Cancer immunotherapy

Abstract

An antibody-drug conjugate (ADC) comprising an anti-human SEMA4A humanized or human antibody, a linker, and a cytotoxin.

Description

[Background technology]

[0001] 2. Background of the Invention Cancer immunotherapy, the induction of the immune system to attack tumor cells, has a long history and has seen a recent resurgence of interest. This has been driven by both immune checkpoint blockade and the success of cancer-directed immunotherapy. The latter, which utilizes the targeting of cell surface proteins on cancer cells, is exemplified by monoclonal antibody therapies (e.g., rituximab, trastuzumab), introduced at the end of the 20th century, and chimeric antigen receptor-T (CAR-T) cells, introduced at the beginning of the 21st century. However, resistance to single-agent immunotherapy is frequent and is generally driven by antigen escape, i.e., downregulation of antigenic targets by cancer cells. Antigen escape can be driven by intratumoral heterogeneity, where subclones expressing low levels of or not expressing the target protein gain a competitive growth advantage during immunotherapy. To overcome this resistance, novel immunotherapies should be targeted against antigens essential for cancer cell survival, and combination therapies can be used. A strategy that combines multiple monoclonal antibodies is particularly attractive, in part because of their low toxicity, and in part because immunophenotyping of cancer cells can reveal sensitive and resistant subpopulations and lead to rational therapeutic decisions. Thus, there is an urgent need to identify novel target proteins on the surface of cancer cells.

[0002] A complete understanding of the surface proteome of cancer cells would be particularly beneficial to advance treatment. However, this knowledge has proven elusive with available technologies. The correlation between RNA and protein expression is modest, making transcriptomic approaches inefficient. Comprehensive identification of cell surface proteins by whole-cell proteomic approaches is limited by their low abundance, hydrophobicity, and lack of protease cleavage sites, as well as inaccurate quantification. To overcome these limitations, various techniques have been used to isolate and identify cancer cell surface proteins. However, these approaches have mainly focused on cancer cell lines rather than primary cells, fail to capture the entire cell surface protein, and / or are not quantitative. As a result, these types of proteomic studies have failed to yield relevant cancer immunotherapy targets.

[0003] WO2021195536 describes the analysis of expression of cell surface candidate targets in multiple myeloma (MM). Surface proteins of seven different MM cell lines were biotinylated and subjected to spectroscopic analysis, thereby identifying 4761 proteins, and an integrated database was used to generate cell surface molecular annotations, which, combined with exclusion based on expression levels, identified 326 surface proteins for further analysis by STRING. Heat maps revealed protein annotations of 94 selected targets in several normal tissues and organs of the whole body. Molecules with high expression in any normal tissue except hematopoietic tissues and molecules with annotations in less than two of the three proteomic databases were excluded. Venn diagram overlap of targets with potential biological and therapeutic relevance revealed 67 common targets, of which 24 were selected based on their expression profiles in primary patients and used for validation in patient samples, including CCR1, CD28, CD320, FCRL3, IFNGR1, IL12RB1, IL27RA, IL2RG, IL6R, ITGA4, KCNN4, LAX1, LILRB1, LILRB4, LRRC8A, LRRC8D, PLXNA3, PLXNC1, S1PR4, SELPLG, SEMA4A, SLAMF6, TLR1 and BCMA.

[0004] Anderson et al. (2022) describe plasma membrane profiling of primary human myeloma cells to identify cell surface proteins of primary cancers. Using a novel approach to prioritize immunotherapy targets, they identified semaphorin-4A (SEMA4A), a cell surface protein not previously implicated in myeloma. Expression of SEMA4A was shown to be essential for normal myeloma cell growth in vitro, indicating that myeloma cells are unable to downregulate the protein to avoid detection, and it was also shown that SEMA4A is not identified as a myeloma therapeutic target by standard CRISPR / Cas9 knockout screening due to exon skipping. Targeting of SEMA4A using mouse antibody-drug conjugates was demonstrated in vitro and in vivo. Summary of the Invention [Problem to be solved by the invention]

[0005] MM is a cancer of plasma cells that causes fatigue, bone pain, pathological fractures, immunosuppression, and renal failure. Despite the recent introduction of novel therapies, including monoclonal antibody therapy and CAR-T cell therapy, resistance to treatment is inevitable. Thus, MM remains invariably fatal, and novel therapies are urgently needed. [Means for solving the problem]

[0006] STATEMENT OF THE INVENT The present invention provides the following: 1. An antibody-drug conjugate (ADC) comprising an anti-human-SEMA4A human or humanized antibody, or an antigen-binding fragment thereof, a linker, and a cytotoxin.

[0007] 2. The antibody comprises a V domain comprising HCDR1, HCDR2 and HCDR3. H and V including LCDR1, LCDR2 and LCDR3 L and the CDRs are (a) 5E3 (SEQ ID NOs: 3, 4, 5, 12, 13, 14); (b) 5Hg1-5Lg1 (SEQ ID NOs: 21, 22, 23, 30, 31, 32); (c) 5Hg1-5Lg2 (SEQ ID NOs: 39, 40, 41, 48, 49, 50); (d) 5Hg2-5Lg1 (SEQ ID NOs: 57, 58, 59, 66, 67, 68); (e) 5Hg2-5Lg2 (SEQ ID NOs: 75, 76, 77, 84, 85, 86); (f) 5Hg2-5Lg3 (SEQ ID NOs: 93, 94, 95, 102, 103, 104); (g) 5Hg2-5Lg5 (SEQ ID NOs: 111, 112, 113, 120, 121, 122); (h) 5Hg2-5Lg6 (SEQ ID NOs: 129, 130, 131, 138, 139, 140); (i) 5Hg4-5Lg1 (SEQ ID NOs: 147, 148, 149, 156, 157, 158); (j) 5Hg4-5Lg2 (SEQ ID NOs: 165, 166, 167, 174, 175, 176); (k) 5Hg4-5Lg4 (SEQ ID NOs: 183, 184, 185, 192, 193, 194); (l) 5Hg4-5Lg5 (SEQ ID NOs: 201, 202, 203, 210, 211, 212); (m) 5Hg5-5Lg2 (SEQ ID NOs: 219, 220, 221, 228, 229, 230); (n) C0120903 (SEQ ID NO: 237, 238, 239, 246, 247, 248); (o) C0120904 (SEQ ID NO: 255, 256, 257, 264, 265, 266); (p)C0120905 (SEQ ID NO: 273, 274, 275, 282, 283, 284); (q) C0120906 (SEQ ID NO: 291, 292, 293, 300, 301, 302); (r) C0120910 (SEQ ID NO: 309, 310, 311, 318, 319, 320); (s)C0120913 (SEQ ID NO: 327, 328, 329, 336, 337, 338); (t)C0120914 (SEQ ID NO: 345, 346, 347, 354, 355, 356); (u)C0120917 (SEQ ID NO: 363, 364, 365, 372, 373, 374); (v) C0120918 (sequence numbers: 381, 382, ​​383, 390, 391, 392); (w)C0120919 (SEQ ID NOs: 399, 400, 401, 408, 409, 410); (x) C0120920 (SEQ ID NO: 417, 418, 419, 426, 427, 428); (y) C0120921 (SEQ ID NO: 435, 436, 437, 444, 445, 446); (z) C0120922 (SEQ ID NO: 453, 453, 455, 462, 463, 464); (aa)C0120923 (SEQ ID NOs: 471, 472, 473, 480, 481, 482); (bb) C0120924 (SEQ ID NOs: 489, 490, 491, 498, 499, 500); (cc)C0120925 (SEQ ID NO: 507, 508, 509, 516, 517, 518); (dd) C0120926 (SEQ ID NOs: 525, 526, 527, 534, 535, 536); (ee) C0120927 (SEQ ID NOs: 543, 544, 545, 552, 553, 554); (ff)C0120928 (SEQ ID NO: 561, 562, 563, 570, 571, 572); (gg) C0120929 (sequence numbers: 579, 580, 581, 588, 589, 590) and (hh) C0120930 (SEQ ID NO: 597, 598, 599, 606, 607, 608) and CDRs are defined according to the Kabat nomenclature: An ADC as described in clause 1.

[0008] 3.Antibody, a.5Hg1-5Lg1 (SEQ ID NO:20 and 29); b.5Hg1-5Lg2 (SEQ ID NOs:38 and 47); c.5Hg2-5Lg1 (SEQ ID NOs:56 and 65); d.5Hg2-5Lg2 (SEQ ID NOs:74 and 83); e.5Hg2-5Lg3 (SEQ ID NOs:92 and 101); f.5Hg2-5Lg5 (SEQ ID NOs:110 and 119); g.5Hg2-5Lg6 (SEQ ID NOs:128 and 137); h.5Hg4-5Lg1 (SEQ ID NOs:146 and 155); i.5Hg4-5Lg2 (SEQ ID NOs: 164 and 173); j.5Hg4-5Lg4 (SEQ ID NOs:182 and 191); k.5Hg4-5Lg5 (SEQ ID NO:200 and 209); l.5Hg5-5Lg2 (SEQ ID NOs:218 and 227); m.C0120903 (SEQ ID NO:236 and 245); n.C0120904 (SEQ ID NO:254 and 263); o.C0120905 (SEQ ID NO:272 and 281); p.C0120906 (SEQ ID NO: 290 and 299); q.C0120910 (sequence numbers: 308 and 317); r.C0120913 (SEQ ID NOs:326 and 335); s.C0120914 (SEQ ID NOs: 344 and 353); t.C0120917 (SEQ ID NO:362 and 371); u.C0120918 (SEQ ID NO:380 and 389); v.C0120919 (SEQ ID NO: 398 and 407); w.C0120920 (SEQ ID NOs: 416 and 425); x.C0120921 (SEQ ID NO: 434 and 443); y.C0120922 (SEQ ID NO: 452 and 461); z.C0120923 (SEQ ID NO: 470 and 479); aa.C0120924 (SEQ ID NOs: 488 and 497); bb.C0120925 (SEQ ID NOs: 506 and 515); cc.C0120926 (SEQ ID NO:524 and 533); dd.C0120927 (SEQ ID NO:542 and 551); ee.C0120928 (SEQ ID NOs:560 and 569); ff.C0120929 (SEQ ID NOs: 578 and 587); and gg.C0120930 (SEQ ID NOs: 596 and 605); V H and V L Selected from V H and V L wherein the sequence is defined by the Kabat nomenclature.

[0009] 4. The ADC of any one of clauses 1-3, wherein the cytotoxin is a microtubule inhibitor (e.g., a maytansinoid or an auristatin).

[0010] 5. The ADC of any one of clauses 1-4, wherein the cytotoxin is selected from monomethylauristatin F (MMAF), monomethylauristatin E (MMAE, vedotin) and mertansine (DM1, e.g., as emtansine with an SMCC linker).

[0011] 6. The ADC of any one of clauses 1-5, wherein the linker is a non-cleavable linker or a cleavable linker.

[0012] 7. The ADC of any one of clauses 1-6, wherein the cytotoxin is monomethylauristatin E (MMAE) and the linker is mc-vcPAB linker (maleimide-based linker, cysteine ​​linked), or the cytotoxin is mertansine (DM1) and the linker is SMCC linker (NHS ester-based, lysine).

[0013] 8. The ADC of any one of clauses 1-7, wherein the ADC bound to SEMA4A can be internalized into a cell expressing human SEMA4A on its surface.

[0014] 9. The ADC of any one of clauses 1-8, wherein the ADC bound to SEMA4A is capable of internalization into a cell expressing human SEMA4A on its surface, and wherein internalization of the ADC into the cell results in cell death.

[0015] 10. V containing HCDR1, HCDR2 and HCDR3 H and V including LCDR1, LCDR2 and LCDR3 L A human or humanized anti-human SEMA4A antibody comprising the CDRs: (a) 5E3 (SEQ ID NOs: 3, 4, 5, 12, 13, 14); (b) 5Hg1-5Lg1 (SEQ ID NOs: 21, 22, 23, 30, 31, 32); (c) 5Hg1-5Lg2 (SEQ ID NOs: 39, 40, 41, 48, 49, 50); (d) 5Hg2-5Lg1 (SEQ ID NOs: 57, 58, 59, 66, 67, 68); (e) 5Hg2-5Lg2 (SEQ ID NOs: 75, 76, 77, 84, 85, 86); (f) 5Hg2-5Lg3 (SEQ ID NOs: 93, 94, 95, 102, 103, 104); (g) 5Hg2-5Lg5 (SEQ ID NOs: 111, 112, 113, 120, 121, 122); (h) 5Hg2-5Lg6 (SEQ ID NOs: 129, 130, 131, 138, 139, 140); (i) 5Hg4-5Lg1 (SEQ ID NOs: 147, 148, 149, 156, 157, 158); (j) 5Hg4-5Lg2 (SEQ ID NOs: 165, 166, 167, 174, 175, 176); (k) 5Hg4-5Lg4 (SEQ ID NOs: 183, 184, 185, 192, 193, 194); (l) 5Hg4-5Lg5 (SEQ ID NOs: 201, 202, 203, 210, 211, 212); (m) 5Hg5-5Lg2 (SEQ ID NOs: 219, 220, 221, 228, 229, 230); (n) C0120903 (SEQ ID NO: 237, 238, 239, 246, 247, 248); (o) C0120904 (SEQ ID NO: 255, 256, 257, 264, 265, 266); (p)C0120905 (SEQ ID NO: 273, 274, 275, 282, 283, 284); (q) C0120906 (SEQ ID NO: 291, 292, 293, 300, 301, 302); (r) C0120910 (SEQ ID NO: 309, 310, 311, 318, 319, 320); (s)C0120913 (SEQ ID NO: 327, 328, 329, 336, 337, 338); (t)C0120914 (SEQ ID NO: 345, 346, 347, 354, 355, 356); (u)C0120917 (SEQ ID NO: 363, 364, 365, 372, 373, 374); (v) C0120918 (sequence numbers: 381, 382, ​​383, 390, 391, 392); (w)C0120919 (SEQ ID NOs: 399, 400, 401, 408, 409, 410); (x) C0120920 (SEQ ID NO: 417, 418, 419, 426, 427, 428); (y) C0120921 (SEQ ID NO: 435, 436, 437, 444, 445, 446); (z) C0120922 (SEQ ID NO: 453, 454, 455, 462, 463, 464); (aa)C0120923 (SEQ ID NOs: 471, 472, 473, 480, 481, 482); (bb) C0120924 (SEQ ID NOs: 489, 490, 491, 498, 499, 500); (cc)C0120925 (SEQ ID NO: 507, 508, 509, 516, 517, 518); (dd) C0120926 (SEQ ID NOs: 525, 526, 527, 534, 535, 536); (ee) C0120927 (SEQ ID NOs: 543, 544, 545, 552, 553, 554); (ff)C0120928 (SEQ ID NO: 561, 562, 563, 570, 571, 572); (gg) C0120929 (sequence numbers: 579, 580, 581, 588, 589, 590) and (hh) C0120930 (SEQ ID NO: 597, 598, 599, 606, 607, 608) and wherein the CDR sequences are defined by the Kabat nomenclature.

[0016] 11.Antibody, (a) 5Hg1-5Lg1 (SEQ ID NOs: 20 and 29); (b) 5Hg1-5Lg2 (SEQ ID NOs:38 and 47); (c) 5Hg2-5Lg1 (SEQ ID NOs:56 and 65); (d) 5Hg2-5Lg2 (SEQ ID NOs:74 and 83); (e) 5Hg2-5Lg3 (SEQ ID NOs:92 and 101); (f) 5Hg2-5Lg5 (SEQ ID NOs:110 and 119); (g) 5Hg2-5Lg6 (SEQ ID NOs:128 and 137); (h) 5Hg4-5Lg1 (SEQ ID NOs:146 and 155); (i) 5Hg4-5Lg2 (SEQ ID NOs: 164 and 173); (j) 5Hg4-5Lg4 (SEQ ID NOs: 182 and 191); (k) 5Hg4-5Lg5 (SEQ ID NOs: 200 and 209); (l) 5Hg5-5Lg2 (SEQ ID NOs: 218 and 227); (m) C0120903 (SEQ ID NO: 236 and 245); (n) C0120904 (sequence numbers: 254 and 263); (o) C0120905 (SEQ ID NO: 272 and 281); (p)C0120906 (SEQ ID NO: 290 and 299); (q) C0120910 (sequence numbers: 308 and 317); (r) C0120913 (SEQ ID NO: 326 and 335); (s) C0120914 (sequence numbers: 344 and 353); (t) C0120917 (SEQ ID NO: 362 and 371); (u) C0120918 (SEQ ID NO: 380 and 389); (v) C0120919 (sequence numbers: 398 and 407); (w) C0120920 (SEQ ID NO: 416 and 425); (x) C0120921 (sequence numbers: 434 and 443); (y) C0120922 (SEQ ID NO: 452 and 461); (z) C0120923 (sequence numbers: 470 and 479); (aa) C0120924 (SEQ ID NOs: 488 and 497); (bb) C0120925 (SEQ ID NOs: 506 and 515); (cc) C0120926 (SEQ ID NOs: 524 and 533); (dd) C0120927 (SEQ ID NOs: 542 and 551); (ee) C0120928 (SEQ ID NOs: 560 and 569); (ff) C0120929 (SEQ ID NO: 578 and 587) and (gg) C0120930 (SEQ ID NO: 596 and 605) V H and V L Selected from V H and V L Including V H Sequence and V L The antibody of clause 10, wherein the sequence is defined by the Kabat nomenclature.

[0017] 12. A chimeric antigen receptor (CAR) comprising the antigen-binding domain of the monoclonal antibody of any one of clauses 10 or 11 linked to a T cell activation moiety.

[0018] 13. The CAR of clause 12, wherein the antigen-binding domain comprises a single-chain Fv (scFv) fragment of the monoclonal antibody of any one of clauses 10 or 11.

[0019] 14. A composition comprising an ADC according to any one of clauses 1 to 9, or an anti-human SEMA4A antibody according to any one of clauses 10 or 11, or a CAR according to any one of clauses 12 and 13, and a diluent.

[0020] 15. a. For use as a medicine; b. For use as a medicine to treat cancer; c. For use in the treatment of blood cancers. d. For use in the treatment of a hematological cancer selected from multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL); e. For use in the manufacture of a medicament for the therapeutic treatment of cancer; f. For use in the manufacture of a medicament for the therapeutic treatment of hematological cancers. g. For use in the manufacture of a medicament for the therapeutic treatment of a hematological cancer selected from MM, NHL, AML, DLBCL and FL, or h. To induce cell death in cells expressing SEMA4A on the cell surface. The ADC of any one of clauses 1 to 9, the antibody of any one of clauses 10 or 11, the CAR of any one of clauses 12 or 13, or the composition of clause 14.

[0021] 16. A method for treating cancer, such as a hematological cancer, e.g. a hematological cancer selected from MM, NHL, AML, DLBCL and FL, comprising administering to a subject the ADC of any one of clauses 1 to 9, the antibody of any one of clauses 10 or 11, the CAR of any one of clauses 12 or 13, or the composition of clause 14.

[0022] 17. A method for producing an ADC according to any one of clauses 1 to 9, comprising conjugating an antibody according to any one of clauses 10 or 11 to a cytotoxin via a linker.

[0023] 18. An isolated recombinant DNA or RNA sequence comprising a sequence encoding the isolated antibody or antigen-binding fragment thereof according to any one of clauses 10 or 11.

[0024] 19. The isolated recombinant DNA sequence according to clause 18, which is a vector.

[0025] 20. An isolated recombinant DNA sequence according to clause 18 or clause 19 which is an expression vector.

[0026] 21. An isolated recombinant DNA sequence according to any one of clauses 18 to 20, encoding an antibody or antigen-binding fragment thereof according to any one of clauses 10 or 11 under the control of a promoter.

[0027] 22. A host cell comprising a DNA or RNA sequence according to any one of clauses 18 to 21.

[0028] 23. A host cell according to clause 22, capable of expressing the isolated antibody or antigen-binding fragment thereof according to any one of clauses 10 or 11.

[0029] 24. A method for producing an isolated antibody or antigen-binding fragment thereof according to clause 10 or 11, comprising culturing a host cell according to any one of clauses 22 or 23 under conditions suitable for expression of the isolated antibody or antigen-binding fragment thereof.

[0030] The present invention provides an antibody-drug conjugate (ADC) comprising a human or humanized antibody against human semaphorin 4A (SEMA4A) or an antigen-binding fragment thereof conjugated to a cytotoxin. As used herein, the term "antibody-drug conjugate" refers to a compound comprising an antibody, such as a humanized or human monoclonal antibody (mAb) or an antigen-binding fragment thereof, linked to a cytotoxic drug (generally a small molecule drug with high systemic toxicity) via a chemical linker. In some embodiments, the ADC may comprise a small molecule cytotoxin chemically modified to contain a linker. The linker is then used to conjugate the cytotoxin to the antibody or its antigen-binding fragment. In some embodiments, upon binding to a target antigen on the surface of a cell, the cytotoxin can be released by cleavage or proteolysis of the linker, and the cytotoxin can then bind to its target and induce cell death.

[0031] The ADCs described herein may comprise whole antibodies or antibody fragments. Whole antibodies typically consist of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide. Each heavy chain contains one N-terminal variable (V H ) domain and three C-terminal constant (CH1, CH2 and CH3) domains, and each light chain contains one N-terminal variable (V L The variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody. H and V L The regions have the same general structure, each containing four framework regions, the sequences of which are relatively conserved. The framework regions are linked by three complementarity determining regions (CDRs). The three CDRs, known as CDR1, CDR2 and CDR3, form the "hypervariable region" of each variable domain that is responsible for antigen binding.

[0032] The ADC may comprise an antigen-binding fragment of a humanized or human antibody. The terms "antibody fragment," "antigen-binding fragment," "functional fragment of an antibody," and "antigen-binding portion" are used interchangeably herein and refer to one or more fragments or portions of an antibody that retain the ability to specifically bind to an antigen. An antibody fragment may comprise, for example, one or more CDRs, a variable region (or a portion thereof), or a combination thereof, optionally further combined with a constant region (or a portion thereof). Examples of antibody fragments include, but are not limited to, (i) a V L , V H (ii) a Fab fragment, which is a monovalent fragment consisting of the V, CL, and CH1 domains; (iii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; (iv) a V(ab')2 fragment, which is a bivalent fragment containing the V, CL, and CH2 domains of an antibody. L Domain and V H (iv) an Fv fragment consisting of two domains (i.e., the V domain) of an Fv fragment linked by a synthetic linker that allows the two domains to be synthesized as a single polypeptide chain; L and V H (v) single-chain Fv (scFv), which is a monovalent molecule consisting of a V polypeptide chain, and (v) diabodies, which are dimers of polypeptide chains, each of which is a V polypeptide chain. H and V L V due to a peptide linker that is too short to allow pairing between L V connected to H , which allows for different V H -V L Antibody fragments drive pairing between complementary domains on the polypeptide chains to produce a dimeric molecule having two functional antigen-binding sites. Antibody fragments are known in the art and are described in further detail, for example, in U.S. Patent Application Publication No. 2009 / 0093024A1.

[0033] The ADCs of the invention comprising a humanized antibody of the invention or comprising a human antibody of the invention can specifically bind to recombinant human SEMA4A (UniProt ID Q9H3S1) and may also bind to recombinant cynomolgus monkey SEMA4A (UniProt ID G7NV79) and / or recombinant mouse SEMA4A (UniProt ID Q62178).

[0034] In one embodiment, the ADC comprises the variable region of a humanized or fully human anti-human SEMA4A antibody. In this regard, the ADC may comprise the light chain variable region, the heavy chain variable region, or both the light chain variable region and the heavy chain variable region of an anti-human SEMA4A monoclonal antibody. Preferably, the ADC comprises the light chain variable region and the heavy chain variable region of an anti-human SEMA4A antibody. The terms "cytotoxin" and "cytotoxic agent" refer to any molecule that inhibits or prevents the function of a cell and / or causes the destruction of a cell (cell death) and / or exerts an anti-proliferative effect. It will be understood that the cytotoxin or cytotoxic agent of an ADC is also referred to in the art as the "payload" or "warhead" of the ADC. Numerous classes of cytotoxic agents are known in the art to have potential utility in ADC molecules and can be used in the ADCs described herein. Exemplary classes of cytotoxic agents include anti-microtubule agents such as tubulysins, maytansinoids, auristatins, or derivatives thereof. More specifically, the cytotoxic agent may be, for example, MMAF, MMAE or DM1, DM4.

[0035] In one embodiment, the cytotoxic agent may be an anti-microtubule agent. The terms "anti-microtubule agent" and "microtubule targeting agent" are synonymous and refer to agents that inhibit cell division by disrupting microtubules. Tubulysins are members of a class of natural products isolated from myxobacterial species (Sasse et al., 2000) that inhibit tubulin polymerization and act as mitotic poisons leading to cell cycle arrest and apoptosis (Steinmetz et al., 2004; Khalil et al., 2006; Kaur et al., 2006). Examples of tubulysins are disclosed, for example, in WO 2015 / 157594, WO 2004 / 005326, WO 2012 / 019123, WO 2009 / 134279, WO 2009 / 055562, WO 2004 / 005327; U.S. Pat. Nos. 7,776,841, 7,754,885, and 7,816,377; and U.S. Patent Application Publication Nos. 2010 / 0240701, 2011 / 0021568, and 2011 / 0263650.

[0036] Maytansinoids inhibit the polymerization of the microtubule protein tubulin, thereby preventing the formation of microtubules (see, e.g., U.S. Pat. No. 6,441,163 and Remillard et al., 1975). Maytansinoids have been shown to inhibit tumor cell growth in vitro using cell culture models and in vivo using experimental animal systems. Furthermore, the cytotoxicity of maytansinoids is 1,000 times greater than that of conventional chemotherapeutic agents such as, for example, methotrexate, daunorubicin, and vincristine (see, e.g., U.S. Pat. No. 5,208,020). Maytansinoids include maytansine, maytansinol, C-3 esters of maytansinol, and other maytansinol analogs and derivatives (see, e.g., U.S. Pat. Nos. 5,208,020 and 6,441,163). The C-3 esters of maytansinol can be naturally occurring or synthetically derived. Furthermore, both naturally occurring and synthetic C-3 maytansinol esters can be classified as C-3 esters with simple carboxylic acids or C-3 esters with derivatives of N-methyl-L-alanine, the latter being more cytotoxic than the former. Synthetic maytansinoid analogs are also known in the art and are described, for example, in Kupchan et al., 1978. Methods for producing maytansinol and its analogs and derivatives are described, for example, in U.S. Pat. No. 4,151,042. Examples of maytansinoids that may be used in conjunction with the ADCs described herein include, but are not limited to, N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1) and N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).

[0037] Auristatins represent a class of highly potent antimitotic agents that have demonstrated substantial preclinical activity at well-tolerated doses (Law et al., 2006; Ma et al., 2006; Tse et al., 2006; Oflazoglu et al., 2008, Oflazoglu et al., 2008). Auristatin ADCs are currently being evaluated in preclinical and clinical trials. Examples of auristatins that may be used in conjunction with the ADCs described herein include, but are not limited to, monomethyl auristatin E (MMAE) and the related molecule monomethyl auristatin F (MMAF) (see, e.g., Doronina et al., 2003; Doronina et al., 2006).

[0038] The SEMA4A humanized or human monoclonal antibody, or antigen-binding fragment thereof, may be conjugated to a cytotoxin using any suitable method known in the art, including site-specific or non-site-specific conjugation methods. Conventional conjugation strategies for antibodies typically rely on stochastic random (i.e., non-specific) conjugation of a payload to an antibody, or antigen-binding fragment thereof, via lysine or cysteine. Thus, in some embodiments, an antibody or antigen-binding fragment thereof is randomly conjugated to a cytotoxic drug, for example, by partial reduction of the antibody or antibody fragment, followed by reaction with a desired drug, with or without a linker moiety attached. For example, an antibody or antigen-binding fragment thereof may be reduced using dithiothreitol (DTT) or a similar reducing agent. A cytotoxic drug, with or without a linker moiety attached thereto, may then be added in molar excess to the reduced antibody or antibody fragment in the presence of dimethylsulfoxide (DMSO). After conjugation, an excess of free cysteine ​​may be added to quench unreacted drug. The reaction mixture can then be purified and buffer exchanged into phosphate buffered saline (PBS).

[0039] In other embodiments, cytotoxic drugs can be conjugated to SEMA4A monoclonal antibodies using site-specific conjugation methods at specific reactive amino acid residues, resulting in homogenous ADC preparations with uniform stoichiometry. Site-specific conjugation can be via cysteine ​​residues or unnatural amino acids. In one embodiment, cytotoxic drugs can be conjugated to the antibody or antigen-binding fragment thereof via at least one cysteine ​​residue. In particular, for example, cytotoxic drugs can be chemically conjugated to the side chain of an amino acid at a specific Kabat position (Kabat et al., 1991) in the Fc region of the SEMA4A monoclonal antibody. In this regard, cytotoxic drugs can be conjugated to SEMA4A monoclonal antibodies via a cysteine ​​residue at any suitable position in the Fc region of the antibody. Alternatively, cytotoxic drugs can be conjugated to SEMA4A monoclonal antibodies or antigen-binding fragments thereof via thiol-maleimide bonds, such as via sulfhydryl reactive groups in the hinge and heavy and light chains.

[0040] Although the SEMA4A humanized or human monoclonal ADCs described herein contain at least one cytotoxin molecule conjugated thereto, the SEMA4A humanized or human monoclonal antibody may contain any suitable number of cytotoxin molecules (e.g., 1, 2, 3, 4 or more cytotoxin molecules) conjugated thereto to achieve a desired therapeutic effect. Thus, the ADCs of the present invention may have a drug-antibody ratio (DAR) of, for example, 1, 2, 3, 4, 5, 6, 7, or 8. The DAR is the average drug (cytotoxin)-to-antibody ratio of a given preparation of the ADC. The DAR is a measure of the drug loading of the ADC.

[0041] In some embodiments, the present invention provides humanized or human monoclonal antibodies, or antigen-binding fragments thereof, directed against human SEMA4A as described above, independent of ADC.

[0042] The humanized and human antibodies of the invention, as well as ADCs or CARs comprising such antibodies, can specifically bind to recombinant human SEMA4A (UniProt ID Q9H3S1, SEQ ID NO: 613) and may also bind to recombinant cynomolgus monkey SEMA4A (UniProt ID G7NV79, SEQ ID NO: 614) and / or recombinant mouse SEMA4A (UniProt ID Q62178, SEQ ID NO: 615).

[0043] The humanized or human antibody, or antigen-binding fragment thereof, against human SEMA4A may comprise any suitable binding affinity for human SEMA4A or an epitope thereof. The term "affinity" refers to the equilibrium constant for the reversible binding of two agents and is expressed as the dissociation constant (K D ). The affinity of an antibody or antigen-binding fragment thereof for an antigen or epitope of interest can be measured using any method known in the art. Such methods include, for example, fluorescence activated cell sorting (FACS), surface plasmon resonance (e.g., Biacore, ProteOn), biolayer interferometry (BLI, e.g., Octet), kinetic exclusion assays (e.g., KinExA), separable beads (e.g., magnetic beads), antigen panning, and / or enzyme-linked immunosorbent assay (ELISA) (Janeway et al., 2001). It is known in the art that the binding affinity of a particular antibody varies depending on the method used to analyze the binding affinity.

[0044] The affinity of a binding agent for a ligand, e.g., the affinity of an antibody for an epitope, can be, for example, from about 1 nM to about 100 nM. In one embodiment, the monoclonal antibody or antigen-binding fragment thereof has a K of 500, 400, 300, 200, or 100 nanomolar (e.g., 100 nM, about 90 nM, about 80 nM, about 70 nM, about 60 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, or about 10 nM, or a range defined by any two of the foregoing values) or less. DIn another embodiment, the monoclonal antibody may bind to human SEMA4A with a K of 10 nanomolar (e.g., about 9 nM, about 8.5 nM, about 8 nM, about 7.5 nM, about 7 nM, about 6.5 nM, about 6 nM, about 5.5 nM, about 5 nM, about 4.5 nM, about 4 nM, about 3.5 nM, about 3 nM, about 2.5 nM, about 2 nM, about 1.5 nM, about 1 nM, or a range defined by any two of the foregoing values). D can bind to human SEMA4A at

[0045] The antigen-binding portion or fragment of a humanized or human antibody of the invention can be of any size, so long as the portion binds to human SEMA4A.

[0046] The antibodies or antigen-binding fragments thereof of the invention may be produced by recombinant means. A "recombinant antibody" is an antibody produced by a recombinantly engineered host cell. The antibodies or antigen-binding fragments thereof according to the invention are optionally isolated or purified. The term "antibody" or "antibody molecule" refers to a natural or partially or fully synthetically produced immunoglobulin. The antigen-binding proteins of the invention may be antibodies, preferably monoclonal antibodies, which may be human or non-human, chimeric or humanized.

[0047] The antibody molecule is preferably a monoclonal antibody molecule. Examples of antibodies are immunoglobulin isotypes such as immunoglobulin G and their isotype subclasses such as IgG1, IgG2, IgG3 and IgG4, and fragments thereof. Although the four human subclasses (IgG1, IgG2, IgG3 and IgG4) each contain a different heavy chain, they are highly homologous and differ mainly in the hinge region and the extent to which they activate the host immune system. IgG1 and IgG4 contain two interchain disulfide bonds in the hinge region, IgG2 has four interchain disulfide bonds, and IgG3 has eleven interchain disulfide bonds.

[0048] The terms "antibody" and "antibody molecule" as used herein include antibody fragments, such as Fab and scFv fragments, where the fragments contain a CDR-based antigen-binding site for an epitope of human SEMA4A. Examples of antibody fragments include, but are not limited to, Fv, Fab, F(ab'), Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv) and domain antibodies (sdAbs). Thus, unless the context requires otherwise, the terms "antigen-binding protein", "antibody" or "antibody molecule" as used herein are equivalent to "antibody or antigen-binding fragment thereof".

[0049] Antibodies are immunoglobulins with the same basic structure consisting of two heavy chains and two light chains forming two Fab arms, which contain an identical domain, the Fc domain, connected to the stem of the antibody by a flexible hinge region, giving it the classic "Y" shape. The Fab domain consists of two variable domains and two constant domains, and the heavy chains are connected to the variable heavy chain (V H ) domain and constant heavy chain 1 (CH1) domain, and the light chain has a variable light chain (V L ) domain and the constant light (CL) domain. There are two variable domains (V H and V L ) form the variable fragment (Fv) that provides the CDR-based antigen specificity of the antibody, and the constant domains (CH1 and V L ) acts as a structural framework. Each variable domain contains three hypervariable loops known as complementarity determining regions (CDRs). H and V L In each domain, the three CDRs (CDR1, CDR2 and CDR3) are flanked by four less variable framework (FW) regions (FW1, FW2, FW3 and FW4) giving the structure FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4. The CDRs provide a specific antigen recognition site on the surface of the antibody.

[0050] Both Kabat and ImMunoGeneTics (IMGT) numbering nomenclature can be used herein. In general, unless otherwise indicated, amino acid residues are numbered herein according to the Kabat numbering scheme (Kabat et al., 1991). When the IMGT numbering scheme is used, amino acid residues are numbered herein according to the ImMunoGeneTics (IMGT) numbering scheme described in Lefranc et al., 2005.

[0051] It is possible to use monoclonal and other antibodies, using techniques of recombinant DNA technology, to produce other antibodies or chimeric molecules that generally retain the specificity of the original antibody. Such techniques may involve introducing CDRs into different immunoglobulin frameworks or grafting variable regions into different immunoglobulin constant regions. The introduction of CDRs of one immunoglobulin into another is described, for example, in EP-A-184187, GB-A-2188638A or EP-A-239400. Alternatively, the hybridoma or other cells producing the antibody molecules may be subjected to genetic mutations or other changes, which may or may not alter the binding specificity of the antibody produced.

[0052] Antibody humanization involves the transfer or "grafting" of key non-human amino acids into a human antibody framework. Primarily, this involves the grafting of amino acids in the complementarity determining regions (CDRs), but also in the V and VF domains. H -V LOther framework amino acids important for the interface and orientation of the CDRs may also be included. Humanization seeks to introduce human content to reduce the risk of immunogenicity while retaining the original binding activity of the non-human parent antibody. The term "humanized antibody" is intended to refer to an antibody in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences, optionally with further framework region modifications being made within the human framework sequences. The term "humanized antibody" includes antibodies in which CDR sequences derived from the germline of another mammalian species have been grafted onto human framework sequences and optimized, for example, by modification (e.g., by affinity maturation) of one or more amino acid residues in one or more of the CDRs and / or one or more framework sequences to modulate or improve the biological properties of the humanized antibody, for example to increase the affinity or to modulate the on-rate and / or off-rate of the binding of the antibody to its target epitope.

[0053] The variable domains used in the present invention can be obtained or derived from any germline or rearranged human variable domain, or can be synthetic variable domains based on consensus sequences or actual sequences of known human variable domains. A repertoire of variable domains can be displayed in a suitable host system, such as the phage display system of WO 92 / 01047, incorporated herein by reference in its entirety, or any of the extensive literature thereafter, including Kay, Winter & McCafferty [Kay, BK, Winter, J., and McCafferty, J. (1996) Phage Display of Peptides and Proteins: A Laboratory Manual, San Diego: Academic Press], so that suitable binding members can be selected. Other suitable host systems include, but are not limited to, yeast display, bacterial display, T7 display, viral display, cell display, ribosome display and covalent display.

[0054] Because antibodies can be modified in a number of ways, the term "antigen binding protein" or "antibody" should be interpreted to encompass antibody fragments, derivatives, functional equivalents and homologs of antibodies, including any polypeptide containing an immunoglobulin binding domain, an aptamer, an affimer, or a bicyclic peptide, whether natural or wholly or partially synthetic.

[0055] Thus, chimeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are included. Cloning and expression of chimeric antibodies is described in EP-A-0120694 and EP-A-0125023.

[0056] An example of an antibody fragment containing both CDR sequences and a CH3 domain is a minibody, which contains an scFv linked to a CH3 domain (Hu et al., 1996).

[0057] A domain (single domain) antibody is a heavy chain antibody or an IgG containing only one variable domain (V H ), a peptide usually about 110 amino acids long. Single domain antibodies (sdAbs) (e.g. nanobodies) are antibody fragments consisting of a single monomeric variable antibody domain. Like whole antibodies (containing two heavy and two light chains), they are antigen-binding proteins that can selectively bind to a specific antigen. Domain antibodies have a molecular weight of only 12-15 kDa and are therefore much smaller than antibodies (150-160 kDa) which are composed of two protein heavy and two light chains, and domain antibodies are even smaller than Fab fragments (about 50 kDa, one light chain and half a heavy chain) and single chain variable fragments (about 25 kDa, two variable domains, one from the light chain and the other from the heavy chain). Single domain antibodies have been engineered from heavy chain antibodies found in camelids, which are V HH Cartilaginous fish also have heavy chain antibodies (IgNAR, "immunoglobulin novel antigen receptor") from which single domain antibodies, called VNAR fragments, can be derived. HOr V L The domain antibody may be of human or murine origin. H Or V L Most single domain antibodies are heavy chain variable domains, but light chain single domain antibodies (V L ) have also been shown to specifically bind to target epitopes.

[0058] Protein scaffolds have a relatively defined three-dimensional structure and typically contain one or more regions that are amenable to specific or random amino acid sequence variation to generate antigen-binding regions within the scaffold that are capable of binding to an antigen.

[0059] The humanized or human antibody or antigen-binding fragment of the present invention specifically binds to human SEMA4A. The term "specific" may refer to the situation where the antibody molecule does not show significant binding to molecules other than its specific binding partner, here the epitope of human SEMA4A. The term "specific" may also be applicable when the antibody is specific to a particular epitope, such as an epitope of human SEMA4A carried by multiple antigens, in which case the antibody molecule can bind to various antigens carrying the epitope. The epitope may be present in human SEMA4A expressed on the cell surface or in soluble SEMA4A (sSEMA4A) shed from the cell surface or recombinantly expressed. In some embodiments, the humanized or human antibody or antigen-binding fragment of the present invention specifically binds to human SEMA4A and binds to cynomolgus monkey and / or mouse SEMA4A, and thus the humanized or human antibody or antigen-binding fragment of the present invention may specifically bind to human SEMA4A and may be cross-reactive with cynomolgus monkey and / or mouse SEMA4A.

[0060] In some embodiments of the present invention, the humanized antibodies and antigen-binding fragments thereof are humanized versions of mouse 5E3 mAb (catalog number: 148402, BioLegend, Inc., USA) which comprise a set of six CDRs of mouse 5E3 (sequence numbers 3, 4, 5, 12, 13 and 14) and human framework sequences.

[0061] In some aspects of the invention, the humanized and human antibodies and their antigen-binding fragments of the invention bind to an epitope bound by murine 5E3 mAb (catalog number: 148402, BioLegend, Inc., USA) or compete with murine mAb 5E3 for binding to an epitope on SEMA4A, preferably human SEMA4A.

[0062] Amino acids may be referred to by their one-letter or three-letter codes or by their full names. The one- and three-letter codes and full names for each of the 20 standard amino acids are set forth in Table 1 below.

[0063] [Table 1]

[0064] In a preferred embodiment, the present invention comprises: a) 5E3 (SEQ ID NOs: 3, 4, 5, 12, 13, 14); b) 5Hg1-5Lg1 (SEQ ID NOs: 21, 22, 23, 30, 31, 32); c) 5Hg1-5Lg2 (SEQ ID NOs: 39, 40, 41, 48, 49, 50); d) 5Hg2-5Lg1 (SEQ ID NOs: 57, 58, 59, 66, 67, 68); e) 5Hg2-5Lg2 (SEQ ID NOs: 75, 76, 77, 84, 85, 86); f) 5Hg2-5Lg3 (SEQ ID NOs: 93, 94, 95, 102, 103, 104); g) 5Hg2-5Lg5 (SEQ ID NOs: 111, 112, 113, 120, 121, 122); h) 5Hg2-5Lg6 (SEQ ID NOs: 129, 130, 131, 138, 139, 140); i) 5Hg4-5Lg1 (SEQ ID NOs: 147, 148, 149, 156, 157, 158); j) 5Hg4-5Lg2 (SEQ ID NOs: 165, 166, 167, 174, 175, 176); k) 5Hg4-5Lg4 (SEQ ID NOs: 183, 184, 185, 192, 193, 194); l) 5Hg4-5Lg5 (SEQ ID NOs: 201, 202, 203, 210, 211, 212); m) 5Hg5-5Lg2 (SEQ ID NOs: 219, 220, 221, 228, 229, 230); n) C0120903 (SEQ ID NO: 237, 238, 239, 246, 247, 248); o) C0120904 (SEQ ID NO: 255, 256, 257, 264, 265, 266); p) C0120905 (SEQ ID NO: 273, 274, 275, 282, 283, 284); q) C0120906 (SEQ ID NO: 291, 292, 293, 300, 301, 302); r) C0120910 (SEQ ID NO: 309, 310, 311, 318, 319, 320); s) C0120913 (SEQ ID NO: 327, 328, 329, 336, 337, 338); t) C0120914 (SEQ ID NO: 345, 346, 347, 354, 355, 356); u) C0120917 (SEQ ID NO: 363, 364, 365, 372, 373, 374); v) C0120918 (SEQ ID NO: 381, 382, ​​383, 390, 391, 392); w) C0120919 (SEQ ID NO: 399, 400, 401, 408, 409, 410); x) C0120920 (SEQ ID NO: 417, 418, 419, 426, 427, 428); y) C0120921 (SEQ ID NOs: 435, 436, 437, 444, 445, 446); z) C0120922 (SEQ ID NO: 453, 453, 455, 462, 463, 464); aa) C0120923 (SEQ ID NO: 471, 472, 473, 480, 481, 482); bb) C0120924 (SEQ ID NOs: 489, 490, 491, 498, 499, 500); cc) C0120925 (SEQ ID NO: 507, 508, 509, 516, 517, 518); dd) C0120926 (SEQ ID NO: 525, 526, 527, 534, 535, 536); ee) C0120927 (SEQ ID NOs: 543, 544, 545, 552, 553, 554); ff) C0120928 (SEQ ID NOs: 561, 562, 563, 570, 571, 572); gg) C0120929 (SEQ ID NOs: 579, 580, 581, 588, 589, 590) and hh) C0120930 (SEQ ID NO: 597, 598, 599, 606, 607, 608) providing a humanized or human antibody or an antigen-binding fragment thereof comprising a set of six CDRs HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of a clone selected from Here, the CDRs are defined according to Kabat.

[0065] For each clone, the CDRs are spaced by framework regions FW1, FW2, FW3 and FW4 giving a structure of the form FW1-CDR1-FW2-CDR2-FW3-CDR3-FW4.

[0066] The present invention relates to a) 5Hg1-5Lg1 (SEQ ID NOs:20 and 29); b) 5Hg1-5Lg2 (SEQ ID NOs:38 and 47); c) 5Hg2-5Lg1 (SEQ ID NOs:56 and 65); d) 5Hg2-5Lg2 (SEQ ID NOs:74 and 83); e) 5Hg2-5Lg3 (SEQ ID NOs:92 and 101); f) 5Hg2-5Lg5 (SEQ ID NOs:110 and 119); g) 5Hg2-5Lg6 (SEQ ID NOs:128 and 137); h) 5Hg4-5Lg1 (SEQ ID NOs:146 and 155); i) 5Hg4-5Lg2 (SEQ ID NOs:164 and 173); j) 5Hg4-5Lg4 (SEQ ID NOs:182 and 191); k) 5Hg4-5Lg5 (SEQ ID NOs: 200 and 209); l) 5Hg5-5Lg2 (SEQ ID NOs: 218 and 227); m) C0120903 (SEQ ID NO: 236 and 245); n) C0120904 (SEQ ID NO: 254 and 263); o) C0120905 (SEQ ID NO: 272 and 281); p) C0120906 (SEQ ID NO: 290 and 299); q) C0120910 (SEQ ID NO: 308 and 317); r) C0120913 (SEQ ID NO: 326 and 335); s) C0120914 (SEQ ID NO: 344 and 353); t) C0120917 (SEQ ID NO: 362 and 371); u) C0120918 (SEQ ID NO: 380 and 389); v) C0120919 (SEQ ID NOs: 398 and 407); w) C0120920 (SEQ ID NO: 416 and 425); x) C0120921 (SEQ ID NO: 434 and 443); y) C0120922 (SEQ ID NO: 452 and 461); z) C0120923 (SEQ ID NO: 470 and 479); aa) C0120924 (SEQ ID NO: 488 and 497); bb) C0120925 (SEQ ID NOs: 506 and 515); cc) C0120926 (SEQ ID NOs: 524 and 533); dd) C0120927 (SEQ ID NOs: 542 and 551); ee) C0120928 (SEQ ID NOs: 560 and 569); ff) C0120929 (SEQ ID NOs: 578 and 587); and gg) C0120930 (SEQ ID NOs: 596 and 605); V of a clone selected from H and / or V Lproviding a human or humanized antibody or antigen-binding fragment thereof comprising the sequence Here, sequences are defined according to Kabat.

[0067] In some instances, the antibodies were selected for germline. H and V L The amino acid sequences of the domains were compared to human germline V, D and J regions accessible through the IMGT (ImMunoGeneTics; www.imgt.org) or ImmuneDiscover databases, and the closest germline was identified by sequence similarity. H and V L This consisted of changing framework residues in the domains back to the closest germline sequence to match identically to the human antibody.

[0068] The antibody or antigen-binding fragment thereof of the present invention may be used in any manner as long as the functional properties of the antibody are maintained. H and / or V L The sequence may include one or more, for example 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, further amino acid modifications.

[0069] The modification may be an amino acid substitution, deletion or insertion, preferably the modification is a substitution. In preferred embodiments where one or more amino acids are replaced with another amino acid, the substitution may be a conservative substitution, for example according to Table 2. In some embodiments, amino acids in the same category in the middle column are replaced with each other, i.e., a non-polar amino acid is replaced with another non-polar amino acid. In some embodiments, amino acids in the same row in the right-most column are replaced with each other.

[0070] [Table 2]

[0071] In some embodiments, substitutions may be functionally conservative, i.e., in some embodiments, the substitution may not affect (or may not substantially affect) one or more functional properties (e.g., binding affinity) of an antibody molecule that contains the substitution, compared to an equivalent unsubstituted antibody molecule.

[0072] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises the V H and / or V L V has one or more amino acid sequence changes (addition, deletion, substitution and / or insertion of amino acid residues) compared to the sequence, preferably 20 or less changes, 15 or less changes, 10 or less changes, 5 or less changes, 4 or less changes, 3 or less changes, 2 or less changes, or 1 change. H and / or V L It may include a domain sequence.

[0073] In a preferred embodiment, the present invention provides a method for the preparation of a medicament for the treatment of cancer, comprising administering to the patient a medicament for which the medicament for the treatment of cancer is administered: a) 5E3 (SEQ ID NOs:2 and 11); b) 5Hg1-5Lg1 (SEQ ID NOs:20 and 29); c) 5Hg1-5Lg2 (SEQ ID NOs:38 and 47); d) 5Hg2-5Lg1 (SEQ ID NOs:56 and 65); e) 5Hg2-5Lg2 (SEQ ID NOs:74 and 83); f) 5Hg2-5Lg3 (SEQ ID NOs:92 and 101); g) 5Hg2-5Lg5 (SEQ ID NOs:110 and 119); h) 5Hg2-5Lg6 (SEQ ID NOs:128 and 137); i) 5Hg4-5Lg1 (SEQ ID NOs:146 and 155); j) 5Hg4-5Lg2 (SEQ ID NOs:164 and 173); k) 5Hg4-5Lg4 (SEQ ID NOs: 182 and 191); l) 5Hg4-5Lg5 (SEQ ID NOs: 200 and 209); m) 5Hg5-5Lg2 (SEQ ID NOs: 218 and 227); n) C0120903 (SEQ ID NO: 236 and 245); o) C0120904 (SEQ ID NO: 254 and 263); p) C0120905 (SEQ ID NO: 272 and 281); q) C0120906 (SEQ ID NO: 290 and 299); r) C0120910 (SEQ ID NO: 308 and 317); s) C0120913 (SEQ ID NO: 326 and 335); t) C0120914 (SEQ ID NO: 344 and 353); u) C0120917 (SEQ ID NO: 362 and 371); v) C0120918 (SEQ ID NO:380 and 389); w) C0120919 (SEQ ID NOs: 398 and 407); x) C0120920 (SEQ ID NO: 416 and 425); y) C0120921 (SEQ ID NO: 434 and 443); z) C0120922 (SEQ ID NO: 452 and 461); aa) C0120923 (SEQ ID NO: 470 and 479); bb) C0120924 (SEQ ID NOs: 488 and 497); cc) C0120925 (SEQ ID NOs: 506 and 515); dd) C0120926 (SEQ ID NOs: 524 and 533); ee) C0120927 (SEQ ID NOs: 542 and 551); ff) C0120928 (SEQ ID NOs: 560 and 569); gg) C0120929 (SEQ ID NOs: 578 and 587); and hh) C0120930 (SEQ ID NOs: 596 and 605); V of a clone selected from H and / or V LV having an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with the amino acid sequence H and / or V L The present invention provides a humanized or human antibody or antigen-binding fragment thereof comprising the domain.

[0074] In a preferred embodiment of the invention, the humanized or human antibody, or antigen-binding fragment thereof, of the invention comprises: (a) 5E3 (SEQ ID NOs: 3, 4, 5, 12, 13, 14); (b) 5Hg1-5Lg1 (SEQ ID NOs: 21, 22, 23, 30, 31, 32); (c) 5Hg1-5Lg2 (SEQ ID NOs: 39, 40, 41, 48, 49, 50); (d) 5Hg2-5Lg1 (SEQ ID NOs: 57, 58, 59, 66, 67, 68); (e) 5Hg2-5Lg2 (SEQ ID NOs: 75, 76, 77, 84, 85, 86); (f) 5Hg2-5Lg3 (SEQ ID NOs: 93, 94, 95, 102, 103, 104); (g) 5Hg2-5Lg5 (SEQ ID NOs: 111, 112, 113, 120, 121, 122); (h) 5Hg2-5Lg6 (SEQ ID NOs: 129, 130, 131, 138, 139, 140); (i) 5Hg4-5Lg1 (SEQ ID NOs: 147, 148, 149, 156, 157, 158); (j) 5Hg4-5Lg2 (SEQ ID NOs: 165, 166, 167, 174, 175, 176); (k) 5Hg4-5Lg4 (SEQ ID NOs: 183, 184, 185, 192, 193, 194); (l) 5Hg4-5Lg5 (SEQ ID NOs: 201, 202, 203, 210, 211, 212); (m) 5Hg5-5Lg2 (SEQ ID NOs: 219, 220, 221, 228, 229, 230); (n) C0120903 (SEQ ID NO: 237, 238, 239, 246, 247, 248); (o) C0120904 (SEQ ID NO: 255, 256, 257, 264, 265, 266); (p)C0120905 (SEQ ID NO: 273, 274, 275, 282, 283, 284); (q) C0120906 (SEQ ID NO: 291, 292, 293, 300, 301, 302); (r) C0120910 (SEQ ID NO: 309, 310, 311, 318, 319, 320); (s)C0120913 (SEQ ID NO: 327, 328, 329, 336, 337, 338); (t)C0120914 (SEQ ID NO: 345, 346, 347, 354, 355, 356); (u)C0120917 (SEQ ID NO: 363, 364, 365, 372, 373, 374); (v) C0120918 (sequence numbers: 381, 382, ​​383, 390, 391, 392); (w)C0120919 (SEQ ID NOs: 399, 400, 401, 408, 409, 410); (x) C0120920 (SEQ ID NO: 417, 418, 419, 426, 427, 428); (y) C0120921 (SEQ ID NO: 435, 436, 437, 444, 445, 446); (z) C0120922 (SEQ ID NO: 453, 453, 455, 462, 463, 464); (aa)C0120923 (SEQ ID NOs: 471, 472, 473, 480, 481, 482); (bb) C0120924 (SEQ ID NOs: 489, 490, 491, 498, 499, 500); (cc)C0120925 (SEQ ID NO: 507, 508, 509, 516, 517, 518); (dd) C0120926 (SEQ ID NOs: 525, 526, 527, 534, 535, 536); (ee) C0120927 (SEQ ID NOs: 543, 544, 545, 552, 553, 554); (ff)C0120928 (SEQ ID NO: 561, 562, 563, 570, 571, 572); (gg) C0120929 (sequence numbers: 579, 580, 581, 588, 589, 590) and (hh) C0120930 (SEQ ID NO: 597, 598, 599, 606, 607, 608) A set of HCDRs of a clone selected from: V comprising HCDR1, HCDR2, and HCDR3 H Domain amino acid sequence and / or set of LCDRs of the clone: ​​V including LCDR1, LCDR2, and LCDR3 L comprising a domain amino acid sequence, where CDRs are defined according to Kabat and V H and / or V L A domain, as defined by the Kabat nomenclature, is a.5E3 (SEQ ID NO:2 and 11); b.5Hg1-5Lg1 (SEQ ID NO:20 and 29); c.5Hg1-5Lg2 (SEQ ID NOs:38 and 47); d.5Hg2-5Lg1 (SEQ ID NOs:56 and 65); e.5Hg2-5Lg2 (SEQ ID NOs:74 and 83); f.5Hg2-5Lg3 (SEQ ID NOs:92 and 101); g.5Hg2-5Lg5 (SEQ ID NOs:110 and 119); h.5Hg2-5Lg6 (SEQ ID NOs:128 and 137); i.5Hg4-5Lg1 (SEQ ID NOs: 146 and 155); j.5Hg4-5Lg2 (SEQ ID NOs:164 and 173); k.5Hg4-5Lg4 (SEQ ID NOs:182 and 191); l.5Hg4-5Lg5 (SEQ ID NOs: 200 and 209); m.5Hg5-5Lg2 (SEQ ID NOs:218 and 227); n.C0120903 (SEQ ID NO: 236 and 245); o.C0120904 (SEQ ID NO:254 and 263); p.C0120905 (SEQ ID NO:272 and 281); q.C0120906 (SEQ ID NO:290 and 299); r.C0120910 (SEQ ID NO:308 and 317); s.C0120913 (SEQ ID NOs: 326 and 335); t.C0120914 (sequence numbers: 344 and 353); u.C0120917 (SEQ ID NO:362 and 371); v.C0120918 (SEQ ID NO:380 and 389); w.C0120919 (SEQ ID NOs: 398 and 407); x.C0120920 (sequence numbers: 416 and 425); y.C0120921 (SEQ ID NO: 434 and 443); z.C0120922 (SEQ ID NOs: 452 and 461); aa.C0120923 (SEQ ID NO: 470 and 479); bb.C0120924 (SEQ ID NOs: 488 and 497); cc.C0120925 (sequence numbers: 506 and 515); dd.C0120926 (SEQ ID NO:524 and 533); ee.C0120927 (SEQ ID NOs:542 and 551); ff.C0120928 (SEQ ID NO:560 and 569); gg.C0120929 (SEQ ID NOs: 578 and 587); and hh.C0120930 (SEQ ID NO: 596 and 605) and having an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the sequence of a clone selected from the

[0075] Sequence identity is generally defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximizing the number of matches and minimizing the number of gaps. Generally, default parameters are used, with a gap creation penalty of 12 and a gap extension penalty equal to 4. Although the use of GAP may be preferred, other algorithms can be used, such as BLAST (using the method of Altschul et al., 1990), FASTA (using the method of Pearson and Lipman 1988), or the Smith-Waterman algorithm (Smith and Waterman 1981), or the TBLASTN program of Altschul et al., (1990), supra, generally using default parameters. In particular, the psi-Blast algorithm can be used (Altschul et al., 1997). Sequence alignment can also be performed using the CLUSTAL(W) algorithm.

[0076] The antibody may comprise a CH2 domain. The CH2 domain is preferably located N-terminal to the CH3 domain, as in human IgG molecules. The CH2 domain of the antibody is preferably a human IgG1, IgG2, IgG3, or IgG4 CH2 domain, more preferably a human IgG1 CH2 domain. The sequences of human IgG domains are known in the art.

[0077] The antibody may comprise an immunoglobulin hinge region or a portion thereof at the N-terminus of the CH2 domain. The immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate and form a dimer. Preferably, the hinge region or a portion thereof is a human IgG1, IgG2, IgG3 or IgG4 hinge region or a portion thereof. More preferably, the hinge region or a portion thereof is an IgG1 hinge region or a portion thereof.

[0078] The sequence of the CH3 domain is not particularly limited. Preferably, the CH3 domain is a human immunoglobulin G domain, such as a human IgG1, IgG2, IgG3, or IgG4 CH3 domain, and most preferably a human IgG1 CH3 domain.

[0079] The antibody of the present invention may comprise a human IgG1, IgG2, IgG3, or IgG4 constant region. The sequences of human IgG1, IgG2, IgG3, or IgG4 CH3 domains are known in the art. The antibody of the present invention may comprise a human IgG constant region, for example a human IgG1 constant region. The antibody of the present invention may comprise a human IgG Fc modified to allow conjugation with a linker and a cytotoxin.

[0080] The antibodies of the invention may comprise a human IgG heavy chain, e.g., a human IgG1 heavy chain, engineered to contain one or more, e.g., two site-specific engineered cysteines that allow for conjugation of a cytotoxin to the antibody via a linker in a controlled manner as described in Dimasi et al., 2017; Li et al., 2016 and Gallagher et al., 2019 (e.g., a human IgG1 heavy chain having 239iCys and S442C (EU numbering) in the CH2 and CH3 Fc domains, respectively), generating a drug-to-antibody ratio of 4 (human IgG1 Alya). The antibodies of the invention may comprise a human lambda light chain constant domain or a human kappa light chain constant domain.

[0081] The antibodies of the invention may comprise a human IgG Fc that has effector functions.

[0082] Fc receptors (FcR) are key immunoregulatory receptors that link antibody-mediated (humoral) immune responses to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcγR (IgG), FcεRI (IgE), FcαRI (IgA), FcμR (IgM) and FcδR (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64 (FcγRI), CD32 (FcγRIIa, FcγRIIb and FcγRIIc) and CD16 (FcγRIIIa and FcγRIIIb). FcγRI is classified as a high affinity receptor (nanomolar range affinity), whereas FcγRII and FcγRIII are low to intermediate affinity (micromolar range affinity).

[0083] In antibody-dependent cell-mediated cytotoxicity (ADCC), FcγRs on the surface of effector cells (natural killer cells, macrophages, monocytes and eosinophils) bind to the Fc region of IgG, which itself is bound to a target cell. Binding triggers signaling pathways that result in the secretion of various substances, such as lytic enzymes, perforin, granzymes and tumor necrosis factor, that mediate the destruction of the target cell. The level of ADCC effector function varies depending on the particular IgG subtype. The level of variation depends on the allotype and the specific FcγR, but in brief, ADCC effector function is high for human IgG1 and IgG3 and low for IgG2 and IgG4. See Table 3 below for IgG subtype variations in effector function ranked in decreasing potency.

[0084] [Table 3]

[0085] FcγRs bind IgG asymmetrically across the hinge and upper CH2 regions. Knowledge of the binding site has led to engineering efforts to modulate IgG effector functions.

[0086] The antibodies of the invention may have an Fc with effector function, enhanced effector function or reduced effector function.

[0087] Antibody potency can be increased by enhancing the ability to mediate cytotoxic functions such as ADCC and antibody-dependent cell-mediated phagocytosis (ADCP). A number of mutations in the Fc domain have been identified that directly or indirectly enhance Fc receptor binding and significantly enhance cytotoxicity: mutations S239D / A330L / I332E ("3M"), F243L or G236A. Alternatively, enhancement of effector function can be achieved by modifying the glycosylation of the Fc domain, where FcγR interacts with carbohydrates on the CH2 domain and the glycan composition has a substantial effect on effector function activity. Afucosylated (non-fucosylated) antibodies show a significant improvement in ADCC activity through increased binding to FcγRIIIa.

[0088] While activation of ADCC and CDC may be desirable for some therapeutic antibodies, in some embodiments antibodies that do not activate effector functions are preferred.

[0089] Due to their lack of effector functions, IgG4 antibodies are the preferred IgG subclass for receptor blockade without cell depletion. However, IgG4 molecules can exchange half molecules in a dynamic process called Fab arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous IgG4. The S228P mutation has been shown to interfere with this recombination process, allowing the design of IgG4 antibodies with reduced propensity for Fab arm exchange.

[0090] Fc engineering approaches have been used to determine the key interaction sites of IgG1 Fc domain with Fcγ receptors and C1q, and then mutate these positions to reduce or eliminate binding. Alanine scanning identified the binding site of C1q to the region covering the hinge and upper CH2 of the Fc domain. The CH2 domain of the antibody or fragment of the invention may contain one or more mutations to reduce or eliminate binding of the CH2 domain to one or more FcγRs, e.g., FcγRI, FcγRlla, FcγRllb, FcγRIII and / or complement. The CH2 domain of a human IgG domain normally binds FcγRs and complement, and reduced binding to FcγRs is expected to reduce antibody-dependent cell-mediated cytotoxicity (ADCC), and reduced binding to complement is expected to reduce complement-dependent cytotoxicity (CDC) activity of the antibody molecule. Mutations that reduce or eliminate binding of the CH2 domain to one or more FcγRs and / or complement are known in the art. The antibody molecules of the invention may comprise an Fc with the modifications K322A / L234A / L235A or L234F / L235E / P331S ("TM") that almost completely abolish FcγR and C1q binding. The antibody molecules of the invention may comprise a CH2 domain that comprises alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 according to the IMGT numbering) ("LALA mutation"). Furthermore, complement activation and ADCC can be reduced by mutation of P329 (positions according to EU numbering), e.g., to either P329A or P329G. The antibody molecule of the invention may comprise a CH2 domain comprising an alanine residue at EU position 234 and 235 (positions 1.3 and 1.2 according to the IMGT numbering) and an alanine (LALA-PA) or glycine (LALA-PG) at EU position 329 (position 114 according to the IMGT numbering). Additionally or alternatively, the antibody molecule of the invention may comprise an alanine, glutamine or glycine at EU position 297 (position 84.4 according to the IMGT numbering).

[0091] Modification of glycosylation on asparagine 297 of the Fc domain, known to be necessary for optimal FcγR interaction, can result in loss of binding to FcγR, and loss of binding to FcγR has been observed with N297 point mutation. The antibody molecule of the present invention may comprise an Fc having an N297A, N297G or N297Q mutation. The antibody molecule of the present invention having an aglycosylated Fc domain can be obtained by enzymatic deglycosylation, by recombinant expression in the presence of a glycosylation inhibitor, or after expression of the Fc domain in bacteria.

[0092] IgG naturally persists for long periods in serum due to FcRn-mediated recycling, giving IgG a typical half-life of about 21 days. Half-life can be extended by manipulating the pH-dependent interactions of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4. The T250Q / M428L variant conferred an approximately two-fold increase in IgG half-life (assessed in rhesus monkeys), while the M252Y / S254T / T256E variant ("YTE") conferred an approximately four-fold increase in IgG half-life (assessed in cynomolgus monkeys). Extending the half-life may allow the possibility of reducing dosing frequency while maintaining or improving efficacy.

[0093] Immunoglobulins are known to have a modular structure with discrete domains that can be combined in many different ways to create multispecific, e.g., bispecific, trispecific, or tetraspecific antibody formats. Exemplary multispecific antibody formats are described, for example, in Spiess et al., 2015; Kontermann 2012. The antibodies of the present invention can be used in such multispecific formats.

[0094] The present invention relates to an antibody of the invention described herein (e.g., comprising the set of HCDRs and LCDRs of 5E3 (SEQ ID NOs: 3, 4, 5, 12, 13, 14) as defined by the Kabat nomenclature) and / or clone 5E3 for binding to an isolated recombinant human SEMA4A (SEQ ID NO: 613) peptide comprising an epitope, as assessed in a competition assay. H and V L Provided are humanized or human antibodies or antigen-binding fragments thereof capable of competing with humanized variants of the amino acid sequences (SEQ ID NOs: 2 and 11).

[0095] The present invention relates to a.5E3 (SEQ ID NO: 2 and 11) b.5Hg1-5Lg1 (SEQ ID NO:20 and 29); c.5Hg1-5Lg2 (SEQ ID NOs:38 and 47); d.5Hg2-5Lg1 (SEQ ID NOs:56 and 65); e.5Hg2-5Lg2 (SEQ ID NOs:74 and 83); f.5Hg2-5Lg3 (SEQ ID NOs:92 and 101); g.5Hg2-5Lg5 (SEQ ID NOs:110 and 119); h.5Hg2-5Lg6 (SEQ ID NOs:128 and 137); i.5Hg4-5Lg1 (SEQ ID NOs: 146 and 155); j.5Hg4-5Lg2 (SEQ ID NOs:164 and 173); k.5Hg4-5Lg4 (SEQ ID NOs:182 and 191); l.5Hg4-5Lg5 (SEQ ID NOs: 200 and 209); m.5Hg5-5Lg2 (SEQ ID NOs:218 and 227); n.C0120903 (SEQ ID NO: 236 and 245); o.C0120904 (SEQ ID NO:254 and 263); p.C0120905 (SEQ ID NO:272 and 281); q.C0120906 (SEQ ID NO:290 and 299); r.C0120910 (SEQ ID NO:308 and 317); s.C0120913 (SEQ ID NOs: 326 and 335); t.C0120914 (sequence numbers: 344 and 353); u.C0120917 (SEQ ID NO:362 and 371); v.C0120918 (SEQ ID NO:380 and 389); w.C0120919 (SEQ ID NOs: 398 and 407); x.C0120920 (sequence numbers: 416 and 425); y.C0120921 (SEQ ID NO: 434 and 443); z.C0120922 (SEQ ID NOs: 452 and 461); aa.C0120923 (SEQ ID NO: 470 and 479); bb.C0120924 (SEQ ID NOs: 488 and 497); cc.C0120925 (sequence numbers: 506 and 515); dd.C0120926 (SEQ ID NO:524 and 533); ee.C0120927 (SEQ ID NOs:542 and 551); ff.C0120928 (SEQ ID NO:560 and 569); gg.C0120929 (SEQ ID NOs: 578 and 587); and hh.C0120930 (SEQ ID NO: 596 and 605) and providing a humanized or human antibody or antigen-binding fragment thereof capable of competing for binding to an isolated recombinant human SEMA4A (SEQ ID NO: 613) peptide comprising an epitope having a clone selected from: Here, sequences are defined according to Kabat nomenclature and competition for binding is assessed in competition assays.

[0096] The competitive assay may be selected from cell-based and cell-free binding assays including immunoassays such as ELISA, homogeneous time-resolved fluorescence (HTRF), flow cytometry, fluorescence micro-volume assay technology (FMAT) assays, Mirrorball, high content imaging based fluorescent immunoassays, radioligand binding assays, biolayer interferometry (BLI), surface plasmon resonance (SPR) and thermal shift assays.

[0097] An antibody that binds to the same epitope as a reference antibody or an overlapping epitope refers to an antibody that blocks the binding of the reference antibody to its binding partner (e.g., antigen or "target") by 50% or more in a competitive assay, and / or conversely, blocks the binding of the antibody to its binding partner by 50% or more in a competitive assay. Such antibodies are said to compete for binding to the epitope of interest. An antibody may compete by binding to the same epitope as the epitope of the reference antibody or an overlapping epitope.

[0098] In an embodiment of the invention, the anti-human-SEMA4A humanized or human antibodies described herein can be used to generate antibody single-chain variable fragments, which can then be used to prepare chimeric antigen receptors (CARs). Antibody single-chain variable fragments consist of immunoglobulin light chains (V) linked by a short linker peptide. L ) and heavy chain (V H In some embodiments, the V L Area and V H The linker between the regions is composed of hydrophilic residues, including glycine and serine, which confer flexibility, and glutamate and lysine, which confer solubility. In some embodiments, the antibody single chain variable fragment can be covalently linked to an intracellular immune cell signaling domain, typically via a transmembrane domain, to generate a CAR. The immune cell signaling domain can be a T cell, NK cell, macrophage, and / or myeloid cell domain.

[0099] Anti-human-SEMA4A humanized or human antibody single chain variable fragments can be covalently linked to an intracellular T cell signaling or activation domain, for example, via a transmembrane domain, to create a CAR. When the CAR is expressed in a T cell, it provides the T cell with the ability to target SEMA4A, particularly human SEMA4A. Thus, the present invention provides CAR T cells comprising a CAR comprising an anti-human SEMA4A antibody single chain variable fragment of an antibody described herein that specifically binds to SEMA4A, particularly human SEMA4A, for use in treating patients with hematological cancer, such as MM, NHL, AML, DLBCL or FL, and a method of treating a patient with hematological cancer, such as MM, NHL, AML, DLBCL or FL, can include administering such a CAR T cell to a patient in need of treatment.

[0100] The transmembrane domain of the CAR may include a hydrophobic alpha helix that spans the cell membrane, anchoring the CAR to the plasma membrane, and bridges the extracellular antigen recognition domain (i.e., a humanized or human antibody single chain variable fragment) with the intracellular signaling region. The CAR may further include a hinge region between the antigen recognition domain and the transmembrane domain. The hinge may help to increase the flexibility of the scFv and reduce spatial constraints between the CAR and its target antigen, SEMA4A. The hinge sequence may be based on membrane proximal regions from immune molecules such as IgG, CD8, and CD28. The CAR of the present disclosure may include a CD3-zeta cytoplasmic domain as a CAR endodomain component. T cells require costimulatory molecules in addition to CD3 signaling to persist after activation. The endodomain of the CAR may include one or more chimeric domains from costimulatory proteins. Signaling domains from a wide variety of costimulatory molecules have been successfully tested and may be selected from CD28, CD27, CD134 (OX40), and CD137. The endodomain of the CAR receptor may comprise a costimulatory domain for enhancing T cell activity, which may be selected from those of CD28 or 4-1BB, CD28-4-1BB or CD28-OX40, and cytokines such as IL-2, IL-5, and IL-12.

[0101] The present invention also provides a nucleic acid or set of nucleic acids encoding the antibody or antigen-binding fragment of the present invention, as well as a vector comprising such a nucleic acid or set of nucleic acids.

[0102] The nucleic acid is a V H and V L In the case of encoding domains or a heavy chain and a light chain, the two domains or chains can be encoded on the same or separate nucleic acid molecules.

[0103] The isolated nucleic acid molecule can be used to express the antibody molecule of the present invention. The nucleic acid is generally provided in the form of a recombinant vector for expression. Thus, another aspect of the present invention provides a vector comprising the above-mentioned nucleic acid. Suitable vectors can be selected or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive expression of the nucleic acid in a host cell. The vector can be a plasmid, such as a virus, a phage or a phagemid, as appropriate.

[0104] The nucleic acid molecules or vectors described herein may be introduced into a host cell. Techniques for introducing nucleic acids or vectors into a host cell are well established in the art and any suitable technique may be used. A range of host cells suitable for the production of recombinant antibody molecules are known in the art and include bacterial, yeast, insect or mammalian host cells. Preferred host cells are mammalian cells, such as CHO, NS0, or HEK cells, such as HEK293 cells.

[0105] Recombinant host cells comprising the nucleic acid or vector of the invention are also provided. Such recombinant host cells may be used to produce antigen binding proteins (e.g., antibodies) of the invention. Thus, a method of producing an antigen binding protein, such as an antibody, of the invention is also provided, comprising culturing a recombinant host cell under conditions suitable for the production of the antigen binding protein, such as an antibody. The method may further comprise the step of isolating and / or purifying the antigen binding protein, such as an antibody.

[0106] Thus, the present invention provides a method of producing an antigen binding protein, e.g., an antibody, of the present invention, comprising expressing a nucleic acid encoding the antigen binding protein, e.g., an antibody, in a host cell and, optionally, isolating and / or purifying the antigen binding protein, e.g., antibody, thus produced. Methods for culturing host cells are well known in the art. Techniques for the purification of recombinant antigen binding proteins, e.g., antibodies, are well known in the art and include, for example, high pressure liquid chromatography (HPLC), fast protein liquid chromatography (FPLC) or affinity chromatography (e.g., using Protein A or Protein L). In some embodiments, purification may be performed using an affinity tag on the antigen binding protein, e.g., an antibody. The method may also include formulating the antigen binding protein, e.g., an antibody, into a pharmaceutical composition, optionally with a pharma- ceutically acceptable excipient or other substances as described below.

[0107] The antigen binding proteins, such as antibodies, of the present invention are expected to find therapeutic use, particularly in humans, for example in the treatment of haematological cancers such as MM, NHL, AML, DLBCL or FL.

[0108] Also provided are compositions, e.g., pharmaceutical compositions, comprising an ADC according to the invention, an antigen binding protein, e.g., an antibody, or a CAR, and an excipient, e.g., a pharma- ceutically acceptable diluent.

[0109] The present invention further provides an ADC, an antigen-binding protein, such as an antibody, or a CAR of the present invention for use in a method of treatment. Also provided is a method of treating a patient, comprising administering to the patient a therapeutically effective amount of an ADC, an antigen-binding protein, such as an antibody, or a CAR according to the present invention. Further provided is the use of an ADC, an antigen-binding protein, such as an antibody, or a CAR according to the present invention for use in the manufacture of a medicament. The patient referred to herein is preferably a human patient.

[0110] The invention also provides an ADC, antigen binding protein, e.g., an antibody, or a CAR of the invention for use in a method of treating a hematological cancer, such as MM, NHL, AML, DLBCL or FL, comprising administering to a patient a therapeutically effective amount of an ADC, antigen binding protein, e.g., an antibody, or a CAR according to the invention. There is further provided the use of an ADC, antigen binding protein, e.g., an antibody, or a CAR according to the invention for use in the manufacture of a medicament for treating a hematological cancer, such as multiple myeloma MM, NHL, AML, DLBCL or FL in a patient.

[0111] In another aspect, the invention relates to an ADC, antigen binding protein, e.g., an antibody, or a CAR of the invention for use in a) treating, b) delaying progression, c) prolonging survival, and / or (d) providing palliation of symptoms in a patient suffering from a hematological cancer, such as MM, NHL, AML, DLBCL, or FL.

[0112] Thus, the ADCs, antigen binding proteins, e.g., antibodies, or CARs described herein may be for therapeutic use, particularly for use in the treatment of hematological cancers such as MM, NHL, AML, DLBCL, or FL.

[0113] The ADCs, antigen binding proteins, e.g., antibodies, or CARs described herein can be used in methods of treatment of the human or animal body. (i) an ADC, antigen binding protein, e.g., an antibody, or a CAR, as described herein for use as a medicament; (ii) an ADC, antigen binding protein, e.g., an antibody, or a CAR, as described herein for use in a method of treating a disease or disorder; (iii) use of an ADC, antigen binding protein, e.g., an antibody, or a CAR described herein in the manufacture of a medicament for use in the treatment of a disease or disorder, and (iv) Methods of treating a disease or disorder in an individual are provided, comprising administering to the individual a therapeutically effective amount of an ADC, antigen binding protein, e.g., an antibody, or CAR described herein.

[0114] The individual may be a patient, preferably a human patient.

[0115] Treatment can be any treatment or therapy that achieves some desired therapeutic effect, for example, inhibiting or slowing the progression of the condition, including slowing the rate of progression, halting the rate of progression, improving the condition, curing or ameliorating (whether partial or total) the condition, preventing, ameliorating, slowing, alleviating or halting one or more symptoms and / or signs of the condition, or prolonging the survival of an individual or patient beyond that expected in the absence of treatment.

[0116] Treatment as a preventative measure (i.e., prophylaxis) is also included. For example, an individual who is susceptible to or at risk of recurrence of a hematological cancer, such as MM, NHL, AML, DLBCL, or FL, can be treated as described herein. Such treatment can prevent or delay recurrence of disease in an individual.

[0117] Although an ADC, an antigen binding protein, such as an antibody, or a CAR can be administered alone, an ADC, an antigen binding protein, such as an antibody, or a CAR is usually administered in the form of a pharmaceutical composition that may contain at least one component in addition to the ADC, the antigen binding protein, such as an antibody, or a CAR. Thus, another aspect of the present invention provides a pharmaceutical composition comprising an ADC, an antigen binding protein, such as an antibody, or a CAR as described herein. Methods are also provided that include formulating an ADC, an antigen binding protein, such as an antibody, or a CAR into a pharmaceutical composition.

[0118] A pharmaceutical composition may include, in addition to an ADC, an antigen-binding protein, e.g., an antibody, or a CAR, pharma- ceutically acceptable excipients, carriers, buffers, stabilizers, or other materials known to those skilled in the art. As used herein, the term "pharma- ceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with the tissues of a subject (e.g., a human) without undue toxicity, irritation, allergic response, or other problem or complication, within the scope of sound medical judgment and commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the formulation. The precise nature of the carrier or other material will depend on the route of administration, which may be by infusion, injection, or any other suitable route, as discussed below.

[0119] For parenteral administration, e.g., subcutaneous administration or intravenous administration, e.g., by injection, a pharmaceutical composition comprising an ADC, antigen binding protein, e.g., an antibody, or a CAR, can be in the form of a parenterally acceptable aqueous solution that is pyrogen-free and has suitable pH, isotonicity and stability. In some embodiments, the ADC, antigen binding protein, e.g., an antibody, or a CAR of the invention can be provided in lyophilized form for reconstitution prior to administration. For example, a lyophilized antigen binding protein, e.g., an antibody, can be reconstituted with sterile water or saline prior to administration to an individual.

[0120] The administration may be a "therapeutically effective amount", which is sufficient to show benefit to the individual. The actual amount administered, as well as the rate and time course of administration, will depend on the nature and severity of what is being treated, the particular individual being treated, the clinical condition of the individual, the cause of the disorder, the site of delivery of the composition, the type of ADC, antigen binding protein, e.g., antibody, or CAR, the method of administration, the schedule of administration, and other factors. The treatment regimen may depend on the severity and / or progression of the symptoms of the disease being treated. The therapeutically effective amount or appropriate dose of an ADC, antigen binding protein, e.g., antibody, or CAR can be determined by comparing the in vitro activity and the in vivo activity in an animal model. Methods for extrapolating effective doses in mice and other test animals to humans are known. The exact dose will depend on many factors, including the size and location of the area being treated, and the exact nature of the ADC, antigen binding protein, e.g., antibody, or CAR.

[0121] Hematological malignancies are a diverse group of blood cancers that affect the blood, bone marrow and lymphatic system. The major categories are lymphoma, leukemia, myeloma, myelodysplastic syndrome and myeloproliferative neoplasms. In a preferred embodiment, the ADC, antigen binding protein, e.g., antibody, or CAR described herein may be for use in a method of treating a hematological cancer, such as MM, NHL, AML, DLBCL or FL. Drawing List [Brief description of the drawings]

[0122] [Figure 1] Figure 2: Primary screening of 70 unpurified humanized IgGs in the Fab-ZAP cell killing assay of NCI-H929 cells identified 18 clones that showed 30% or more cell killing. All 70 unpurified clones were screened at a final concentration of 1.56%. Twelve of the 18 hit clones (●) were re-expressed and purified for further characterization. Ten of the 12 clones selected for progression also showed inhibition in the 5E3 epitope competition assay (see Figure 2). [Diagram 2]Figure 1: Primary screening of 320 unpurified humanized IgGs in an epitope competition assay on MM.1S cells identified 17 clones that showed 30% or more inhibition. All 320 unpurified clones were screened at a final concentration of 25%. Ten of the 17 hit clones (●) were re-expressed and purified for further characterization. Two clones that showed less than 30% inhibition: 5Hg2 / 5Lg6 and 5Hg4 / 5Lg4, were also re-expressed and purified for further characterization. All 12 clones selected for further characterization were also identified as hits in the Fab-ZAP cell killing assay. [Diagram 3] Figure 1: Primary screening of unpurified fully human phage-displayed IgG in the Fab-ZAP cell killing assay of NCI-H929 cells identified a panel of clones that showed ≥15% cell killing. All unpurified clones were screened at a final assay concentration of 3.13%. Ten clones (●) were re-expressed and purified for further characterization. [Figure 4] Figure 3: Primary screening of unpurified fully human phage-displayed IgG in binding assays on MM.1S cells identified clones that bound with a fluorescence intensity of over 1 million FLU. All unpurified clones were tested at a final assay concentration of 1.56%. The binding assay data was used in conjunction with the Fab-ZAP assay data (Figure 3) to select a panel of 10 clones (●) that were hits in the Fab-ZAP assay and showed good binding (>1 million FLU) to MM.1S cells. C0120910 went on to show binding when tested at a final assay concentration of 25% (data not shown). [Figure 5A]Figure 5A shows the measurement of binding specificity of humanized leads expressed in human IgG1 Alya vectors. Figure 5 shows the ability of leads to bind to expi293 cells transiently transfected with human SEMA4A (i), mouse SEMA4A (ii), cynomolgus monkey SEMA4A (iii), human SEMA4B (iv) and mock-transfected expi293 cells (v) measured using Mirrorball. Lead binding was compared to mouse 5E3 expressed as human IgG1 Alya. C0021144 IgG was included as a positive control for binding to mock-transfected expi293 cells. A commercially available anti-SEMA4B polyclonal antibody (R&D Systems, AF5485) was included as a positive control for binding to human SEMA4B-transfected expi293 cells. See Example 4.1 for description of human IgG1 Alya vectors. [Figure 5B] FIG. 5B depicts measurements of binding specificity of fully human phage display leads. [Figure 6A] Figure 6A shows the measurement of the ability of humanized leads expressed in human IgG1 Alya vectors to kill NCI-H929 cells in a Fab-ZAP cell killing assay. Figure 6 compares the cell killing ability of all clones to mouse 5E3 in human IgG1 Alya vectors. Many humanized clones showed IC50 values ​​comparable to the mouse 5E3 antibody (VH and VL SEQ ID NOs: 2 and 11). Some humanized clones did not show complete cell killing. For a description of the human Alya IgG1 vector, see Example 4.1. [Figure 6B] FIG. 6B shows measurement of the ability of fully human leads expressed in human IgG1 Alya vectors to kill NCI1-H929 cells in a Fab-ZAP cell killing assay. [Figure 7]Figure 1 shows the testing of humanized leads expressed in human IgG1 Alya vectors in an epitope competition assay. Increasing concentrations of humanized and fully human phage display leads expressed in human IgG1 Alya vectors were used to measure their ability to inhibit binding of a fixed concentration of mouse 5E3 to SEMA4A endogenously expressed on MM.1S cells. The fixed concentration of 5E3 was mouse IgG1 isotype and the binding was detected using an anti-mouse antibody labeled with Alexa Fluor 647. Mouse 5E3 expressed as a human isotype (human IgG1 Alya) was competed in the assay and the test clones were compared to this. All humanized clones showed some level of competition with 5E3. [Figure 8A] Figure 8 shows epitope binning. Epitope binning was performed using an OctetRED (Pall ForteBio) instrument as described in section 4.6 to group lead anti-human SEMA4A clones into bins based on binding to recombinant human SEMA4A. In a tandem format, antigen (biotinylated recombinant human SEMA4A) immobilized on a streptavidin biosensor was presented to two competing analytes in successive steps. Binding to distinct, non-overlapping epitopes was indicated when saturation with the first Fab fragment (mouse 5E3) did not block binding of the second fully human phage display-derived IgG. All humanized leads were shown in section 3.2 to compete with the 5E3 epitope. Figure 8A represents data for fully human clones with epitopes that overlap or compete with 5E3. [Figure 8B] FIG. 8B shows data corresponding to fully human clones with non-overlapping or non-competing epitopes with 5E3. [Figure 8C] FIG. 8C shows data corresponding to fully human clones with non-overlapping or non-competing epitopes with 5E3. [Figure 8D] FIG. 8D shows data corresponding to fully human clones with non-overlapping or non-competing epitopes. [Figure 9A]Figure 9A measures the binding specificity of humanized leads expressed in human IgG1 Alya vectors and conjugated to McMMAF. Figure 9 measures the ability of leads to bind expi293 cells transiently transfected with human SEMA4A (i), human SEMA4B (ii) and mock-transfected expi293 cells (iii) using Mirrorball. Binding was compared to human IgG1 expressed in Alya vectors (Alya IgG1) and mouse 5E3 as McMMAF bound. C0021144 IgG was included as a positive control for binding to mock-transfected expi293 cells; C0021144 was not conjugated to McMMAF. A commercially available anti-SEMA4B polyclonal antibody (R&D Systems, AF5485) was included as a positive control for binding to human SEMA4B transfected expi293 cells; AF5485 was not conjugated to McMMAF. See Example 4.1 for a description of the human IgG1 Alya vector. [Figure 9B] FIG. 9B measures the binding specificity of fully human leads expressed in human IgG1 Alya vectors and conjugated to McMMAF. [Figure 10A] Figure 10A shows the ability of humanized leads conjugated to McMMAF expressed in human IgG1 Alya vector to kill NCI-H929 cells measured in a cell killing assay. Figure 10 compares the cell killing ability of all clones to mouse 5E3 as Alya IgG1 conjugated to McMMAF. Humanized clones showed a range of IC50 values ​​across cell lines, and no cell killing was observed in K562 cells, a negative control cell line for SEMA4A expression. See Example 4.1 for description of human Alya IgG1 vector. J6M0 anti-BCMA monoclonal antibody control (Tai et al., 2014). [Figure 10B]FIG. 10B shows the ability of humanized leads conjugated to McMMAF expressed in human IgG1 Alya vectors to kill MM1.S cells measured in a cell killing assay. [Figure 10C] FIG. 10C shows the ability of humanized leads conjugated to McMMAF expressed in human IgG1 Alya vector to kill KARPAS 25 cells measured in a cell killing assay. [Figure 10D] FIG. 10D shows the ability of humanized leads conjugated to McMMAF expressed in human IgG1 Alya vector to kill K562 cells measured in a cell killing assay. [Figure 11A] Figure 11A shows the measurement of the ability of fully human leads conjugated to McMMAF expressed in human Alya IgG1 vector to kill NCI-H929 cells in a cell killing assay. Figure 11 compares the cell killing ability of all clones to mouse 5E3 as Alya IgG1 conjugated to McMMAF. Fully human clones showed a range of IC50 values, some of which were slightly more potent than mouse 5E3 as Alya IgG1 conjugated to McMMAF. As expected, the clones did not induce cell killing of the negative control cell line for SEMA4A expression in K562 cells. See Example 4.1 for a description of the human Alya IgG1 vector. J6M0 anti-BCMA monoclonal antibody control (Tai et al., 2014). [Figure 11B] FIG. 11B shows measurement of the ability of fully human leads conjugated to McMMAF expressed in human Alya IgG1 vectors to kill MM1.S cells in a cell killing assay. [Figure 11C] FIG. 11C shows measurement of the ability of fully human leads conjugated to McMMAF expressed in human Alya IgG1 vectors to kill K562 cells in a cell killing assay. [Figure 12A]Figure 12 shows cellular internalization of McMMAF-conjugated humanized and fully human leads expressed in human Alya IgG1 vector. Internalization of each lead and control antibody was assessed over a range of concentrations over 24 hours. For description of human Alya IgG1 vector, see Example 4.1. Figure 12A plots the percentage of cells scored as positive for Fab-pHast over time for each concentration of each antibody in the same manner as shown here for mouse 5E3 human Alya IgG1. [Figure 12B] As shown in FIG. 12B for the 2.5 nM 5E3 (mouse 5E3 human Alya IgG1 McMMAF binding) data, each time course was subjected to area under the curve (AUC) analysis. [Figure 12C] FIG. 12C shows internalization titration curves plotted from AUC analysis for a panel of humanized lead and fully human clone (all human Alya IgG1 McMMAF conjugate format) monoclonals. [Figure 12D] FIG. 12D shows that the mean integrated staining intensity for each concentration and time point was also extracted and plotted as fold change in signal relative to baseline. [Figure 13] Figure 1 shows the determination of binding affinity (KD) of SEMA4A-specific human antibody (Alya IgG1 conjugated to McMMMAF) by flow cytometry. KD values ​​of all clones were compared to human Alya IgG1 and murine 5E3 bound by McMMAF. Humanized leads, 5Hg1 / 5Lg1 and 5Hg2 / 5Lg2, showed similar KD values ​​to murine 5E3. Fully human phage display derived leads had lower affinity than murine 5E3. See Example 4.1 for description of human Alya IgG1 vector. [Figure 14]Figure 1: Measurement of binding specificity of fully human germlined leads expressed in human Alya IgG1 vector. The ability of germlined leads to bind to expi293 cells transiently transfected with human SEMA4A (i), mouse SEMA4A (ii), cynomolgus monkey SEMA4A (iii), human SEMA4B (iv), and mock-transfected expi293 cells (v) was measured using Mirrorball. Binding was compared to mouse 5E3 (human Alya IgG1 not conjugated to McMMAF). C0021144 IgG was included as a positive control for binding to mock-transfected expi293 cells. A commercially available anti-SEMA4B polyclonal antibody (R&D Systems, AF5485) was included as a positive control for binding to human SEMA4B-transfected expi293 cells. All six fully human germline clones tested showed strong binding to human, mouse and cynomolgus SEMA4A, and no binding to human SEMA4B and mock-transfected expi293 cells. See Example 4.1 for a description of the human Alya IgG1 vector. [Figure 15A] Figure 15 shows low expression of soluble SEMA4A in serum in both healthy and myeloma patients. Figure 15A shows the standard curve of an ELISA using recombinant human SEMA4A (rSEMA4A). The lower limit of detection (LLOD) was determined as the mean of the blank samples plus three standard deviations of the blank. The detection limit was calculated as the lowest concentration tested above the LLOD (1.56 ng / mL). [Figure 15B] FIG. 15B shows that sSEMA4A was detected by ELISA in the serum of healthy and myeloma patients and found to be 3.3 ng / ml and 8.1 ng / ml, respectively. [Figure 16]Figure 1 shows an evaluation of the effect of soluble SEMA4A on the potency of mouse 5E3 and fully human leads in a cell killing assay using NCI-H929 cells. Concentrations of sSEMA4A up to 100ng / ml did not significantly affect the ability of either mouse 5E3 or fully human clones as Alya IgG1 conjugated with McMMAF, respectively, to kill NCI-H929 cells in terms of IC50 values. For a description of the human Alya IgG1 vector, see Example 4.1. [Figure 17] FIG. 1 shows comparative sequence alignment of SEMA4A lead panel VH domains aligned with mouse 5E3 VH sequence. Individual VH CDR sequences are in bold, on a grey background, and highlighted with black bars. SEQ ID NO: as defined by Kabat and Wu (1991). [Figure 18] FIG. 1 shows comparative sequence alignment of SEMA4A lead panel VL domains aligned with mouse 5E3 VL sequence. Individual VL CDR sequences are highlighted in bold, grey background and black bars. SEQ ID NO: defined by Kabat and Wu (1991). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0123] Working Example Example 1: Humanization of 5E3 mAb 1.1 Rational design of 5E3 humanized germline variants Anti-human SEMA4A monoclonal mouse IgG1(lambda)5E3 was purchased from BioLegend® (catalog number: 148402) and its primary amino acid sequence was reverse engineered. Specifically, 5E3 was digested with either trypsin, chymotrypsin, or a combination of trypsin and AspN. Each of the three digests was analyzed by LC / MS / MS using an Orbitrap Fusion mass spectrometer (Thermofisher Scientific). The resulting data was searched using Peaks software and a database of mouse germline sequences, followed by manual interpretation.

[0124] 10V H and 7 V L A panel of human germline sequences was selected as a framework for mouse 5E3 humanization. The selection of human germlines was guided by aligning the original non-humanized mouse 5E3 sequence to all human germline sequences listed in the IMGT® database (www.IMGT.org). Human germlines with the highest amino acid sequence homology to the mouse 5E3 sequence were prioritized for subsequent 5E3 CDR grafting. Specifically, the human germlines were selected from the "3xV H CDR Graft, 3xV L CDR graft" or "1xV H Each V was engineered to be either "CDR grafted" or "CDR grafted" (Table 4). H and V L The gene sequences were synthesized de novo and cloned into an appropriate IgG expression vector (Persic, et al., 1997) using standard molecular biology techniques.

[0125] [Table 4]

[0126] [Table 5]

[0127] 1.2 High-throughput expression of crude IgG A panel of humanized 5E3 clones was expressed as huIgG1 in a high-throughput manner (screening in production format or SiPF) using Expi293TMF cells according to the Life Technologies Expi293 Expression System protocol for 96-well microtiter plates (protocol number CO25793 0912). Ten humanized 5E3 V clones cloned into pEU1.3 were Hand seven humanized 5E3 V cloned into either the pEU3.4 (kappa) or pEU4.4 (lambda) vectors. L Purified DNA from each of the constructs was analyzed using 70 possible V H xV L The combined DNA was diluted in OptiMEM and used to transfect Expi293TMF cells in a 96 deepwell block (Merck, Cat# AXYPDW20CS) using Expi293TMF as the transfection reagent. Cells were grown at 37°C / 8%CO2 / 80% humidity with shaking at 1000 rpm and fed (with Enhance 1 and Enhance 2) 18 hours after transfection. IgG was harvested 4 days after transfection and used as unpurified undiluted IgG in subsequent assays.

[0128] Example 2: Isolation of fully human anti-SMA4A antibodies from a naive library by phage display 2.1 Production of Recombinant Human, Cynomolgus and Mouse SEMA4A Recombinant human SEMA4A (UniProt ID Q9H3S1, amino acids 33-683), recombinant cynomolgus SEMA4A (UniProt ID G7NV79, amino acids 32-679) and recombinant mouse SEMA4A (UniProt ID Q62178, amino acids 33-682) with C-terminal Avi-tag and His10-tag (SEQ ID NOs: 616, 618, 617, respectively) were cloned into the pcDNA3.1 vector and transiently expressed in the Expi293 expression system. Proteins were subsequently purified using a HisTrap HP His-tag protein purification column (40 mM imidazole wash, 400 mM imidazole elution). Proteins (75 kDa human SEMA4A, 79 kDa cynomolgus SEMA4A and 79 kDa mouse SEMA4A) were buffer exchanged into 50 mM bicine pH 8.3 and biotinylated using BirA biotin ligase (3 mg biotin ligase per 10 nmol substrate protein). Finally, proteins were buffer exchanged into Dulbecco's phosphate buffered saline (DPBS) containing 5% (w / v) trehalose (pH 7).

[0129] [Table 6]

[0130] [Table 7]

[0131] 2.2 Phage display selection to isolate SEMA4A-specific fully human scFv Soluble phage display selections were performed using five naive libraries (nFL, DP47, CS, BMV, and EG3) cloned into a phagemid vector based on the filamentous phage M13 (Vaughan et al., 1996; Lloyd et al., 2008). Anti-SEMA4A scFv antibodies were isolated from the phage display library using a series of selection cycles against recombinant biotinylated human and mouse SEMA4A proteins (avi-SEMA4A-His10, in-house) essentially as previously described (Hawkins et al., 1992; Vaughan et al., 1996). Briefly, for the initial solution-phase selection, biotinylated human SEMA4A in DPBS pH 7 was added to purified phage particles pre-incubated for 1 h in Marvel-PBS (3% w / v) containing streptavidin-conjugated paramagnetic beads (Dynabeads® M280, Invitrogen Life Sciences, UK) (final concentration 100 nM biotinylated human SEMA4A). The streptavidin beads were removed before the addition of antigen. Phage particles bound to biotinylated human SEMA4A were captured using fresh streptavidin-conjugated paramagnetic beads, and weakly bound phage were removed by a series of washing cycles with PBS-Tween (0.1% v / v). Bound phage particles were eluted from the beads using trypsin (final concentration of 10 μg / ml diluted in 0.1 M sodium phosphate buffer; pH 7), infected into E. coli TG1 bacteria, and rescued for the next round of selection (Vaughan et al., 1996). Two subsequent rounds of selection were performed as described above, but with reduced concentrations of biotinylated SEMA4A antigen; specifically, 50 nM biotinylated human or mouse SEMA4A in round 2 and 25 nM biotinylated human SEMA4A in round 3. Mouse antigen was introduced in round 2 to drive mouse cross-reactivity.

[0132] 2.3 Identification of SEMA4A-specific fully human scFv (unpurified) using a direct binding assay on cells (Mirrorball) To determine whether the unpurified scFvs from the periplasmic preparations could bind to cell surface-expressed human SEMA4A, they were incubated with expi293 cells transiently transfected with human SEMA4A (Origene, US; Catalog No. SC323738). Binding of the unpurified scFvs was detected using two antibodies: a mouse anti-c-myc antibody (Bio-Rad, US; Catalog No. MCA2200GA) that binds to the myc tag expressed on the scFv, followed by an anti-mouse antibody conjugated to Alexa Fluor 647 (Invitrogen, US; Catalog No. A21235).

[0133] Selection outputs were screened at a single concentration as crude bacterial periplasmic extracts containing scFvs, prepared in 200 mM Tris buffer pH 7.4, 0.5 mM EDTA and 0.5 M sucrose. 10 μl of crude scFv samples were added to a Greiner® 384 well assay plate (Greiner Bio-one, UK; Catalogue No: 781906). This was followed by the addition of 10 μl of antibody detection mix containing 8 nM mouse anti-c-myc antibody conjugated to Alexa Fluor 647 and 8 nM anti-mouse antibody, and 20 μl of cell suspension at 75,000 cells / ml.

[0134] Non-specific binding wells (negative controls) were defined for each plate by substituting a negative control unpurified scFv for the test scFv samples. Non-specific binding clones were identified using a parallel assay with mock-transfected expi293 cells (i.e., human SEMA4A DNA was omitted during transfection).

[0135] All dilutions were performed in Hanks Balanced Salt Solution (Sigma, UK; Catalogue No: H8264) (assay buffer) containing 0.1% bovine serum albumin (BSA) (Sigma, UK; Catalogue No: A9576). Assay plates were incubated in the dark at room temperature for 4 hours before being read on a Mirrorball fluorescence cytometer (SPT Labtech, UK) using a 640 nm laser to measure excitation and emission in the FL-4 channel (650 and 690 nm). Data were analysed as counts x median mean intensity (xFLU).

[0136] 226 unique sequence hits were identified in direct binding assays reformatted from scFv to human IgG1 as described in section 2.4.

[0137] 2.4 Reformatting of SEMA4A-binding antibodies from fully human scFv to IgG1 (unpurified IgG) 226 crude scFv periplasmic extracts that showed specific binding to human SEMA4A by Mirrorball assay were subjected to DNA sequencing (Osbourn et al., 1996; Vaughan et al., 1996). 226 unique scFvs were pooled into lambda and kappa pools based on their sequences. The total output was further subjected to the following cloning strategy. A two-step strategy was used to batch convert the scFvs to IgG, first replacing the (G4S)3scFv linker with a DNA segment encoding the hinge and constant domains of the heavy chain (HC) and the promoter and signal sequence of the light chain (LC), and then replacing the scFv backbone with the LC constant domain and HC signal sequence in an IgG vector (as described in Xiao et al., 2017). The final expression vector is bicistronic with both HC and LC under the control of the CMV promoter. The converted IgG was then expressed as crude IgG as described in section 3.2.

[0138] Example 3: Functional screening of crude 5E3 humanized and fully human, phage display derived, IgG1 in primary screening Example 3 describes the primary screening of unpurified IgG1 (section 1.2) from the 5E3 humanization approach (Example 1) and the phage display approach (Example 2). Both sets of IgG were screened for function in a Fab-ZAP cell killing assay. The 5E3 humanized IgGs were also screened for their ability to compete for binding to the 5E3 epitope in a mouse 5E3 epitope competition assay. Fully human phage display derived clones were also screened for their ability to bind to MM.1S multiple myeloma cells that endogenously express human SEMA4A.

[0139] 3.1 Discovery of mAbs capable of internalizing and killing NCI-H929 cells using the Fab-ZAP assay The Fab-ZAP assay assessed the ability of unpurified IgG to bind, internalize, and kill NCI-H929 multiple myeloma cells in vitro using a secondary saporin conjugate, Fab-ZAP (Advanced Targeting Systems, San Diego, CA, IT-51), CellTiter Glo 2.0 (Promega, Madison, WI, G9248), and a Pherastar plate reader (BMG Labtech, Germany). Fab-ZAP is an anti-human Fab conjugated to a warhead saporin. Unpurified humanized 5E3 and fully human phage display-derived human IgG1 were prediluted 8-fold and 4-fold, respectively. Unpurified human IgG1 was contained in Expi293 expression medium (Gibco, UK, A1435101). After 8-fold dilution into the assay, the final assay concentrations were 1.56 and 3.13%, respectively. 5 μl of pre-diluted unpurified human IgG1 was pre-incubated with 5 μl of 80 nM Fab-ZAP for 30 min at room temperature in a 384-well assay plate (Greiner Bio-one, UK, 781091). 2,000 NCI-H929 cells were added to the assay wells in a volume of 30 μl. The assay plate was incubated for 72 h at 37° C. / 5% CO2 / 95% humidity. Cell death was assessed by adding 20 μl of CellTiter Glo 2.0 followed by a 10 min incubation before reading the plate for luminescence on a Pherastar. All dilutions were performed in RPMI 1640 (Gibco, UK, A10491-01) supplemented with 10% fetal bovine serum (FBS) (Gibco, UK, 10500-064) and 55 μM β-mercaptoethanol (Gibco, UK, 21985-023). Maximum assay signal was defined by wells containing only cells. Background assay signal was defined by wells containing only buffer, i.e., no cells. Data were analyzed and graphed using GraphPad Prism software (GraphPad Software, Inc, La Jolla, CA). Results of this experiment are shown in Figures 1 and 3.

[0140] 3.2 Use an IgG epitope competition assay to demonstrate that the mAb has an overlapping epitope with mouse 5E3 The 5E3 epitope competition assay assessed the ability of unpurified IgG expressed in a human IgG1 backbone to compete for binding to the mouse 5E3 epitope on human SEMA4A endogenously expressed by MM.1S cells, a human multiple myeloma cell line. The assay utilized a secondary Alexa Fluor 647 conjugate, 5E3 expressed in a mouse IgG1 (lambda) backbone (BioLegend®, Diego, CA, 148402), and a Mirrorball fluorescence cytometer (SPT Labtech, UK). Binding of 5E3 mouse IgG1 to MM.1S cells was measured via a secondary Alexa Fluor 647 (Invitrogen, A21235) labeled anti-mouse in the presence of a single concentration of each unpurified human IgG1. Occupancy of the 5E3 epitope on SEMA4A by unpurified IgG resulted in a decrease in the fluorescent signal as measured by Mirrorball. 10 μl of undiluted unpurified IgG in Expi293 expression medium (Gibco, UK, A1435101) was added to an assay plate (Greiner Bio-one, UK, 781906), followed by 10 μl of 1.2 nM 5E3 mouse IgG1 containing 1,500 cells spiked with 4 nM anti-mouse Alexa Fluor 647 and 20 μl of MM.1S cell suspension. The assay plate was incubated at room temperature for 4 h in the dark and then read on a Mirrorball fluorescence cytometer (SPT Labtech, UK) using a 640 nm laser to measure excitation and emission in the FL-4 channel (650-690 nm). Data were analyzed as counts x median mean intensity (xFLU). All dilutions were performed in Hanks Balanced Salt Solution (Sigma, UK; Catalogue No.: H8264) containing 0.1% (w / v) BSA (Sigma, UK; Catalogue No.: A9576). Maximum binding signal was determined by analyzing the binding of 5E3 mouse IgG1 to MM.1S cells in the absence of competitor unpurified human IgG1. Background binding signal was determined by analyzing the fluorescence generated in the absence of 5E3 mouse IgG1.Data were analyzed and graphed using GraphPad Prism software (GraphPad Software, Inc, La Jolla, Calif.) The results of this experiment are shown in FIG.

[0141] 3.3 Demonstration of specific binding of mAbs to the MM1.S cell line using the Mirrorball binding assay The MM.1S binding assay assessed the ability of unpurified human IgG1 to directly bind to human SEMA4A endogenously expressed by MM.1S cells. Direct binding of a single concentration of unpurified human IgG1 to MM.1S cells was measured via an anti-human secondary antibody labeled with Alexa Fluor 647 (Invitrogen, A21445). 10 μl of undiluted unpurified IgG in Expi293 expression medium (Gibco, UK, A1435101) was added to an assay plate (Greiner Bio-one, UK, 781906), followed by the addition of 10 μl of 8 nM anti-human Alexa Fluor 647 and 20 μl of MM.1S cell suspension containing 1,500 cells. The assay plates were incubated in the dark for 4 h at room temperature and then read on a Mirrorball fluorescence cytometer (SPT Labtech, UK) using a 640 nm laser to measure excitation and emission in the FL-4 channel (650-690 nm). Data were analyzed as counts x median mean intensity (xFLU). All dilutions were performed in Hanks' balanced salt solution (Sigma, UK; Catalogue number: H8264) containing 0.1% (w / v) BSA (Sigma, UK; Catalogue number: A9576). Data were analyzed and graphed using GraphPad Prism software (GraphPad Software, Inc, La Jolla, CA). The results of this experiment are shown in Figure 4.

[0142] Eighteen humanized clones were identified as hits in the Fab-ZAP assay (cell killing > 30%) and 17 humanized clones were identified as hits in the mouse 5E3 epitope competition assay (inhibition > 30%). Twelve clones that were hits in both assays and two clones (5Hg2 / 5Lg6 and 5Hg4 / 5Lg4) that were only Fab-ZAP hits were re-expressed and purified for further characterization.

[0143] Forty-nine fully human phage display derived clones were identified as hits in the Fab-ZAP assay (cell killing > 15%). The 49 clones were further triaged based on whether they bound to MM.1S cells that endogenously express human SEMA4A. This resulted in a panel of 10 clones that were hits in the Fab-ZAP assay and bound to MM.1S cells. These 10 clones were reformatted from human IgG1 to human Alya IgG1, expressed and purified for further characterization as described in section 4.1.

[0144] Example 4: Secondary screening of purified human Alya IgG1 4.1 Reformatting SEMA4A-binding antibodies into human Alya IgG1 The specific variable fragment expresses a human IgG1-ADC isotype (Alya IgG1) consisting of a human IgG1 constant chain with modifications to introduce two cysteines into each heavy chain fragment to which drug molecules can be covalently conjugated, resulting in a "drug-to-antibody" ratio of 4 (4DAR). H ) and variable light chain (V L) domains are introduced into vectors expressing the heavy and light chains of a fully human antibody, respectively. The variable heavy chain was cloned into a mammalian expression vector (pEU1.22, Alya vector) containing the human heavy chain constant domain and regulatory elements to express the entire IgG1 heavy chain in mammalian cells. pEU1.22 is engineered to contain two site-specific cysteines (239iCys and S442C (EU numbering) in the CH2 and CH3 Fc domains, respectively) that allow for controlled drug conjugation. Similarly, the variable light chain domain was cloned into a mammalian expression vector for expressing the human lambda light chain constant domain (pEU4.4) or the human kappa light chain constant domain (pEU3.4) and regulatory elements to express the entire IgG light chain in mammalian cells. The vector for the expression of the heavy chain was first described in Thompson et al., 2016. The vector for the expression of the light chain was first described in Persic, et al., 1997.

[0145] To obtain clones as IgG, heavy and light chain IgG expression vectors were transiently transfected into ExpiCHO (ThermoScientific UK; Catalogue No: A29133) cells where the antibodies were expressed and secreted into the medium. The harvested medium was filtered before purification. IgG was purified using Protein A chromatography (HiTrap Fibro PrismA, Cytiva, UK). Culture supernatants were loaded onto appropriate Protein A columns pre-equilibrated in 25 mM Tris pH 7.4, 50 mM NaCl. Bound IgG was eluted from the column using 0.1 M sodium citrate pH 3.0, 100 mM NaCl. IgG was buffer exchanged into PBS. Purified IgG was passed through a 0.2 μm filter and the concentration of IgG was determined by absorbance at 280 nm using an extinction coefficient based on the amino acid sequence of IgG. Purified IgG was analyzed for aggregation or degradation using SEC-HPLC and SDS-PAGE techniques.

[0146] 4.2 Confirmation of the binding profile of SEMA4A-specific human Alya IgG (purified, unconjugated) using a direct binding assay (Mirrorball) Hits from the primary screen were reformatted into the human Alya IgG1 vector, re-expressed and purified as described in section 4.1. H A single clone was identified that contained an asparagine deamidation motif (Asn Gly) within CDR2. The reliability of this potential sequence was confirmed by the de novo two-clonal variant V H The risk was eliminated by synthesizing the genes (Asn Ala and Gln Gly) and cloning these sequences into a human Alya IgG1 expression vector. To determine the specificity profile of each purified human Alya IgG1, a dilution series was tested for binding to human SEMA4A, mouse SEMA4A, cynomolgus monkey SEMA4A, human SEMA4B and mock transfected cells (transient transfection set up in expi 293 cells). Five assays were performed using the methods described in section 2.3, using the appropriate cells and substituting the recombinant human Alya IgG1 dilution series for the unpurified scFv periplasmic preparation. Data were analyzed as counts x median mean intensity (xFLU).

[0147] A further modification of the assay was to replace the mouse anti-c-myc and anti-mouse antibodies conjugated to Alexa Fluor 647 with anti-human Alexa Fluor 647 detection reagent (ThermoFisher Scientific, UK; Catalogue number: A21445), as recombinant human Alya IgG1 did not have a c-myc tag.

[0148] The required binding profile was binding to human SEMA4A, mouse SEMA4A, cynomolgus monkey SEMA4A, but not to human SEMA4B and mock-transfected expi293 cells. The results of this experiment are shown in Figure 5.

[0149] 4.3 Confirmation of cell killing capacity of SEMA4A-specific human Alya IgG1 (purified, unconjugated) using Fab-ZAP cell killing assay Hits from the primary screen that were reformatted into human Alya IgG1 vector and re-expressed and purified as described in section 4.1 were tested in the Fab-ZAP cell killing assay. The Fab-ZAP assay method described in Example 3.1 was used to test purified human Alya IgG1. Purified human Alya IgG1 was diluted to an 8x stock and then serially diluted using 4x dilutions to generate a 12-point concentration response curve. 5μl of the concentration response curve was transferred in duplicate to the assay plate in place of unpurified IgG. The ability of humanized and fully human leads expressed in human Alya IgG1 vector to kill NCI-H929 cells in the Fab-ZAP cell killing assay is shown in Figures 6A and 6B, respectively. Table 6 shows the cell killing ability of humanized lead clones compared to mouse 5E3 in human IgG1 Alya vector. A number of humanized clones showed ICs comparable to 5E3. 50 The values ​​were shown.

[0150] [Table 8]

[0151] 4.4 IgG competition assay to assess epitope binding of SEMA4A-specific Alya IgG (purified, unconjugated) Hits from the primary screen were reformatted into the human Alya IgG1 vector and re-expressed and purified as described in section 4.1. The 5E3 epitope competition assay method described in section 3.2 was used to test purified human Alya IgG1. Purified human Alya IgG1 was diluted to a 4x stock and then serially diluted using 4x dilutions to generate a 10-point concentration response curve. 10 μl of the concentration response curve was transferred in duplicate to the assay plate in place of unpurified IgG. The results of this experiment are shown in Figure 7 and Table 7.

[0152] [Table 9]

[0153] 4.5 Generation of Fab fragments to enable improved variant kinetic profiling Equivalent to the conversion of scFvs to IgG1-ADCs (section 4.1), the scFvs were also converted to Fab fragments, containing the variable heavy chains (V H ) and variable light chain (V L The variable heavy chain was subcloned into vectors expressing portions of human antibody heavy and full light chains, respectively. The variable heavy chain was cloned into a mammalian expression vector (pEU1.3 Fab) containing the CH1 human heavy chain constant domain, a portion of the hinge region, and the necessary regulatory elements to express Fab heavy chain fragments in mammalian cells. Similarly, the variable light chain domain was cloned into a mammalian expression vector for expressing the human lambda light chain constant domain (pEU4.4) or the human kappa light chain constant domain (pEU3.4) and regulatory elements to express full IgG light chains in mammalian cells.

[0154] To obtain clones as Fabs, Fab heavy and light chain IgG expression vectors were transiently transfected into ExpiCHO cells (ThermoScientific UK; Catalogue number: A29133), where the Fab fragments were expressed and secreted into the culture medium. The harvested medium was filtered before purification. The Fab fragments were purified using an affinity matrix (Capture Select CH1-XL Columns, ThermoFisher Scientific, UK) that recognizes the CH1 domain of human IgG antibodies. The culture supernatant was loaded onto the appropriate Capture Select column pre-equilibrated in PBS. The bound Fab fragments were eluted from the column using 50 mM sodium acetate buffer pH 4.0-4.5. The Fab fragments were buffer exchanged into PBS. The concentration of the Fab fragments was determined by passing the purified Fab fragments through a 0.2 μm filter and measuring the absorbance at 280 nm using an extinction coefficient based on the amino acid sequence of the Fab fragment. The purified Fab fragments were analyzed for aggregation or degradation using SEC-HPLC and SDS-PAGE techniques.

[0155] 4.6 Determining the Binding Affinities of the 5E3 Humanized and Fully Human Lead Panel to Human SEMA4A and Cynomolgus SEMA4A Using Biolayer Interferometry The OctetRED (Pall ForteBio) instrument was used to evaluate the kinetic parameters of the interaction between the lead anti-SEMA4A IgG and recombinantly produced human SEMA4A and cynomolgus SEMA4A. The Octet biosensor uses an optical analysis technique that analyzes the interference pattern of white light reflected from two surfaces: a layer of immobilized protein on the sensor tip and an internal reference layer. Changes in binding at the biosensor tip result in a shift in the interference pattern, which can be measured in real time. Molecules that associate with or dissociate from the ligand at the biosensor tip shift the interference pattern and generate a response on the Octet system that is recorded by the acquisition software.

[0156] Typically, a given concentration of analyte is contacted with the coupled ligand and any binding is detected as an increase in signal (association phase). This is followed by a buffer rinse during which dissociation of the analyte from the surface-immobilized ligand can be observed as a decrease in signal (dissociation phase). This is repeated with a range of analyte concentrations to provide data for analysis of binding kinetics.

[0157] Octet Kinetics buffer (PBS containing 0.01% (v / v) BSA and 0.002% (v / v) Tween 20) is typically used as dilution buffer for analyte samples and as flow buffer during the dissociation phase. Experimental data are recorded as a shift in the interference pattern (nm) over time, which is directly proportional to the optical thickness of the biosensor tip and is in turn an approximate measure of the mass of bound analyte. The proprietary Octet Data Analysis software package can then be used to process the data and fit binding models to the data sets. The back association (ka, M-1s-1) and dissociation (kd, s-1) rate constants are expressed as the dissociation (K D , M) affinity constants can be calculated.

[0158] Binding affinities between analytes (human Alya IgG1 and humanized and phage display derived IgG as Fab) and human SEMA4A and cynomolgus SEMA4A were estimated using an assay in which biotinylated SEMA4A antigen was captured on a streptavidin sensor tip. A new sensor chip was used for each measurement and no regeneration was used. A series of dilutions (3.25-240 nM) of lead Fab and human Alya IgG1 were individually contacted with the ligand surface for a sufficient time (typically 5 min) to observe sensorgrams that could be fitted to a reliable and appropriate binding model, followed by an appropriate dissociation time (typically 10 min). Blank reference (0 nM analyte) data were subtracted from each data set to reduce the impact of any buffering artifacts or non-specific binding effects. A 1:1 binding model was then fitted simultaneously to the data from each analyte titration using Octet Evaluation software (Table 8).

[0159] [Table 10]

[0160] 4.7 Epitope Binning of Antibodies from Humanized and Fully Human Antibody Panels Using Octet Epitope binning was performed using an OctetRED (Pall ForteBio) instrument as described in section 4.6 to group lead anti-human SEMA4A clones into bins based on their binding to recombinant human SEMA4A. This grouping was performed using a cross-competition assay to characterize the competitive binding of analyte pairs to a specific antigen. In this in-tandem format, an antigen (biotinylated recombinant human SEMA4A) immobilized on a streptavidin biosensor is presented to two competing analytes in successive steps. Binding to distinct, non-overlapping epitopes is indicated if saturation with the first Fab fragment (mouse 5E3) does not block binding of the second IgG.

[0161] A predefined concentration of analyte is contacted with the coupled ligand and any binding is detected as an increase in signal (association phase). Epitope binning is achieved by saturating this first association (typically measured for 5 min during affinity measurements) followed by a second association step in which the sensor is immersed in a well containing both the first analyte (mouse 5E3 Fab) and the second analyte (naive SEMA4A IgG) (typically 5 min). The first analyte is included to maintain saturation while the second analyte attempts to bind. This is followed by a suitable length of dissociation time (typically 10 min). A new sensor chip was used for each measurement and no regeneration was used.

[0162] The results of this experiment are shown in Figure 8. Figure 8A represents data generated from two clones with overlapping or competing epitopes, while Figures 8B-D show data corresponding to clones with non-overlapping epitopes.

[0163] Example 5: Conjugation of linker and warhead (McMMAF) to human Alya IgG1 5.1 Conjugation of SEMA4A-specific human Alya IgG1 to McMMAF Humanized and fully human leads were cloned into the human Alya IgG1 vector, expressed, purified, and concentrated to 10 mg / ml in DPBS, pH 7. Each protein was gently reduced using TCEP (Tris-(2-carboxyethyl)-phosphine hydrochloride, Sigma Cat. No.: 75259) at 40-fold molar excess of TCEP to antibody for 3 hours at 37° C. After desalting into PBS, pH 7.2, 1 mM EDTA (Zeba™ Spin Desalting Columns, ThermoFisher Scientific, Cat. No.: 89882), IgG was oxidized using dhAA (dehydroascorbic acid, Sigma Cat. No.: 261556) at 20-fold molar excess of dhAA to antibody for 4 hours at room temperature. MMAF conjugation was performed overnight at 4° C. using McMMAF (MedChemExpress, Cat. No.: HY-15578) at 12-fold molar excess of MMAF to antibody. The reaction was stopped using a 4-fold molar excess of NAC (N-acetyl-L-cysteine, Sigma-Aldrich, Catalog No.: A7250) for 15 min at room temperature. Finally, each conjugated SEMA4A IgG (human Alya IgG1) was buffer exchanged into PBS, pH 7.2, 1 mM EDTA, concentrated, and purified using Superose 6 Increase 10 / 300 GL (Sigma-Aldrich, Catalog No.: GE29-0915-96) with PBS, pH 7.2, 1 mM EDTA. The IgGs, together with each unconjugated SEMA4A human Alya IgG1, were run on SDS PAGE (NuPAGE 4-12% BisTris gels (12 wells), ThermoFisher Scientific, Catalog No.: NP0322PK2) at 200V for 60 min to confirm conjugation.

[0164] 5.2 Reconfirmation of binding / specificity of lead panel (human Alya IgG1 conjugated to McMMAF) to SEMA4A (Mirrorball) Human Alya IgG1 clones were conjugated to McMMAF as described in section 5.1. After conjugation, it was necessary to retest these clones to reconfirm their binding profiles to human SEMA4A, human SEMA4B and mock transfected cells (transient transfection set up in expi 293 cells).

[0165] To determine the specificity profile of each purified human Alya IgG1 conjugated to McMMAF, a dilution series was tested for binding to human SEMA4A, human SEMA4B and mock transfected cells (transient transfection set up in expi 293 cells). These three assays used the methods described in section 2.3, using the appropriate cells and substituting the unpurified scFv periplasmic preparation with human Alya IgG1 conjugated to McMMAF. Data were analyzed as counts x median mean intensity (xFLU).

[0166] A further modification of the assay was to replace the mouse anti-c-myc and anti-mouse antibodies conjugated to Alexa Fluor 647 with anti-human Alexa Fluor 647 detection reagents (ThermoFisher Scientific, UK; Catalogue number: A21445) as the recombinant IgG did not have a c-myc tag.

[0167] The required binding profile was for binding to human SEMA4A, but not for binding to human SEMA4B and mock-transfected expi293 cells. The results of this experiment are shown in Figure 9.

[0168] 5.3 Cell killing assays using the SEMA4A lead panel (human Alya IgG1 conjugated to McMMAF) benchmarked against J6M0 in MM.1S, NCI-H929 and Karpas-25 cell lines The ability of SEMA4A-specific human Alya IgG1 conjugated to McMMAF to bind, internalize, and kill MM.1S, NCI-H929, and Karpas-25 multiple myeloma cells and K562 leukemia cells in vitro was assessed by cell killing assays using CellTiter Glo 2.0 (Promega, Madison, WI, G9248) and a Pherastar plate reader (BMG Labtech, Germany). The ability of the conjugates to kill all four cell lines was tested and benchmarked against McMMAF-conjugated BCMA-specific Alya IgG1, known as J6M0 (Tai et al., 2014). For these assays, three cell lines were selected as high (MM.1S), medium (NCI-H929), and low (Karpas-25) expression of human SEMA4A. K562 cells do not express human SEMA4A.

[0169] In comparing SEMA4A and BCMA expression levels, NCI-H929 cells were shown to express comparable levels of both targets (3E5 antibody binding sites per cell for both SEMA4A and BCMA as determined using Quantum™ Simply Cellular® anti-Mouse IgG beads, Catalog No: 815, Bangs Laboratories, Inc, Indiana, USA). This cell line was considered to be a suitable line to understand the efficacy of the SEMA4A panel of mAbs vs. J6M0 on cell killing and internalization rates.

[0170] McMMAF conjugates were diluted to 4x stocks and then serially diluted using 4x dilutions to generate 12-point concentration-response curves. 10 μl of the concentration-response curve was added to an assay plate (Greiner Bio-one, UK, 781091) followed by 30 μl of cell suspension containing 2,000 MM.1S, NCI-H929, Karpas-25 or K562 cells. Assay plates were incubated at 37° C. / 5% CO2 / 95% humidity for 96 hours. Cell death was assessed by adding 20 μl of CellTiter Glo 2.0 followed by a 10 minute incubation before reading the plates for luminescence on a Pherastar. All dilutions were performed in RPMI 1640 (Gibco, UK, A10491-01) supplemented with 10% FBS (Gibco, UK, 10500-064), except for assays testing Karpas-25 cells, where the assay medium was also supplemented with 55 μM β-mercaptoethanol (Gibco, UK, 21985-023). Maximum assay signal was defined by wells containing only cells. Background assay signal was defined by wells containing only buffer, i.e., no cells. Data was analyzed and graphed using GraphPad Prism software (GraphPad Software, Inc, La Jolla, CA). Results of this experiment are shown in Figure 10 and Table 9, and Figure 11 and Table 10.

[0171] [Table 11]

[0172] [Table 12]

[0173] 5.4 Internalization of Humanized and Fully Human Lead Panel (Human Alya IgG1 Conjugated to McMMAF) Benchmarked Against J6M0 in NCI-H929 Cell Line The humanized and fully human lead panel as human Alya IgG1 clones were conjugated to McMMAF as described in section 5.1. The ability of these conjugated IgGs to be internalized into a SEMA4A-expressing multiple myeloma cell line was tested and benchmarked against a McMMAF-conjugated BCMA-specific human Alya IgG1 known as J6M0 (Tai et al., 2014). BCMA (3.3x10 per cell) was expressed in 100% of the BCMA-specific cells. 5 antibody binding sites) and SEMA4A (3.2x10 per cell 5 The NCI-H929 cell line, which has equal expression of all three antibody binding sites, was chosen for this purpose.

[0174] Internalization into NCI-H929 cells was visualized using a Fab-pHast internalization assay, which was set up using known concentrations of McMMAF-conjugated Alya IgG, Fab-pHast (Advanced Targeting Systems, San Diego, CA, PH-01), Hoechst 33342 (Invitrogen, UK, H3570) and an ImageXpress Micro high content imaging system (Molecular Devices, San Jose, CA).

[0175] Fab-pHast is an anti-human secondary antibody conjugated to a pH-sensitive fluorescent label. The neutral pH of tissue culture medium quenches Fab-pHast fluorescence. Significant Fab-pHast fluorescence can only be detected when the antibody-secondary complex is internalized into an acidic compartment of the endocytic pathway.

[0176] Humanized and fully human lead panels as McMMAF-conjugated human Alya IgG1 were labeled by diluting to known concentrations in 10x Fab-pHast solution. Serial dilutions were set up in the same solution to generate titration curves before incubating the labeling reactions for 20 min at room temperature. Labeled antibodies were added at a 1:10 ratio to NCI-H929 cells pre-seeded in serum-free RPMI 1640 (Gibco, UK, A10491-01) on 96-well tissue culture plates coated with poly-D-lysine. Treated cells were incubated at 37°C / 5% CO2 / 95% humidity and imaged in the blue and yellow channels of an ImageXpress Micro at 3, 6 and 24 hours. These images were processed using the MetaXpress 6 software package (Molecular Devices, San Jose, CA) to identify objects in the blue (total nuclear Hoescht staining) and yellow (internalized Fab-pHast label) channels based on defined thresholds. The software reported both the percentage of Fab-pHast positive cells and their average integrated intensity (defined as the sum of all Fab-pHast positive pixel intensities divided by the total number of Hoescht positive nuclei). These data were analyzed and graphed using GraphPad Prism software (GraphPad Software, Inc, La Jolla, CA). Representative results from this experiment are shown in FIG. 12, and the average results of duplicate experiments are shown in Table 11 below.

[0177] [Table 13]

[0178] 5.5 Determination of Binding Affinity of Lead Panel SEMA4A-Specific IgG (Human Alya IgG1 Conjugated to McMMAF) by Flow Cytometry Binding of SEMA4A-specific IgG to membrane-bound SEMA4A was assessed using flow cytometry in NCI-H929 cells that endogenously express human SEMA4A. Binding assays were performed by incubating anti-SEMA4A antibodies with 200,000 cells for 3 hours at 4°C, followed by two washes with PBS, 1% (w / v) BSA (FACS buffer). A range of antibody concentrations was assessed using a 10-point, 3-fold dilution series. Cells were then incubated with Alexa Fluor 647 conjugated AffiniPure Fab Fragment Goat Anti-Human IgG(H+L) (Jackson Immunoresearch Europe Limited, Ely, Cambridgeshire) diluted 1:100 for 1 hour at 4°C, followed by two washes with FACS buffer. Cells were then stained with LIVE / DEAD™ Fixable Violet Dead Cell Stain (Life Technologies, L34955) for 30 minutes at 4°C, followed by two washes with FACS buffer. Cells were then fixed using BD Cell Fix (BD Biosciences, 340181). Live single-cell fluorescence was measured using a Novocyte™ 3000 flow cytometer and Novoexpress software (Acea Biosciences, Inc, San Diego, CA). Data was analyzed using FlowJo software (FlowJo, LLC, Ashland, OR). Antibody conjugate concentrations and K determined using Prism software (GraphPad Software Inc, La Jolla, CA). D The mean fluorescence intensity values ​​were plotted against the mean fluorescence intensity values. The results of this experiment are shown in Figure 13 and Table 12.

[0179] [Table 14]

[0180] Example 6: Germlining of fully human phage display-derived leads and validation of retained function 6.1 Fully human phage display-derived lead germlining Fully human lead identified from phage display selection were germlined to help mitigate potential immunogenicity risks. Individual antibody sequences were compared to human germline V, D and J regions accessible via IMGT (ImMunoGeneTics; www.imgt.org) or ImmuneDiscover databases. Amino acid residues that differed from the standard human germline sequence that were outside the VHCDR1, VHCDR2, VHCDR3, VLCDR1, VLCDR2, VLCDR3 regions and were not Vernier residues were reverted. Each fully germlined V H and V L The gene sequences were synthesized de novo and cloned into an appropriate IgG expression vector (Persic, et al., 1997) using standard molecular biology techniques.

[0181] 6.2 Confirmation of the binding specificity of germline fully human phage display derived human Alya IgG1 (purified, unconjugated) using a direct binding assay (Mirrorball) To confirm the SEMA4A binding specificity of each germline fully human, the phage display derived human Alya IgG1 dilution series was tested for binding to human SEMA4A, mouse SEMA4A, cynomolgus monkey SEMA4A, human SEMA4B and mock transfected cells (transient transfection set up in expi 293 cells). The five assays used the methods described in section 2.3 using the appropriate cells and substituting the recombinant germline human IgG1 Alya dilution series for the unpurified scFv periplasmic preparation. Data were analyzed as counts x median mean intensity (xFLU).

[0182] A further modification of the assay was to replace the mouse anti-c-myc and anti-mouse antibodies conjugated to Alexa Fluor 647 with anti-human Alexa Fluor 647 detection reagent (ThermoFisher Scientific, UK; Catalogue No: A21445) as the recombinant IgG did not have a c-myc tag. The required binding profile was binding to human SEMA4A, mouse SEMA4A, cynomolgus monkey SEMA4A, but not to human SEMA4B and mock-transfected expi293 cells. The results of this experiment are shown in Figure 14.

[0183] Example 7. Assessing serum levels of SEMA4A in MM patient samples to understand whether soluble SEMA4A (sSEMA4A) influences lead panel cell killing efficacy 7.1 Assessment of SEMA4A levels in patient samples Having determined that SEMA4A is a potential ADC target, we further explored the feasibility of targeting SEMA4A in human MM patients by measuring the serum concentration of SEMA4A. Many cell surface proteins are released into the circulation, and this soluble protein can act as a sink and "bathe" therapeutic antibodies. This means that unacceptable blood concentrations of antibodies are required to achieve efficacy. To understand whether this could interfere with therapeutic targeting of SEMA4A, we designed a sandwich ELISA to detect sSEMA4A.

[0184] White maxisorp plates (ThermoScientific, UK, 436110) were coated with 50 μl of capture antibody (mouse anti-human SEMA4A clone 5E3, BioLegend®, San Diego, CA, 148402) at 5 μg / mL in PBS and incubated overnight at 4°C. Unbound antibody was removed and plates were washed with PBS. Non-specific binding was blocked by incubation with casein (ThermoScientific, UK, 37528) for 1 h at room temperature followed by PBS washes. 50 μl / well of unknown samples or diluted standards (recombinant human SEMA4A (Abcam, UK, ab182683)) were incubated for 1-2 h at room temperature. Samples were removed and plates were washed 3 times with PBS-Tween 20 (0.1% v / v) wash buffer. Captured sSEMA4A was detected by anti-SEMA4A polyclonal antibody (R&D Systems, AF4694, 50 μl at 5 μg / ml in 1:20 casein) with 1 h incubation at room temperature. Plates were washed 3 times with wash buffer and secondary detection antibody (donkey anti-sheep IgG conjugated HRP, Abcam, ab97125) was added at 1 / 5000 in 1:20 casein (50 μl) for 1 h at room temperature. Secondary detection antibody was removed and washed 5 times with wash buffer. 50 μl of Pico ELISA substrate (Thermo Scientific, UK, 37070) was added to the plate according to the manufacturer's guidelines, incubated for 5 min at room temperature and luminescence was detected using an EnVision plate reader (PerkinElmer).

[0185] Dilution studies demonstrated that the assay has a detection limit of 2ng / mL or less (Figure 15). We assayed sSEMA4A in serum of healthy controls and myeloma patients and found these to be 3.3ng / mL and 8.1ng / mL, respectively (Figure 15). Although the difference between the two groups was significant (p=0.0022; t-test), the absolute level of sSEMA4A is highly unlikely to destroy the activity of SEMA4A ADCs in myeloma. For example, successful targeting of TNFRSF17 (BCMA) in myeloma with belantamab-mafodotin or CAR-T cells does not appear to be compromised by the median serum expression level of soluble BCMA of 176ng / mL. Thus, we conclude that shed SEMA4A is highly unlikely to affect the ability to target this cell surface protein.

[0186] 7.2 To determine the effect of sSEMA4A on the ability of fully human SEMA4A-specific human Alya IgG1 (McMMAF conjugate) to kill NCI-H929 cells. Using the cell killing assay described in section 5.3, we evaluated whether the presence of sSEMA4A affects the potency of fully human SEMA4A-specific human Alya IgG conjugated to McMMAF to kill NCI-H929 cells. sSEMA4A was derived from cell culture medium (CCM) of NCI-H929 cells concentrated 100-fold. sSEMA4A levels in CCM were quantified using the ELISA described in Example 7.1. CCM without sSEMA4A was derived from NCI-H929 SEMA4A knockout cells and used at a SEMA4A concentration of 0 ng / ml. The potency of fully human phage display-derived human Alya IgG1 conjugated to McMMAF was tested in the presence of sSEMA4A added to the assay at 0, 8, 25 and 100 ng / ml.

[0187] The results of this experiment (n=2) are shown in Figure 16. Cell killing IC of fully human phage display-derived human Alya IgG1 conjugated to McMMAF 50Values ​​are IC measured in the absence of sSEMA4A. 50 There was no significant change compared with the values.

[0188] Example 8. Expression profiles of SEMA4A, BCMA and CD22 in normal and hematological tissues 8.1 Immunohistochemistry (IHC) method SEMA4A: Sections of formalin-fixed paraffin-embedded (FFPE) tissues were cut at 4 micron thickness. Sections were deparaffinized using a Ventana Discovery autostainer and antigen retrieval was performed in cell conditioning solution or CC1 (low pH) solution at 95°C for 64 minutes. Antibody against SEMA4A (Atlas Antibody, HPA069136, rabbit polyclonal) was applied at a concentration of 0.2 μg / ml for 60 minutes. Detection was completed using the Ventana HQ kit with 3,3'-diaminobenzidine (DAB) as the chromogen and hematoxylin as the counterstain.

[0189] A variety of FFPE cell pellets, including cell lines with known SEMA4A expression, including cells overexpressing the closest family member SEMA4B, were used to optimize the staining protocol and confirm its specificity. Normal human tissues with expected SEMA4A expression (tonsil, spleen) were used to confirm the suitability of the assay for FFPE tissues.

[0190] BCMA: Sections of formalin-fixed paraffin-embedded (FFPE) tissue were cut at 4 micron thickness. Sections were deparaffinized using a Ventana Discovery autostainer and antigen retrieval was performed in CC2 (high pH) solution at 95°C for 48 minutes. Antibody against BCMA (Cell Signaling Technology #88183S, rabbit monoclonal) was applied at a concentration of 1.6 μg / ml for 60 minutes. Detection was completed using the Ventana HQ kit with DAB as chromogen and hematoxylin as counterstain. Various FFPE cell pellets, including cell lines with known BCMA expression, were used to optimize the staining protocol and confirm its specificity.

[0191] CD22: Sections of formalin-fixed paraffin-embedded (FFPE) tissues were cut at 4 micron thickness. Sections were deparaffinized using a Leica Bond autostainer and antigen retrieval was performed in ER2 (high pH) solution at 95°C for 20 minutes. Antibody against CD22 (Cell Signaling Technology #98035, rabbit monoclonal) was applied at a concentration of 0.2 μg / ml for 30 minutes. Detection was completed using the Leica Bond Refine kit with DAB as chromogen and hematoxylin as counterstain. Various FFPE cell pellets, including cell lines with known CD22 expression, were used to optimize the staining protocol and confirm its specificity.

[0192] 8.2 Evaluation of SEMA4A expression The IHC protocol was applied to a collection of normal and tumor tissues to evaluate the prevalence and intensity of expression in different tumor types of interest. Sections were examined by a certified pathologist and staining results were graded for the percentage of positively stained tumor cells as well as the intensity of staining in the majority of cells. For each sample, predominant internalization (membrane, cytoplasmic or nuclear) was also observed. In normal tissues, positive staining of human SEMA4A was observed in immune cells (consistent with B cells and certain cells of the myeloid / dendritic cell lineage) as well as in some cells of the brain.

[0193] In tumor tissues, a high prevalence of positive staining was observed in MM (92% of samples with some positive tumor staining), DLBCL (100%), FL (100%) and AML (74%) samples. Expression intensity was strongest overall in MM and DLBCL. The high prevalence of expression in these tumor indications, coupled with the absence or low expression levels in non-tumor tissues (other than brain), suggested that SEMA4A is a potentially valuable target for specific antibody-based anti-tumor therapeutic approaches.

[0194] SEMA4A expression in multiple myeloma and DLBCL was compared to the expression of other potential therapeutic targets for these tumors, namely BCMA for MM and CD22 for DLBCL. Using the same scoring system, the same samples stained for SEMA4A were stained and scored for BCMA or CD22 expression. High expression was defined as a score of ≧6. Results for MM are shown in Tables 13 and 14, and results for DLBCL are shown in Tables 15 and 16.

[0195] All MM samples had some positivity for both SEMA4A and BCMA. The overall expression intensity appeared to be similar between SEMA4A and BCMA, but expression tended to be more membrane-localized for SEMA4A and often predominate in the cytoplasm for BCMA. No strong correlation was found between the intensity of SEMA4A expression and the intensity of BCMA expression in individual samples (Table 13).

[0196] [Table 15]

[0197] [Table 16]

[0198] In DLBCL samples, all cases had some positivity for SEMA4A, but 19 / 20 (95%) for CD22. Overall expression intensity tended to be higher for SEMA4A than for CD22, with higher scores and a higher percentage of high-expressing cases (Table 15). For MM cases, SEMA4A expression was predominantly membranous, whereas CD22 was often more predominantly cytoplasmic. There appeared to be no association between the intensity of SEMA4A expression and CD22 expression in individual samples (Table 16).

[0199] [Table 17]

[0200] [Table 18]

[0201] Example 9. In vivo model of efficacy Human MM NCI-H929, MM.1S, and MM.1R xenograft models (among others) were implanted in the flank at 5x10 6 The JJN-3 xenograft model of human MM is established in 4-6 week old female CB-17 mice (or appropriate species) by subcutaneously implanting 10x10 cells (for example) into the flank of female athymic nude mice (Envigo, Frederick, MD). 6 Tumors are established by subcutaneous implantation of 150 mm cells (for example). Tumor growth can be monitored over the course of the study and tumor volume is calculated as [length (mm) x width (mm) x width (mm)] / 2. 3 ~200mm 3Once the CI is reached, mice are randomly assigned to groups (n=5, 6, 8, or 10 animals per group for NCI-H929, MM.1S, MM.1R, and JJN-3 models, respectively) using a deterministic randomization method implemented in (e.g.) the Study Director software package (Studylog Systems, South San Francisco, CA) prior to administration of a single intravenous injection of the lead antibody or IgG isotype ADC control. Treatment information is not blinded during tumor measurements. Mouse weights and tumor measurements are determined twice weekly for the duration of the study. Sample size estimates for 100% regression in tumor volume (relative to control) are calculated using nQuery version 4.0 (e.g.) (Statistical Solutions Ltd., Cork, Ireland, 2015). A two-group t-test of equal means is used with a power of 80% at a significance level of 0.05 (one-sided).

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[0203]

Table 19

[0204]

Table 20

[0205]

Table 21

[0206]

Table 22

[0207]

Table 23

[0208] [Table 24]

[0209] SEQ ID NO: 613 Recombinant human SEMA4A (UniProt ID Q9H3S1, isoform 1)

[0210] [Table 25]

[0211] SEQ ID NO: 614 Recombinant Cynomolgus SEMA4A (UniProt ID G7NV79)

[0212] [Table 26]

[0213] SEQ ID NO: 615 recombinant mouse SEMA4A (UniProt ID Q62178)

[0214] [Table 27]

[0215] [Table 28]

[0216] [Table 29]

Claims

1. An antibody-drug conjugate (ADC) comprising an anti-human-SEMA4A human or humanized antibody, or an antigen-binding fragment thereof, a linker, and a cytotoxin.

2. The antibody comprises a V Ｈ and V comprising LCDR1, LCDR2 and LCDR3. Ｌ wherein the CDRs comprise: (a) 5E3 (SEQ ID NOs: 3, 4, 5, 12, 13, 14); (b) 5Hg1-5Lg1 (SEQ ID NOs: 21, 22, 23, 30, 31, 32); (c) 5Hg1-5Lg2 (SEQ ID NOs: 39, 40, 41, 48, 49, 50); (d) 5Hg2-5Lg1 (SEQ ID NOs: 57, 58, 59, 66, 67, 68); (e) 5Hg2-5Lg2 (SEQ ID NOs: 75, 76, 77, 84, 85, 86); (f) 5Hg2-5Lg3 (SEQ ID NOs: 93, 94, 95, 102, 103, 104); (g) 5Hg2-5Lg5 (SEQ ID NOs: 111, 112, 113, 120, 121, 122); (h) 5Hg2-5Lg6 (SEQ ID NOs: 129, 130, 131, 138, 139, 140); (i) 5Hg4-5Lg1 (SEQ ID NOs: 147, 148, 149, 156, 157, 158); (j) 5Hg4-5Lg2 (SEQ ID NOs: 165, 166, 167, 174, 175, 176); (k) 5Hg4-5Lg4 (SEQ ID NOs: 183, 184, 185, 192, 193, 194); (l) 5Hg4-5Lg5 (SEQ ID NOs: 201, 202, 203, 210, 211, 212); (m) 5Hg5-5Lg2 (SEQ ID NOs: 219, 220, 221, 228, 229, 230); (n) C0120903 (sequence numbers: 237, 238, 239, 246, 247, 248); (o) C0120904 (SEQ ID NOs: 255, 256, 257, 264, 265, 266); (p)C0120905 (SEQ ID NOs: 273, 274, 275, 282, 283, 284); (q) C0120906 (SEQ ID NOs: 291, 292, 293, 300, 301, 302); (r) C0120910 (SEQ ID NOs: 309, 310, 311, 318, 319, 320); (s) C0120913 (SEQ ID NOs: 327, 328, 329, 336, 337, 338); (t)C0120914 (SEQ ID NOs: 345, 346, 347, 354, 355, 356); (u) C0120917 (SEQ ID NOs: 363, 364, 365, 372, 373, 374); (v) C0120918 (SEQ ID NOs: 381, 382, ​​383, 390, 391, 392); (w) C0120919 (SEQ ID NOs: 399, 400, 401, 408, 409, 410); (x) C0120920 (sequence numbers: 417, 418, 419, 426, 427, 428); (y) C0120921 (SEQ ID NOs: 435, 436, 437, 444, 445, 446); (z) C0120922 (SEQ ID NOs: 453, 453, 455, 462, 463, 464); (aa) C0120923 (SEQ ID NOs: 471, 472, 473, 480, 481, 482); (bb) C0120924 (SEQ ID NOs: 489, 490, 491, 498, 499, 500); (cc) C0120925 (SEQ ID NOs: 507, 508, 509, 516, 517, 518); (dd)C0120926 (SEQ ID NOs: 525, 526, 527, 534, 535, 536); (ee) C0120927 (SEQ ID NOs: 543, 544, 545, 552, 553, 554); (ff) C0120928 (SEQ ID NOs: 561, 562, 563, 570, 571, 572); (gg)C0120929 (SEQ ID NOs: 579, 580, 581, 588, 589, 590) and (hh) C0120930 (SEQ ID NO: 597, 598, 599, 606, 607, 608) and The ADC of claim 1, wherein the CDRs are defined according to the Kabat nomenclature.

3. The antibody, a. 5Hg1-5Lg1 (SEQ ID NOs:20 and 29); b. 5Hg1-5Lg2 (SEQ ID NOs:38 and 47); c. 5Hg2-5Lg1 (SEQ ID NOs:56 and 65); d. 5Hg2-5Lg2 (SEQ ID NOs:74 and 83); e. 5Hg2-5Lg3 (SEQ ID NOs:92 and 101); f. 5Hg2-5Lg5 (SEQ ID NOs:110 and 119); g. 5Hg2-5Lg6 (SEQ ID NOs:128 and 137); h. 5Hg4-5Lg1 (SEQ ID NOs:146 and 155); i. 5Hg4-5Lg2 (SEQ ID NOs:164 and 173); j. 5Hg4-5Lg4 (SEQ ID NOs:182 and 191); k. 5Hg4-5Lg5 (SEQ ID NOs:200 and 209); 1. 5Hg5-5Lg2 (SEQ ID NOs:218 and 227); m.C0120903 (SEQ ID NOs:236 and 245); n.C0120904 (SEQ ID NOs:254 and 263); o.C0120905 (SEQ ID NOs:272 and 281); p.C0120906 (SEQ ID NOs:290 and 299); q.C0120910 (SEQ ID NOs:308 and 317); r.C0120913 (SEQ ID NOs:326 and 335); s.C0120914 (SEQ ID NOs:344 and 353); t.C0120917 (SEQ ID NOs:362 and 371); u.C0120918 (SEQ ID NOs:380 and 389); v. C0120919 (SEQ ID NOs: 398 and 407); w.C0120920 (SEQ ID NOs: 416 and 425); x.C0120921 (SEQ ID NOs: 434 and 443); y.C0120922 (SEQ ID NOs: 452 and 461); z.C0120923 (SEQ ID NOs:470 and 479); aa.C0120924 (SEQ ID NOs: 488 and 497); bb.C0120925 (SEQ ID NOs:506 and 515); cc.C0120926 (SEQ ID NOs:524 and 533); dd.C0120927 (SEQ ID NOs:542 and 551); ee.C0120928 (SEQ ID NOs:560 and 569); ff.C0120929 (SEQ ID NOs:578 and 587); and gg.C0120930 (SEQ ID NOs:596 and 605); V Ｈ and V Ｌ V selected from Ｈ and V Ｌ Including, 3. The ADC of claim 1 or claim 2, wherein the sequence is defined by the Kabat nomenclature.

4. The ADC of any one of claims 1 to 3, wherein the cytotoxin is a microtubule inhibitor (e.g., a maytansinoid or an auristatin).

5. 5. The ADC of any one of claims 1 to 4, wherein the cytotoxin is selected from monomethylauristatin F (MMAF), monomethylauristatin E (MMAE, vedotin) and mertansine (as emtansine with a DM1 or SMCC linker).

6. The ADC of any one of claims 1 to 5, wherein the linker is a non-cleavable linker or a cleavable linker.

7. 7. The ADC of any one of claims 1-6, wherein the cytotoxin is monomethylauristatin E (MMAE) and the linker is a mc-vcPAB linker (maleimide-based linker, cysteine ​​linked), or the cytotoxin is mertansine (DM1) and the linker is a SMCC linker (NHS ester-based, lysine).

8. The ADC of any one of claims 1 to 7, wherein the SEMA4A-bound ADC is capable of being internalized into a cell expressing human SEMA4A on its surface.

9. 9. The ADC of any one of claims 1 to 8, wherein the SEMA4A-bound ADC is capable of internalization into a cell expressing human SEMA4A on its surface, and wherein internalization of the ADC into the cell results in cell death.

10. V comprising HCDR1, HCDR2 and HCDR3 Ｈ and V comprising LCDR1, LCDR2 and LCDR3. Ｌ 1. A human or humanized anti-human SEMA4A antibody comprising: (a) 5E3 (SEQ ID NOs: 3, 4, 5, 12, 13, 14); (b) 5Hg1-5Lg1 (SEQ ID NOs: 21, 22, 23, 30, 31, 32); (c) 5Hg1-5Lg2 (SEQ ID NOs: 39, 40, 41, 48, 49, 50); (d) 5Hg2-5Lg1 (SEQ ID NOs: 57, 58, 59, 66, 67, 68); (e) 5Hg2-5Lg2 (SEQ ID NOs: 75, 76, 77, 84, 85, 86); (f) 5Hg2-5Lg3 (SEQ ID NOs: 93, 94, 95, 102, 103, 104); (g) 5Hg2-5Lg5 (SEQ ID NOs: 111, 112, 113, 120, 121, 122); (h) 5Hg2-5Lg6 (SEQ ID NOs: 129, 130, 131, 138, 139, 140); (i) 5Hg4-5Lg1 (SEQ ID NOs: 147, 148, 149, 156, 157, 158); (j) 5Hg4-5Lg2 (SEQ ID NOs: 165, 166, 167, 174, 175, 176); (k) 5Hg4-5Lg4 (SEQ ID NOs: 183, 184, 185, 192, 193, 194); (l) 5Hg4-5Lg5 (SEQ ID NOs: 201, 202, 203, 210, 211, 212); (m) 5Hg5-5Lg2 (SEQ ID NOs: 219, 220, 221, 228, 229, 230); (n) C0120903 (sequence numbers: 237, 238, 239, 246, 247, 248); (o) C0120904 (SEQ ID NOs: 255, 256, 257, 264, 265, 266); (p)C0120905 (SEQ ID NOs: 273, 274, 275, 282, 283, 284); (q) C0120906 (SEQ ID NOs: 291, 292, 293, 300, 301, 302); (r) C0120910 (SEQ ID NOs: 309, 310, 311, 318, 319, 320); (s) C0120913 (SEQ ID NOs: 327, 328, 329, 336, 337, 338); (t)C0120914 (SEQ ID NOs: 345, 346, 347, 354, 355, 356); (u) C0120917 (SEQ ID NOs: 363, 364, 365, 372, 373, 374); (v) C0120918 (SEQ ID NOs: 381, 382, ​​383, 390, 391, 392); (w) C0120919 (SEQ ID NOs: 399, 400, 401, 408, 409, 410); (x) C0120920 (sequence numbers: 417, 418, 419, 426, 427, 428); (y) C0120921 (SEQ ID NOs: 435, 436, 437, 444, 445, 446); (z) C0120922 (SEQ ID NOs: 453, 454, 455, 462, 463, 464); (aa) C0120923 (SEQ ID NOs: 471, 472, 473, 480, 481, 482); (bb) C0120924 (SEQ ID NOs: 489, 490, 491, 498, 499, 500); (cc) C0120925 (SEQ ID NOs: 507, 508, 509, 516, 517, 518); (dd)C0120926 (SEQ ID NOs: 525, 526, 527, 534, 535, 536); (ee) C0120927 (SEQ ID NOs: 543, 544, 545, 552, 553, 554); (ff) C0120928 (SEQ ID NOs: 561, 562, 563, 570, 571, 572); (gg)C0120929 (SEQ ID NOs: 579, 580, 581, 588, 589, 590) and (hh) C0120930 (SEQ ID NO: 597, 598, 599, 606, 607, 608) and A human or humanized anti-human SEMA4A antibody, wherein the sequences of the CDRs are defined according to the Kabat nomenclature system.

11. The antibody, (a) 5Hg1-5Lg1 (SEQ ID NOs: 20 and 29); (b) 5Hg1-5Lg2 (SEQ ID NOs:38 and 47); (c) 5Hg2-5Lg1 (SEQ ID NOs: 56 and 65); (d) 5Hg2-5Lg2 (SEQ ID NOs:74 and 83); (e) 5Hg2-5Lg3 (SEQ ID NOs: 92 and 101); (f) 5Hg2-5Lg5 (SEQ ID NOs: 110 and 119); (g) 5Hg2-5Lg6 (SEQ ID NOs: 128 and 137); (h) 5Hg4-5Lg1 (SEQ ID NOs: 146 and 155); (i) 5Hg4-5Lg2 (SEQ ID NOs: 164 and 173); (j) 5Hg4-5Lg4 (SEQ ID NOs: 182 and 191); (k) 5Hg4-5Lg5 (SEQ ID NOs: 200 and 209); (l) 5Hg5-5Lg2 (SEQ ID NOs: 218 and 227); (m) C0120903 (sequence numbers: 236 and 245); (n) C0120904 (sequence numbers: 254 and 263); (o) C0120905 (sequence numbers: 272 and 281); (p)C0120906 (SEQ ID NOs: 290 and 299); (q) C0120910 (sequence numbers: 308 and 317); (r) C0120913 (SEQ ID NOs: 326 and 335); (s) C0120914 (sequence numbers: 344 and 353); (t) C0120917 (SEQ ID NOs: 362 and 371); (u) C0120918 (SEQ ID NO: 380 and 389); (v) C0120919 (SEQ ID NOs: 398 and 407); (w) C0120920 (SEQ ID NOs: 416 and 425); (x) C0120921 (sequence numbers: 434 and 443); (y) C0120922 (sequence numbers: 452 and 461); (z) C0120923 (SEQ ID NOs: 470 and 479); (aa) C0120924 (SEQ ID NOs: 488 and 497); (bb) C0120925 (SEQ ID NOs: 506 and 515); (cc) C0120926 (SEQ ID NOs: 524 and 533); (dd) C0120927 (SEQ ID NOs: 542 and 551); (ee) C0120928 (SEQ ID NOs: 560 and 569); (ff) C0120929 (SEQ ID NOs: 578 and 587) and (gg) C0120930 (SEQ ID NOs: 596 and 605) V Ｈ and V Ｌ V selected from Ｈ and V Ｌ Including, The V Ｈ Sequence and V Ｌ The antibody of claim 10, wherein the sequence is defined by the Kabat nomenclature system.

12. 12. A chimeric antigen receptor (CAR) comprising the antigen binding domain of the monoclonal antibody of claim 10 or 11 linked to a T cell activation moiety.

13. The CAR of claim 12, wherein the antigen-binding domain comprises a single-chain Fv (scFv) fragment of the monoclonal antibody of any one of claims 10 or 11.

14. A composition comprising the ADC according to any one of claims 1 to 9, or the anti-human SEMA4A antibody according to any one of claims 10 or 11, or the CAR according to any one of claims 12 and 13, and a diluent.

15. a. For use as a medicine; b. For use as a medicament for treating cancer, c. For use in treating hematological cancers, d. For use in the treatment of a hematological cancer selected from multiple myeloma (MM), non-Hodgkin's lymphoma (NHL), acute myeloid leukemia (AML), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL); e. For use in the manufacture of a medicament for the therapeutic treatment of cancer, f. For use in the manufacture of a medicament for the therapeutic treatment of hematological cancers; g. For use in the manufacture of a medicament for the therapeutic treatment of a hematological cancer selected from MM, NHL, AML, DLBCL and FL; or h. The ADC according to any one of claims 1 to 9, the antibody according to any one of claims 10 or 11, the CAR according to any one of claims 12 or 13, or the composition according to claim 14, for inducing cell death in a cell expressing SEMA4A on the cell surface.

16. A method for treating cancer such as blood cancer, for example, blood cancer selected from MM, NHL, AML, DLBCL and FL, comprising administering to a subject the ADC according to any one of claims 1 to 9, the antibody according to any one of claims 10 or 11, the CAR according to any one of claims 12 or 13, or the composition according to claim 14.

17. A method for producing the ADC of any one of claims 1 to 9, comprising conjugating the antibody of claim 10 or claim 11 to a cytotoxin via a linker.

18. 12. An isolated recombinant DNA or RNA sequence comprising a sequence encoding the isolated antibody or antigen-binding fragment thereof of claim 10 or 11.

19. 20. The isolated recombinant DNA sequence of claim 18, which is a vector.

20. 20. The isolated recombinant DNA sequence of claim 18 or claim 19, which is an expression vector.

21. 21. An isolated recombinant DNA sequence according to any one of claims 18 to 20, encoding an antibody or an antigen-binding fragment thereof according to any one of claims 10 or 11 under the control of a promoter.

22. A host cell comprising a DNA or RNA sequence according to any one of claims 18 to 21.

23. 23. A host cell according to claim 22, capable of expressing the isolated antibody or antigen-binding fragment thereof according to any one of claims 10 or 11.

24. 24. A method for producing an isolated antibody or antigen-binding fragment thereof according to claim 10 or 11, comprising culturing a host cell according to claim 22 or 23 under conditions suitable for expression of the isolated antibody or antigen-binding fragment thereof.

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