-expressing cancer.

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