Antibodies against slam family member 7 and uses thereof

Abstract

Therapeutic blockade of inhibitory immune checkpoints has been one of the most significant recent advances in anti-cancer treatment. There has been increasing interest in blocking the SIRPα-CD47 immune checkpoint to promote phagocytosis and anti-tumor immunity, but one of the limitations of SIRPα-CD47 blockade has been that agents blocking depleted red blood cells, platelets and lymphocytes, leading to anemia, thrombocytopenia and lymphopenia. The present application relates to SLAMF7-binding molecules such as antibodies or antigen-binding fragments thereof that reduce or inhibit the interaction between SLAMF7 and CD47 on the tumor cells, and their uses for increasing the susceptibility to phagocytosis of tumor cells expressing SLAMF7 and CD47 and for treating a cancer comprising such tumor cells. Since red blood cells and platelets do not express SLAMF7, the SLAMF7-binding molecules described herein are unlikely to induce anemia and thrombocytopenia, one of the main side effects of anti-CD47-based therapies.

Description

[0001] ANTIBODIES AGAINST SLAM FAMILY MEMBER 7 AND USES THEREOF

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] The present application claims the benefit of U.S. provisional patent application serial No. 63 / 489,007 filed on March 8, 2023, which is incorporated herein by reference in its entirety.

[0004] SEQUENCE LISTING

[0005] A sequence listing is submitted herewith as an XML file named G12810-00857_Seq Listing.xml, created on March 7, 2024, and having a size of ~55300 bytes. The content of the aforementioned file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] The present invention generally relates to the field of cancer, and more particularly to the treatment of cancers expressing CD47.

[0008] BACKGROUND ART

[0009] Therapeutic blockade of inhibitory immune checkpoints has been one of the most significant recent advances in anti-cancer treatment1 2. Monoclonal antibodies (MAbs) blocking inhibitory receptors expressed on T cells (such as CTLA-4 and PD-1) ortheir ligands have shown significant efficacy against a wide range of cancer types and have been approved for patient care. However, these agents are not effective against many cancers. This limitation prompted researchers to evaluate other inhibitory immune checkpoints as candidates, including receptors expressed on innate immune cells3 9.

[0010] Macrophages, as well as other phagocytes like neutrophils, are amongst the most primitive components of the immune system46 10. Their main functions are phagocytosis leading to destruction of other cells, cytokine production and antigen presentation to T cells. Consequently, they can be highly effective in anti-tumor immunity. The activation of macrophages is controlled by the balance between the engagement of activating and inhibitory cell surface receptors, by ligands expressed or not on potential “target” cells. When activating receptors are predominantly triggered, macrophages are activated and display effector functions. Macrophage activating receptors include receptors for the Fc portion of antibodies (FcRs), the apoptotic cell receptor MerTK, the integrin CD11b (Mac-1) and the homotypic receptor SLAMF758 10 11. However, when inhibitory receptors are predominantly triggered, macrophage activation is suppressed. The best- studied macrophage inhibitory receptor is signal inhibitory regulatory protein (SIRP) a, which recognizes as ligand CD47 often overexpressed on tumor cells3489. There has been increasing interest in blocking the SIRPa-CD47 immune checkpoint to promote phagocytosis and anti-tumor immunity5 7 10 12 13. Blockade of the SIRPa-CD47 interaction, by anti-CD47 MAbs, anti-SIRPa antibodies or soluble SIRPa-Fc fusion proteins, has shown promising results as anti-cancer immunotherapy in pre-clinical studies and phase 1 clinical trials, in particular against hematological malignancies5 7 1314. The ability of SIRPa-CD47 blockade to augment phagocytosis requires co-engagement of activating receptors, such as FcRs or SLAMF7, by ligands on tumor cells3-5-8’10’15.

[0011] FcRs are engaged when tumor-opsonizing antibodies, such as the therapeutic anti-CD20 MAb rituximab, are present6 10 11. In contrast, the homotypic receptor SLAMF7 (that is, the ligand of SLAMF7 is another SLAMF7 molecule expressed on another cell) is engaged when tumor cells, in addition to macrophages, express SLAMF7. SLAMF7 is primarily expressed on hematopoietic tumor cells, such as multiple myeloma cells and lymphoma cells45 15 16. A recent report documented that SLAMF7-negative tumor cells, such as breast cancer cells, can be artificially decorated with SLAMF7, using bispecific nanoconjugates, to unleash SLAMF7-dependent phagocytosis and anti-tumor immunity17. Conversely, the role of SLAMF7 in phagocytosis can be bypassed in response to inflammatory stimuli, due to activation of unconventional pro-phagocytic integrins CD11 a and CD11 c15.

[0012] One of the limitations of SIRPa-CD47 blockade has been that agents blocking depleted red blood cells, platelets and lymphocytes, leading to anemia, thrombocytopenia and lymphopenia, whereas, in one study, agents blocking SIRPa depleted neutrophils, thus causing neutropenia3718-20. The mechanisms involved in these toxicities remain to be clarified. One possibility is that the blocking agents, which bind target molecules also found on normal hematopoietic cells and were capable of engaging FcRs via their intact Fc segments, triggered FcR-mediated elimination of the normal hematopoietic cells. Another possibility is that the blocking agents had yet unappreciated mechanisms of action that interfered with normal hematopoietic cell homeostasis.

[0013] There is thus a need for the development of novel approaches for the treatment of CD47- expressing cancers.

[0014] The present description refers to a number of documents, the content of which is herein incorporated by reference in their entirety.

[0015] SUMMARY

[0016] In various aspects and embodiments, the present disclosure provides the following items 1 to 84 and 1A to 55A:

[0017] 1. A method for increasing the susceptibility to phagocytosis of cells such as tumor cells expressing SLAM family member 7 (SLAMF7) and Cluster of Differentiation 47 (CD47), the comprising contacting the tumor cells with a SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on the tumor cells.

[0018] 2. The method of item 1 , wherein the SLAMF7-binding molecule binds to a domain or epitope comprising residues 172-174, 177 and 181-183 of human SLAMF7 (SEQ ID NO:23).

[0019] 3. The method of item 1 or 2, wherein the SLAMF7-binding molecule is an antibody or an antigen-binding fragment thereof.

[0020] 4. The method of item 3, wherein the antibody or antigen-binding fragment thereof comprises the following complementary determining regions (CDRs): a light chain CDR1 comprising the amino acid sequence KASQDVDTAVA (SEQ ID NO:1), a light chain CDR2 comprising the amino acid sequence WASTRHT (SEQ ID NO:2), a light chain CDR3 comprising the amino acid sequence QQYRSYPFT (SEQ ID NO:3), a heavy chain CDR1 comprising the amino acid sequence GIDFSRY (SEQ ID NO:4), a heavy chain CDR2 comprising the amino acid sequence NPDSST (SEQ ID NO:5), and a heavy chain CDR3 comprising the amino acid sequence PGDYDAWYFDV (SEQ ID NO:6).

[0021] 5. The method of item 4, wherein the antibody or antigen-binding fragment thereof comprises the following framework regions (FRs): a light chain FR1 comprising the amino acid sequence DITMSQSHKFMSTSVGDRVSITC (SEQ ID NO:7), a light chain FR2 comprising the amino acid sequence WYQQKPGQSPKLLIY (SEQ ID NO:8), a light chain FR3 comprising the amino acid sequence GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (SEQ ID NO:9), a light chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NO: 10), a heavy chain FR1 comprising the amino acid sequence EVKLLQSGGGLVQPGGSLKLSCAAS (SEQ ID NO:11), a heavy chain FR2 comprising the amino acid sequence WMSWVRRAPGKGLEWIGEI (SEQ ID NO:12), a heavy chain FR3 comprising the amino acid sequence INYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR (SEQ ID NO:13), and / or a heavy chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NO: 14).

[0022] 6. The method of item 5, wherein the antibody or antigen-binding fragment thereof comprises all the FRs defined in item 5.

[0023] 7. The method of any one of items 4 to 6, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising the amino acid sequence depicted in FIG. 21A (SEQ ID NO:16).

[0024] 8. The method of any one of items 4 to 7, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence depicted in FIG. 22A (SEQ ID NQ:20).

[0025] 9. The method of any one of items 3 to 8, wherein the antibody or antigen-binding fragment thereof further comprises at least one constant domain or a fragment thereof.

[0026] 10. The method of item 9, which comprises a Fragment crystallizable (Fc) fragment of a heavy chain constant region of an antibody. 11 . The method of any one of items 3 to 10, wherein the antibody is a fully human antibody or a chimeric antibody.

[0027] 12. The method of item 11 , wherein the antibody is an IgG 1 antibody.

[0028] 13. The method of any one of items 1 to 12, wherein the tumor cells are hematopoietic tumor cells.

[0029] 14. The method of item 13, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

[0030] 15. The method of any one of items 1 to 14, wherein the method further comprises contacting the tumor cells with a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or an an agent capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

[0031] 16. The method of item 15, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigenbinding fragment thereof, or a soluble SIRPa polypeptide.

[0032] 17. Use of a SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on the tumor cells for increasing the susceptibility to phagocytosis of tumor cells expressing SLAM family member 7 (SLAMF7) and Cluster of Differentiation 47 (CD47).

[0033] 18. Use of a SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on the tumor cells for the manufacture of a medicament for increasing the susceptibility to phagocytosis of tumor cells expressing SLAM family member 7 (SLAMF7) and Cluster of Differentiation 47 (CD47).

[0034] 19. The use of item 17 or 18, wherein the SLAMF7-binding molecule binds to a domain or epitope comprising residues 172-174, 177 and 181-183 of human SLAMF7 (SEQ ID NO:23).

[0035] 20. The use of any one of items 17 to 19, wherein the SLAMF7-binding molecule is an antibody or an antigen-binding fragment thereof.

[0036] 21 . The use of item 20, wherein the antibody or antigen-binding fragment thereof comprises the following complementary determining regions (CDRs): a light chain CDR1 comprising the amino acid sequence KASQDVDTAVA, a light chain CDR2 comprising the amino acid sequence WASTRHT, a light chain CDR3 comprising the amino acid sequence QQYRSYPFT, a heavy chain CDR1 comprising the amino acid sequence GIDFSRY, a heavy chain CDR2 comprising the amino acid sequence NPDSST, and a heavy chain CDR3 comprising the amino acid sequence PGDYDAWYFDV.

[0037] 22. The use of item 21 , wherein the antibody or antigen-binding fragment thereof comprises the following framework regions (FRs): a light chain FR1 comprising the amino acid sequence DITMSQSHKFMSTSVGDRVSITC (SEQ ID NO:7), a light chain FR2 comprising the amino acid sequence WYQQKPGQSPKLLIY (SEQ ID NO:8), a light chain FR3 comprising the amino acid sequence WMSWVRRAPGKGLEWIGEI (SEQ ID NO:9), a light chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NQ:10), a heavy chain FR1 comprising the amino acid sequence EVKLLQSGGGLVQPGGSLKLSCAAS (SEQ ID NO:11), a heavy chain FR2 comprising the amino acid sequence WMSWVRRAPGKGLEWIGEI (SEQ ID NO: 12), a heavy chain FR3 comprising the amino acid sequence

[0038] INYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR (SEQ ID NO:13), and / or a heavy chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NO: 14).

[0039] 23. The use of item 22, wherein the antibody or antigen-binding fragment thereof comprises all the FRs defined in item 22.

[0040] 24. The use of any one of items 20 to 23, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising the amino acid sequence depicted in FIG. 21A (SEQ ID NO:16).

[0041] 25. The use of any one of items 20 to 24, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence depicted in FIG. 22A (SEQ ID NQ:20).

[0042] 26. The use of any one of items 20 to 25, wherein the antibody or antigen-binding fragment thereof further comprises at least one constant domain or a fragment thereof.

[0043] 27. The use of item 26, which comprises a Fragment crystallizable (Fc) fragment of a heavy chain constant region of an antibody.

[0044] 28. The use of any one of items 20 to 27, wherein the antibody is a fully human antibody or a chimeric antibody.

[0045] 29. The use of item 28, wherein the antibody is an IgG 1 antibody.

[0046] 30. The use of any one of items 17 to 29, wherein the tumor cells are hematopoietic tumor cells.

[0047] 31. The use of item 30, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

[0048] 32. The use of any one of items 17 to 31 , wherein the SLAMF7-binding molecule or medicament is for use in combination with a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or with an agent capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

[0049] 33. The use of item 32, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigenbinding fragment thereof, or a soluble SIRPa polypeptide.

[0050] 34. A method for treating a cancer comprising tumor cells expressing SLAM family member 7 (SLAMF7) and Cluster of Differentiation 47 (CD47) in a subject in need thereof, the method comprising administering to the subject an effective amount of a SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on the tumor cells.

[0051] 35. The method of item 34, wherein the SLAMF7-binding molecule binds to a domain or epitope comprising residues 172-174, 177 and 181-183 of human SLAMF7 (SEQ ID NO:23).

[0052] 36. The method of item 34 or 35, wherein the SLAMF7-binding molecule is an antibody or an antigen-binding fragment thereof. 37. The method of item 36, wherein the antibody or antigen-binding fragment thereof comprises the following complementary determining regions (CDRs): a light chain CDR1 comprising the amino acid sequence KASQDVDTAVA (SEQ ID NO:1), a light chain CDR2 comprising the amino acid sequence WASTRHT (SEQ ID NO:2), a light chain CDR3 comprising the amino acid sequence QQYRSYPFT (SEQ ID NO:3), a heavy chain CDR1 comprising the amino acid sequence GIDFSRY (SEQ ID NO:4), a heavy chain CDR2 comprising the amino acid sequence NPDSST (SEQ ID NO:5), and a heavy chain CDR3 comprising the amino acid sequence PGDYDAWYFDV (SEQ ID NO:6).

[0053] 38. The method of item 37, wherein the antibody or antigen-binding fragment thereof comprises the following framework regions (FRs): a light chain FR1 comprising the amino acid sequence DITMSQSHKFMSTSVGDRVSITC (SEQ ID NO:7), a light chain FR2 comprising the amino acid sequence WYQQKPGQSPKLLIY (SEQ ID NO:8), a light chain FR3 comprising the amino acid sequence GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (SEQ ID NO:9), a light chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NO: 10), a heavy chain FR1 comprising the amino acid sequence EVKLLQSGGGLVQPGGSLKLSCAAS (SEQ ID NO:11), a heavy chain FR2 comprising the amino acid sequence WMSWVRRAPGKGLEWIGEI (SEQ ID NO:12), a heavy chain FR3 comprising the amino acid sequence INYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR (SEQ ID NO:13), and / or a heavy chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NO: 14).

[0054] 39. The method of item 38, wherein the antibody or antigen-binding fragment thereof comprises all the FRs defined in item 38.

[0055] 40. The method of any one of items 36 to 39, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising the amino acid sequence depicted in FIG. 21A (SEQ ID NO:16).

[0056] 41 . The method of any one of items 36 to 40, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence depicted in FIG. 22A (SEQ ID NQ:20).

[0057] 42. The method of any one of items 36 to 41 , wherein the antibody or antigen-binding fragment thereof further comprises at least one constant domain or a fragment thereof.

[0058] 43. The method of item 42, which comprises a Fragment crystallizable (Fc) fragment of a heavy chain constant region of an antibody.

[0059] 44. The method of any one of items 36 to 43, wherein the antibody is a fully human antibody or a chimeric antibody.

[0060] 45. The method of item 44, wherein the antibody is an IgG 1 antibody.

[0061] 46. The method of any one of items 34 to 46, wherein the tumor cells are hematopoietic tumor cells. 47. The method of item 46, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

[0062] 48. The method of any one of items 34 to 47, comprising administering to the subject an effective amount of a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or of an agent capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

[0063] 49. The method of item 48, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigenbinding fragment thereof, or a soluble SIRPa polypeptide.

[0064] 50. Use of a SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on the tumor cells for treating a cancer comprising tumor cells expressing SLAM family member 7 (SLAMF7) and Cluster of Differentiation 47 (CD47) in a subject.

[0065] 51. Use of a SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on the tumor cells for the manufacture of a medicament for treating a cancer comprising tumor cells expressing SLAM family member 7 (SLAMF7) and Cluster of Differentiation 47 (CD47) in a subject.

[0066] 52. The use of item 50 or 51 , wherein the SLAMF7-binding molecule binds to a domain or epitope comprising residues 172-174, 177 and 181-183 of human SLAMF7 (SEQ ID NO:23).

[0067] 53. The use of any one of items 50 to 52, wherein the SLAMF7-binding molecule is an antibody or an antigen-binding fragment thereof.

[0068] 54. The use of item 53, wherein the antibody or antigen-binding fragment thereof comprises the following complementary determining regions (CDRs): a light chain CDR1 comprising the amino acid sequence KASQDVDTAVA (SEQ ID NO:1), a light chain CDR2 comprising the amino acid sequence WASTRHT (SEQ ID NO:2), a light chain CDR3 comprising the amino acid sequence QQYRSYPFT (SEQ ID NO:3), a heavy chain CDR1 comprising the amino acid sequence GIDFSRY (SEQ ID NO:4), a heavy chain CDR2 comprising the amino acid sequence NPDSST (SEQ ID NO:5), and a heavy chain CDR3 comprising the amino acid sequence PGDYDAWYFDV (SEQ ID NO:6).

[0069] 55. The use of item 54, wherein the antibody or antigen-binding fragment thereof comprises the following framework regions (FRs): a light chain FR1 comprising the amino acid sequence DITMSQSHKFMSTSVGDRVSITC (SEQ ID NO:7), a light chain FR2 comprising the amino acid sequence WYQQKPGQSPKLLIY (SEQ ID NO:8), a light chain FR3 comprising the amino acid sequence GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (SEQ ID NO:9), a light chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NO: 10), a heavy chain FR1 comprising the amino acid sequence EVKLLQSGGGLVQPGGSLKLSCAAS (SEQ ID NO:11), a heavy chain FR2 comprising the amino acid sequence WMSWVRRAPGKGLEWIGEI (SEQ ID NO:12), a heavy chain FR3 comprising the amino acid sequence INYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR (SEQ ID NO:13), and / or a heavy chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NO: 14).

[0070] 56. The use of item 55, wherein the antibody or antigen-binding fragment thereof comprises all the FRs defined in item 55.

[0071] 57. The use of any one of items 53 to 56, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising the amino acid sequence depicted in FIG. 21A (SEQ ID NO:16).

[0072] 58. The use of any one of items 53 to 57, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence depicted in FIG. 22A (SEQ ID NQ:20).

[0073] 59. The use of any one of items 53 to 58, wherein the antibody or antigen-binding fragment thereof further comprises at least one constant domain or a fragment thereof.

[0074] 60. The use of item 59, which comprises a Fragment crystallizable (Fc) fragment of a heavy chain constant region of an antibody.

[0075] 61 . The use of any one of items 53 to 60, wherein the antibody is a fully human antibody or a chimeric antibody.

[0076] 62. The use of item 61 , wherein the antibody is an IgG 1 antibody.

[0077] 63. The use of any one of items 50 to 62, wherein the tumor cells are hematopoietic tumor cells.

[0078] 64. The use of item 63, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

[0079] 65. The use of any one of items 50 to 64, wherein the SLAMF7-binding molecule or medicament is for use in combination with a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or with an agent capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

[0080] 66. The use of item 65, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigenbinding fragment thereof, or a soluble SIRPa polypeptide.

[0081] 67. A SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on the tumor cells.

[0082] 68. The SLAMF7-binding molecule of item 67, wherein the SLAMF7-binding molecule binds to a domain or epitope comprising residues 172-174, 177 and 181-183 of human SLAMF7 (SEQ ID NO:23).

[0083] 69. The SLAMF7-binding molecule of item 67 or 68, wherein the SLAMF7-binding molecule is an antibody or an antigen-binding fragment thereof.

[0084] 70. The SLAMF7-binding molecule of item 69, wherein the antibody or antigen-binding fragment thereof comprises the following complementary determining regions (CDRs): a light chain CDR1 comprising the amino acid sequence KASQDVDTAVA (SEQ ID NO:1), a light chain CDR2 comprising the amino acid sequence WASTRHT (SEQ ID NO:2), a light chain CDR3 comprising the amino acid sequence QQYRSYPFT (SEQ ID N0:3), a heavy chain CDR1 comprising the amino acid sequence GIDFSRY (SEQ ID NO:4), a heavy chain CDR2 comprising the amino acid sequence NPDSST (SEQ ID NO:5), and a heavy chain CDR3 comprising the amino acid sequence PGDYDAWYFDV (SEQ ID NO:6).

[0085] 71 . The SLAMF7-binding molecule of item 70, wherein the antibody or antigen-binding fragment thereof comprises the following framework regions (FRs): a light chain FR1 comprising the amino acid sequence DITMSQSHKFMSTSVGDRVSITC (SEQ ID NO:7), a light chain FR2 comprising the amino acid sequence WYQQKPGQSPKLLIY (SEQ ID NO:8), a light chain FR3 comprising the amino acid sequence GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (SEQ ID NO:9), a light chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NO: 10), a heavy chain FR1 comprising the amino acid sequence EVKLLQSGGGLVQPGGSLKLSCAAS (SEQ ID NO:11), a heavy chain FR2 comprising the amino acid sequence WMSWVRRAPGKGLEWIGEI (SEQ ID NO:12), a heavy chain FR3 comprising the amino acid sequence INYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR (SEQ ID NO:13), and / or a heavy chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NO: 14).

[0086] 72. The SLAMF7-binding molecule of item 71 , wherein the antibody or antigen-binding fragment thereof comprises all the FRs defined in item 71.

[0087] 73. The SLAMF7-binding molecule of any one of items 69 to 72, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising the amino acid sequence depicted in FIG. 21A (SEQ ID NO:16).

[0088] 74. The SLAMF7-binding molecule of any one of items 69 to 73, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence depicted in FIG. 22A (SEQ ID NQ:20).

[0089] 75. The SLAMF7-binding molecule of any one of items 69 to 74, wherein the antibody or antigen-binding fragment thereof further comprises at least one constant domain or a fragment thereof.

[0090] 76. The SLAMF7-binding molecule of item 75, which comprises a Fragment crystallizable (Fc) fragment of a heavy chain constant region of an antibody.

[0091] 77. The SLAMF7-binding molecule of any one of items 69 to 76, wherein the antibody is a fully human antibody or a chimeric antibody.

[0092] 78. The SLAMF7-binding molecule of item 77, wherein the antibody is an IgG 1 antibody.

[0093] 79. The SLAMF7-binding molecule of any one of items 67 to 78, for use in increasing the susceptibility to phagocytosis of tumor cells expressing SLAM family member 7 (SLAMF7) and Cluster of Differentiation 47 (CD47).

[0094] 80. The SLAMF7-binding molecule of any one of items 67 to 78, for use in the treatment of a cancer comprising tumor cells expressing SLAM family member 7 (SLAMF7) and Cluster of Differentiation 47 (CD47) in a subject. 81 . The SLAMF7-binding molecule for use according to item 79 or 80, wherein the tumor cells are hematopoietic tumor cells.

[0095] 82. The SLAMF7-binding molecule for use according to item 81 , wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

[0096] 83. The SLAMF7-binding molecule for use according to any one of items 79 to 82, wherein the SLAMF7-binding molecule is for use in combination with a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or with an agent capable of inducing antibody-dependent cell- mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

[0097] 84. The SLAMF7-binding molecule for use according to item 83, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigen-binding fragment thereof, or a soluble SIRPa polypeptide.

[0098] 1A. A SLAM family member 7 (SLAMF7)-binding molecule that reduces or inhibits the interaction between SLAMF7 and Cluster of Differentiation 47 (CD47) on a cell.

[0099] 2A. The SLAMF7-binding molecule of claim 1 A, wherein the SLAMF7-binding molecule binds to a domain or epitope comprising residues 172-174, 177 and 181-183 of human SLAMF7.

[0100] 3A. The SLAMF7-binding molecule of claim 1 A or 2A, wherein the SLAMF7-binding molecule is an antibody or an antigen-binding fragment thereof.

[0101] 4A. The SLAMF7-binding molecule of claim 3A, wherein the antibody or antigen-binding fragment thereof comprises the following complementary determining regions (CDRs): a light chain CDR1 comprising the amino acid sequence KASQDVDTAVA (SEQ ID NO:1), a light chain CDR2 comprising the amino acid sequence WASTRHT (SEQ ID NO:2), a light chain CDR3 comprising the amino acid sequence QQYRSYPFT (SEQ ID NO:3), a heavy chain CDR1 comprising the amino acid sequence GIDFSRY (SEQ ID NO:4), a heavy chain CDR2 comprising the amino acid sequence NPDSST (SEQ ID NO:5), and a heavy chain CDR3 comprising the amino acid sequence PGDYDAWYFDV (SEQ ID NO:6).

[0102] 5A. The SLAMF7-binding molecule of claim 4A, wherein the antibody or antigen-binding fragment thereof comprises the following framework regions (FRs): a light chain FR1 comprising the amino acid sequence DITMSQSHKFMSTSVGDRVSITC (SEQ ID NO:7), a light chain FR2 comprising the amino acid sequence WYQQKPGQSPKLLIY (SEQ ID NO:8), a light chain FR3 comprising the amino acid sequence GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (SEQ ID NO:9), a light chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NQ:10), a heavy chain FR1 comprising the amino acid sequence EVKLLQSGGGLVQPGGSLKLSCAAS (SEQ ID NO:11), a heavy chain FR2 comprising the amino acid sequence WMSWVRRAPGKGLEWIGEI (SEQ ID NO: 12), a heavy chain FR3 comprising the amino acid sequence INYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR (SEQ ID NO:13), and / or a heavy chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NO:14).

[0103] 6A. The SLAMF7-binding molecule of claim 5A, wherein the antibody or antigen-binding fragment thereof comprises all the FRs defined in claim 5. 7A. The SLAMF7-binding molecule of any one of claims 3A to 6A, wherein the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO:16.

[0104] 8A. The SLAMF7-binding molecule of any one of claims 3A to 7A, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:20.

[0105] 9A. The SLAMF7-binding molecule of any one of claims 3A to 8A, wherein the antibody or antigen-binding fragment thereof further comprises at least one constant domain or a fragment thereof.

[0106] 10A. The SLAMF7-binding molecule of claim 9A, which comprises a Fragment crystallizable (Fc) fragment of a heavy chain constant region of an antibody.

[0107] 11 A. The SLAMF7-binding molecule of any one of claims 3A to 10A, wherein the antibody is a fully human antibody or a chimeric antibody.

[0108] 12A. The SLAMF7-binding molecule of claim 11 A, wherein the antibody is an IgG 1 antibody.

[0109] 13A. The SLAMF7-binding molecule of any one of claims 1A to 12A, wherein the SLAMF7- binding molecule is conjugated to an antitumor agent.

[0110] 14A. The SLAMF7-binding molecule of claim 13A, wherein the antitumor agent is a chemotherapeutic agent, a radionuclide, or a checkpoint inhibitor.

[0111] 15A. The SLAMF7-binding molecule of any one of claims 1A to 14A, which is a bispecific binding molecule.

[0112] 16A. The SLAMF7-binding molecule of claim 15A, wherein the bispecific binding molecule comprising a binding module that binds to signal inhibitory regulatory protein alpha (SIRPa).

[0113] 17A. The SLAMF7-binding molecule of claim 16A, wherein the binding module that binds to SIRPa is an anti- SIRPa antibody or an antigen-bonding fragment thereof.

[0114] 18A. The SLAMF7-binding molecule of any one of claims 1A to 17A, for use in increasing the susceptibility to phagocytosis of cells expressing SLAMF7 and Cluster of Differentiation 47 (CD47).

[0115] 19A. The SLAMF7-binding molecule for use according to claim 18A, wherein the cells are tumor cells.

[0116] 20A. The SLAMF7-binding molecule of any one of claims 1A to 17A, for use in the treatment of a cancer comprising tumor cells expressing SLAMF7 and CD47 in a subject.

[0117] 21 A. The SLAMF7-binding molecule for use according to claim 19A or 20A, wherein the tumor cells are hematopoietic tumor cells.

[0118] 22A. The SLAMF7-binding molecule for use according to claim 21 A, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

[0119] 23A. The SLAMF7-binding molecule for use according to any one of claims 18Ato 22A, wherein the SLAMF7-binding molecule is for use in combination with a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or with an agent capable of inducing antibody-dependent cell- mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

[0120] 24A. The SLAMF7-binding molecule for use according to claim 23A, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigen-binding fragment thereof, or a soluble SIRPa polypeptide.

[0121] 25A. A method for increasing the susceptibility to phagocytosis of cells expressing SLAM family member 7 (SLAMF7) and Cluster of Differentiation 47 (CD47), the comprising contacting the tumor cells with the SLAMF7-binding molecule defined in any one of claims 1 A to 17A.

[0122] 26A. The method of claim 25A, wherein the cells are tumor cells.

[0123] 27A. The method of claim 26A, wherein the tumor cells are hematopoietic tumor cells.

[0124] 28A. The method of claim 27A, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

[0125] 29A. The method of any one of claims 25A to 28A, wherein the method further comprises contacting the cells with a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or an agent capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

[0126] 30A. The method of claim 29A, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigen-binding fragment thereof, or a soluble SIRPa polypeptide.

[0127] 31A. Use of the SLAMF7-binding molecule defined in any one of claims 1Ato 17A for increasing the susceptibility to phagocytosis of tumor cells expressing SLAMF7 and CD47.

[0128] 32A. Use of the SLAMF7-binding molecule defined in any one of claims 1A to 17A for the manufacture of a medicament for increasing the susceptibility to phagocytosis of tumor cells expressing SLAMF7 and CD47.

[0129] 33A. The use of claim 31 A or 32A, wherein the tumor cells are hematopoietic tumor cells.

[0130] 34A. The use of claim 33A, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

[0131] 35A. The use of any one of claims 31A to 34A, wherein the SLAMF7-binding molecule or medicament is for use in combination with a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or with an agent capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

[0132] 36A. The use of claim 35A, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigenbinding fragment thereof, or a soluble SIRPa polypeptide.

[0133] 37A. A method for treating a cancer comprising tumor cells expressing SLAMF7 and CD47 in a subject in need thereof, the method comprising administering to the subject an effective amount of the SLAMF7-binding molecule defined in any one of claims 1A to 17A.

[0134] 38A. The method of claim 37A, wherein the tumor cells are hematopoietic tumor cells. 39A. The method of claim 38A, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

[0135] 40A. The method of any one of claims 37A to 39A, further comprising administering to the subject an effective amount of a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or of an agent capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

[0136] 41A. The method of claim 40A, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigen-binding fragment thereof, or a soluble SIRPa polypeptide.

[0137] 42A. Use of the SLAMF7-binding molecule defined in any one of claims 1A to 17A for treating a cancer comprising tumor cells expressing SLAMF7 and CD47 in a subject.

[0138] 43A. Use of the SLAMF7-binding molecule defined in any one of claims 1A to 17A for the manufacture of a medicament for treating a cancer comprising tumor cells expressing SLAMF7 and CD47 in a subject.

[0139] 44A. The use of claim 42A or 43A, wherein the tumor cells are hematopoietic tumor cells.

[0140] 45A. The use of claim 44, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

[0141] 46A. The use of any one of claims 42A to 45A, wherein the SLAMF7-binding molecule or medicament is for use in combination with a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or with an agent capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

[0142] 47A. The use of claim 46A, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigenbinding fragment thereof, or a soluble SIRPa polypeptide.

[0143] Other objects, advantages and features of the present disclosure will become more apparent upon reading of the following non-restrictive description of specific embodiments thereof, given by way of example only with reference to the accompanying drawings.

[0144] BRIEF DESCRIPTION OF DRAWINGS

[0145] In the appended drawings:

[0146] FIGs. 1A-G show that blockade of CD47, but not of SIRPa, promotes phagocytosis triggered by pro-phagocytic ligand SLAMF7. FIG. 1A: Schematic representation of phagocytosis assay is shown. Fc, fragment crystallizable. MAbs, monoclonal antibodies. M J-, macrophage. FIG. 1B-C: Phagocytosis of mouse (m) tumor cells L1210, P815 or SP2 / 0 by wild-type (WT) bone marrow-derived macrophages (BMDMs) in the presence of Fc-silent MAbs was assayed by fluorescence microscopy. Ctrl, control; IgG, immunoglobulin G. The Fc portion of the MAbs was from mlgG2a and carried the “LALAPG” mutation, which prevents Fc receptor-binding. FIG. 1D: Sub-cutaneous tumor transplantation assay. Tumor volume over time of L1210 injected subcutaneously in RAG-1 KO mice, in the presence of the indicated MAbs (Fc-silent), was measured using a caliper. FIG. 1E: Same as FIG. 1B, except that activated CD4+T cells were used as targets. FIG. 1F: T cell transfer assay. Residual CD4+T cells in blood of WT mice were enumerated 24 h after injection. Left, schematic representation of the assay; middle, percentage (%) of injected T cells remaining in blood; right, number of remaining T cells per 0.1x106WBCs remaining in blood. WBC, white blood cells; IV, intravenous; CFSE, carboxyfluorescein succinimidyl ester. FIG. 1G: Phagocytosis of human (h) Raji lymphoma cells by human blood- derived macrophages in the presence of Fc-silent blocking MAbs or soluble fusion proteins was assessed by fluorescence microscopy. The Fc portion was from hlgG 1 and carried the LALAPG mutation. All data are means ± s.e.m. ns, not significant; *p < 0.05, **p < 0.01 , ***p < 0.001 , and ****p <0.0001. Results are pooled from a total of 4 (FIG. 1 B) or 3 (FIG. 1C, E) mice studied in independent experiments; mice from 3 independent experiments (FIG. 1D); 6 mice from 3 independent experiments (FIG. 1F); and 4 healthy human donors from 4 independent experiments (FIG. 1G). Each symbol represents one mouse or healthy donor.

[0147] FIGs. 2A-I show that genetic deficiency of CD47 or SIRPa phenocopies the differential impact of CD47 or SIRPa blockade. FIG. 2A: Schematic of the phagocytosis assay. Phagocytosis of L1210 (FIG. 2B), activated CD4+T cells (FIG. 2C), Raji (FIG. 2D) and human lymphoma cells Daudi (FIG. 2E), expressing or not expressing CD47, by WT or SIRPa KO BMDMs was tested. FIG. 2F: As in FIG. 1D, except that control L1210. FIGs. G-l: as in FIG. 1F, except that WT and CD47 KO activated CD4+T cells were injected in WT or SIRPa KO mice. A schematic representation of the assay is shown in FIG. 2G; a representative dot plot is depicted in FIG. 2H; percentages (%) of injected T cells remaining in blood (left) and numbers of remaining T cells per 0.1x106WBCs (right) are shown in FIG. 11. All data are means ± s.e.m. ns, not significant; *p < 0.05, **p < 0.01 , ***p < 0.001 , and ****p < 0.0001. Results are pooled from 6 (FIG. 2B), 4 (FIG. 2C,E), and 3 (FIG. 2D) mice studied in independent experiments; 11 (control L1210) or 10 (CD47 KO L1210) mice from 2 independent experiments (FIG. 2F), one mouse from “RAG-1 KO mice + CD47 KO L1210” group showed no clinically detectable tumor; 8 mice (FIG. 2I) from 3 independent experiments. Each symbol represents one mouse. CD47 KO L1210 cells were injected subcutaneously in RAG-1 KO or RAG-1 -SIRPa double (D) KO mice.

[0148] FIGs. 3A-F show that blockade or loss of either CD47 or SIRPa promotes phagocytosis of antibody-opsonized tumor cells. FIGs. 3A-B: Same as FIG. 1B, except that IgG-opsonized L1210 were used as targets. FIG. 3C: Same as FIG. 1G, except that SIRPa KO BMDMs expressing the V1 or V2 variant of hSIRPa and IgG-opsonized Raji were used. FIG. 3D,E: Same as FIG. 2B, except that IgG-opsonized L1210 (FIG. 3D) or C3bi-opsonized L1210 (FIG. 3E) were used as targets. FIG. 3F: Subcutaneous tumor transplantation assay. As in FIGs. 1D and 2F, except that L1210 derivatives expressing Tac (hCD25) were injected in RAG-1 KO or RAG-1-SIRPa DKO mice. Anti-Tac / hCD25 MAb 7G7, which is a mlgG2a, was used to opsonize the tumor cells. All data are means ± s.e.m. ns, not significant; *p < 0.05, **p < 0.01 , ***p < 0.001 , and ****p < 0.0001 . Results are pooled from a total of 4 (FIG. 3B) and 3 (FIG. 3C-E) mice studied in independent experiments; 15 mice (16 mice for “RAG- 1 KO + Anti-T ac” group) from 3 independent experiments (FIG. 3F). One mouse from “RAG-1 KO + Ctrl IgG” group showed no clinically detectable tumor. Each symbol represents one mouse.

[0149] FIGs. 4A-H show that CD47 physically interacts in cis with pro-phagocytic ligand SLAMF7 on tumor cells. FIG. 4A: Binding of CD47-Fc (empty curves) to WT or SIRPa KO BMDMs was assayed by flow cytometry. Filled curves, Ctrl Fc. FIG. 4B: Mass spectrometry analysis of anti-Flag immunoprecipitates from L1210 cells expressing a Flag-tagged variant of mCD47. The Flag tag was added at the carboxyl terminus of CD47. A schematic representation of the relative abundance, shown with a gradient, of the various potentially associated proteins is depicted. FIG. 4C: Coimmunoprecipitation of CD47 and SLAMF7 in CD47 KO 293T cells expressing a Flag-tagged variant of mCD47 and a Myc-tagged variant of mSLAMF7. Flag and Myc tags were added at the carboxyl terminus of CD47 or SLAMF7, respectively. The abundance of the transfected proteins in total cell lysates was analyzed in parallel. IP, immunoprecipitation. FIGs. 4D-E: FRET assay. (FIG. 4D) CD47 KO 293T cells were transfected with tagged versions of mCD47 and mSLAMF7. After coupling the two proteins to different fluorophores (Dye 647 for CD47 as acceptor and Dye 547 for SLAMF7 as donor), proximity was analyzed by FRET, in the presence of the indicated MAbs. Schematic representations of the experimental conditions are depicted on the left, while representative confocal microscopy images are shown on the right. Pre- and post-bleaching confocal images, as well as the calculated FRET efficiency images and the differential interference contrast (DIC) images, are shown. FIG. 4E: FRET data for 18 independent cells are graphically represented. Scale bars, 5 pm. FIGs. 4F-H: LUV-based FRET assay. Recombinant histidine-tagged versions of the extracellular domain of mCD47 and mSLAMF7, coupled to different fluorophores (Dye 505 for SLAMF7 as donor and Dye TMR for CD47 as acceptor) were sequentially added to nickel-containing LUVs. Changes in donor fluorescence were analyzed over time. FIG. 4F: Schematic representation of assay. FIG. 4G: Time-course of normalized dye 505 fluorescence intensity for mSLAMF7 or hPD-L1. The interaction of hPD-1 and hPD-L1 was used as a positive control. FIG. 4H: Same as FIG. 4G, except that blocking anti-mCD47, blocking anti-mSLAMF7 or control MAbs were added 30 min before the LUV FRET assay. All data are means ± s.e.m. ****p < 0.0001. Results are representative of 5 (FIG. 4A), 3 (FIGs. 4C, D, G), and 2 (FIG. 4H) independent experiments. Results are pooled from 3 (FIGs. 4B, E) independent experiments.

[0150] FIGs. 5A-F show that freeing of SLAMF7 from CD47 correlates with greater phagocytosis. FIGs. 5A-B: The impact of blocking anti-mCD47 MAbs on the ability of mSLAMF7 on tumor cells (BI-141) to interact in trans with mSLAMF7 was tested. A schematic representation of the assay is depicted in (FIG. 5A). BI-141 cells expressing mCD47 and mSLAMF7 were treated with the indicated MAbs, prior to staining with mSLAMF7-Fc or control Fc fusion proteins. The mean fluorescence intensity (MFI) of fusion protein staining was assayed by flow cytometry. Representative experiment is shown in FIG. 5B, left, and quantification of the data is shown in FIG. 5B, right. FIG. 5C: Same as FIGs. 5A-B, except that BI-141 cells without mSLAMF7 expression were used. FIGs. 5D,E: Same as FIG. 2B, using control and CD47 KO L1210, with or without mSLAMF7 overexpression, as targets. Expression of SLAMF7 is depicted in FIG. 5D. empty curves, anti-SLAMF7; filled curves, Ctrl IgG. Phagocytosis assays are shown in FIG. 5E. FIG. 5F: Same as FIG. 5E, except that WT, SIRPa KO, SLAMF7 KO, or SIRPa-SLAMF7 DKO BMDMs were used. All data are means ± s.e.m. ns, not significant; **p < 0.01 , ***p < 0.001 , and ****p < 0.0001. Flow cytometry profiles are representative of 3 (FIGs. 5B-D) independent experiments. Results are pooled from 3 (FIGs. 5B,C) independent experiments; 3 (FIGs. 5E,F) mice studied in independent experiments. Each symbol represents one mouse.

[0151] FIGs. 6A-K show the identification of a first-in-class anti-SLAMF7 MAb with agonistic activity towards phagocytosis. FIGs. 6A,B: Same as FIG. 1B, using SIRPa KO (FIG. 6A) or WT (FIG. 6B) BMDMs, Raji cells and the indicated MAbs. Elo, elotuzumab. FIGs. 6C,D: Same as FIG. 6A, using BMDMs from WT or SIRPa KO mice and target cells expressing or not hSLAMF7 (Daudi, MM.1S, and activated mouse CD8+T cells expressing or not expressing hSLAMF7) were studied. FIG. 6E: Same as FIG. 1G, using human macrophages, Raji cells and the indicated MAbs. FIGs. 6F-H: Same as FIG. 6A, except that SFR-SIRPa DKO BMDMs, expressing or not expressing hSLAMF7 (FIG. 6F), and hSLAMF7-positive target (FIG. 6G, Raji) and mSLAMF7- positive target (FIG. 6H, L1210) as targets were used. Expression of hSLAMF7 (empty curves) was determined by flow cytometry. Filled curves, control IgG. FIG. 6I: RAG-1 KO mice were injected subcutaneously with Raji, in the presence of the indicated MAbs. Tumor volume overtime was measured using a caliper. FIG. 6J: Tumor volume in Rag1 mice injected subcutaneously with Raji cells, and with intraperitoneal injection of Fc-silent mAbs on days 14, 16, 18, 20 and 22, measured using a caliper on days 14, 16, 18, 20, 22 and 24. FIG. 6K: Survival curves of Rag1- / ~ mice injected subcutaneously with Raji cells, and with intraperitoneal injection of Fc-silent mAbs on days 14, 16, 18, 20 and 22. All data are mean t s.e.m. *P < 0.05, **P < 0.01 , ***P < 0.001 and ****p < 0.0001. Flow cytometry profiles are representative of 3 (FIG. 6F) independent experiments. Results are pooled from 3 (FIG. 6A, except 6 for Ctrl IgG and 5 for Z10) and (FIG. 6B-D,G,H) mice studied in independent experiments; 3 healthy human donors from 3 independent experiments (FIG. 6E); 11 (Ctrl IgG) or 12 (anti-hSLAMF7 MAb Z10) mice from 3 independent experiments (FIG. 6I), 18 mice (‘Ctrl IgG (MOPC21)’, ‘SIRPa mAb (no. 27) + SLAMF7 mAb (Z10)’ and ‘CD47 mAb (B6H12)’ groups) from four independent experiments, eight mice (‘SLAMF7 mAb (Elo) and ‘SIRPa mAb (no. 27) + SLAMF7 mAb (Elo)’ groups) and seven mice (‘SLAMF7 mAb (Z10)’ and ‘SIRPa mAb (no. 27)’ groups) from two independent experiments (FIG. 6K); ten mice from two independent experiments (FIG. 6I). Each symbol represents one mouse or healthy donor. FIGs. 7A-E show that the agonistic anti-SLAMF7 MAb is a non-blocking antibody that frees SLAMF7 from CD47. FIGs. 7A-B: Same as FIGs. 4D-E, except that hCD47 and hSLAMF7, in the presence of indicated Fc-silent MAbs, were studied. FIG. 7C: Actin polarization in SIRPa KO BMDMs incubated with Raji was detected by immunofluorescence. The images are representative examples of fully polarized (top images, arrows) and non-polarized (bottom images, arrows) conjugates. Bottom graph, quantitation. Scale bars, 5 pm. FIGs. 7D-E: Mapping of the binding sites of MAb Z10, MAb Z8 and MAb elotuzumab (Elo) on CD47 KO 293T cells expressing hSLAMF7, mSLAMF7, chimeras between hSLAMF7 and mSLAMF7, or mutants of hSLAMF7. The mean fluorescence intensity (MFI) of MAbs Z10, Z8 and Elo staining was assayed by flow cytometry (FIG. 7E). V, variable Ig-like; C, constant Ig-like; +, binding; -, no binding; WT, wild-type; V174A, valine 174-to-alanine 174 mutation; R181A, arginine 181-to-alanine 181 mutation; N182A, asparagine 182-to-alanine 182 mutation; F183A, phenylalanine 183-to-alanine 183 mutation. All data are means ± s.e.m. *p < 0.05, **p < 0.01 , ***p < 0.001 , and ****p < 0.0001. Photographs are representative of 3 (FIGs. 7A,C) independent experiments. Flow cytometry profiles are representative of 6 (FIG. 7D, except 2 for mSLAMF7) independent experiments. Results are pooled from 3 (FIGs. 7B,C,E) independent experiments. Each symbol represents one mouse.

[0152] FIGs. 8A-G show the generation of Fc-silent MAbs and anti-SIRPa MAbs. FIG. 8A: The three mutations (“LALAPG”) introduced in the Fc portion of MAbs to render them Fc-silent are depicted. LALAPG is: L234A (“LA”), leucine 234-to-alanine 234; L235A (“LA”), leucine 235-to- alanine 235; P329G (“PG”), proline 329-to-g lycine 329. FIG. 8B: Binding of Fc-intact and Fc-silent variants of mlgG2a (MAb MOPC21) or hlgG1 (MAb Z10) to FcRs on BMDMs was assessed by flow cytometry. FIG. 8C: Binding of anti-mSIRPa MAbs #23, #27 and MY-1 to EL-4 cells expressing full- length mSIRPa (empty right curves) or a variant of mSIRPa containing only the first Ig-like variable (V) domain (empty left curves) was assessed by flow cytometry. Filled curves, SIRPa-negative cells. FIG. 8D: The ability of anti-mSIRPa MAbs to block binding of a soluble mCD47-Fc fusion protein to EL-4 cells, expressing or not expressing mSIRPa, was studied by flow cytometry. MFI, mean fluorescence intensity. FIG. 8E: Binding of anti-mSIRPa MAbs to mSIRPa-Fc and 3 known isoforms of mSIRP H (a,b,c), an activating receptor related to SIRPa, was determined by ELISA. MY-1 , but not #23 and #27, also bound to SIRP H . FIG. 8F: Binding of Fc-silent anti-hCD47 MAbs or hSIRPa- Fc fusion proteins to CD47 KO and CD47-positive Daudi cells was evaluated by flow cytometry. FIG. 8G: Binding of Fc-silent anti-hSIRPa MAbs to SIRPa KO BMDMs, transduced with retroviruses encoding or not encoding the V1 or V2 version of hSIRPa, was evaluated by flow cytometry. These two variants are the most common variants of SIRPa found in the human population. Some MAbs such as 18D5 recognize only V1 . All data are means ± s.e.m. ****p < 0.0001 . Flow cytometry profiles are representative of 3 (FIGs. 8C,F,G) or 2 (FIG. 8B) independent experiments. Results are pooled from 5 (FIG. 8D) and 3 (FIG. 8E) independent experiments. FIGs. 9A-C show the pHrodo-based phagocytosis assay and sub-cutaneous tumor transplantation assay. FIGs. 9A-B: Same as FIG. 1 B, except that phagocytosis was assessed by flow cytometry using the pHrodo dye. A representative experiment is shown in FIG. 9A and quantification of multiple experiments is depicted in FIG. 9B. Cells with percentages (%) displaying enhanced staining with pHrodo are boxed. FIG. 9C: Tumors from the experiment depicted in FIG. 1D were dissected, weighed, measured and analyzed by flow cytometry. Macrophages were CD1 1 b+F4 / 80+; neutrophils were CD11 b+Ly6G+; and NK cells were CD1 1 b NK1.1+. All data are means ± s.e.m. ns, not significant; **p < 0.01 , ***p < 0.001 , and ****p < 0.0001. Flow cytometry profiles are representative of 3 (FIG. 9A) independent experiments. Results are pooled from 3 (FIG. 9B) mice studied in independent experiments or 14 (FIG. 9C) mice from 3 independent experiments. Each symbol represents one mouse.

[0153] FIGs. 10A-D show the impact of SLAMF7 in phagocytosis in vitro and in vivo. FIG. 10A: Same as FIG. 1 B, except that WT or SLAMF7 KO BMDMs, as well as L1210 (left) or activated CD4+T cells (right) as targets, were used. FIG. 10B: Same as FIG. 1 F, except that WT and SLAMF7 KO mice were used. FIG. 10C: Same as FIG. 1G, except that SIRPa KO BMDMs, expressing or not expressing hSIRPa version V1 or V2, and Raji as target, were used. FIG. 10D: Same as FIG. 1G, using WT BMDMs or SLAM family receptor (SFR) KO BMDMs, and Raji as target. SFR KO mice lack all SFRs, including SLAMF7. All data are means ± s.e.m. ns, not significant; **p < 0.01 , ***p < 0.001 , and ****p < 0.0001 . Results are pooled from a total of 3 (FIGs. 10A,C,D) mice studied in independent experiments or 6 (FIG. 10B) mice from 3 independent experiments. Each symbol represents one mouse.

[0154] FIGs. 11A-B show pHrodo-based phagocytosis and in vivo tumor formation assays. FIG. 11 A: Same as FIG. 2A, except that phagocytosis was assessed by flow cytometry using the pHrodo dye. A representative experiment is shown on the left and quantification of multiple experiments is depicted on the right. Cells with percentages (%) displaying enhanced staining with pHrodo are boxed. FIG. 11 B: Tumors from the experiment depicted in FIG. 2F were dissected, weighed, measured and analyzed by flow cytometry. Macrophages were CD1 1 b+F4 / 80+; neutrophils were CD1 1 b+Ly6G+, and NK cells were CD1 1 b NK1.1+. Data are means ± s.e.m. ns, not significant; *p < 0.05, ***p < 0.001 , and ****p < 0.0001 . Flow cytometry profiles are representative of 4 independent experiments. Results are pooled from a total of 4 (FIG. 11 A) mice studied in independent experiments or 1 1 (control L1210) or 10 (CD47 KO L1210) mice from 2 independent experiments (FIG. 11B), one mouse from “RAG-1 KO mice + CD47 KO L1210” group showed no clinically detectable tumor. Each symbol represents one mouse.

[0155] FIGs. 12A-E show that loss of SIRPa does not alter macrophage differentiation. FIG. 12A: Volcano plot of gene expression differences between SIRPa KO vs WT BMDMs is depicted. RNA from BMDMs was processed for RNA-Seq library preparation. Differential expression analysis was performed with DESeq2 v1 .14.1 from the raw alignment counts calculated with Featurecounts v1 .4.6. Differentially expressed genes were defined as genes with an adjusted p value of < 0.05 and a log2fold change of greater or equal to 1.0 (X axis), -log-io p value is shown on Y axis. FIG. 12B: Expression of various cell surface markers (empty curves) on BMDMs was evaluated by flow cytometry. Filled curves, Ctrl IgG. FIG. 12C: Same as FIG. 12B, except that BMDMs from a second independent SIRPa KO mouse strain (#54) were studied. FIGs. 12D,E: Same as FIG. 2B, except that BMDMs from SIRPa KO mouse strain #54 were studied. All data are means ± s.e.m. ns, not significant; **p < 0.01 , ***p < 0.001 , and ****p < 0.0001. 3 pairs of mice were studied in one experiment (FIG. 12A). Flow cytometry profiles are representative of 3 (FIGs. 12B,C) independent experiments. Results are pooled from a total of 3 (FIGs. 12D,E) mice studied in 3 independent experiments. Each symbol represents one mouse.

[0156] FIG. 13 shows that loss of SIRPa in mice promotes FcR-mediated tumor growth inhibition. Tumors from the experiment depicted in FIG. 3F were dissected, weighed, measured and analyzed by flow cytometry. Macrophages were CD1 1 b+F4 / 80+; neutrophils were CD1 1 b+Ly6G+; and NK cells were CD1 1 b NK1.1+. All data are means ± s.e.m. ns, not significant; ****p < 0.0001. Results are pooled from a total of 15 mice (16 mice for “RAG-1 KO + 7G7” group) from 3 independent experiments. One mouse from “RAG-1 KO + IgG” group showed no clinically detectable tumor. Each symbol represents one mouse.

[0157] FIG. 14A-H show CD47 interacts in cis with SLAMF7. FIG. 14A: Expression of Flag or CD47 (empty curves) on CD47 KO L1210 cells expressing or not expressing mCD47-Flag was assessed by flow cytometry. Permeabilization was used for the anti-Flag staining. Filled curves, Ctrl IgG. FIG. 14B,C: Same as FIG. 4B. A schematic representation of the protocol used for mass spectrometry is depicted in FIG. 14B. Means of the normalized total ion current (TIC) for the potential interactors are shown in FIG. 14C. FIG. 14D: Expression of hCD47 (empty curves) on control or CD47 KO 293T cells was assessed by flow cytometry. Filled curves, Ctrl IgG. FIG. 14E: Binding of soluble mSIRPa-Fc fusion protein and expression of mCD47 on transfected CD47 KO 293T cells were assessed by flow cytometry. FIG. 14F: Binding of soluble mSLAMF7-Fc fusion protein and expression of mSLAMF7 on transfected CD47 KO 293T cells were assessed by flow cytometry. FIGs. 14G,H: Same as FIGs. 4D,E, except that CD47 KO 293T cells were transfected with a variety of mSLAMF7 and mCD47 constructs. All data are means ± s.e.m. ns, not significant; ****p < 0.0001 . Flow cytometry profiles are representative of 3 (FIGs. 14E,F) or 2 (FIGs. 14A,D) independent experiments. Results are pooled from 3 (FIGs. 14C,H) independent experiments.

[0158] FIGs. 15A-H show structure-function analyses of CD47 and SLAMF7. FIG. 15A: Expression of mCD47 and mSLAMF7 and binding of mSIRPa-Fc (empty curves) on L1210 derivatives were assessed by flow cytometry. Filled curves, Ctrl IgG or Fc fusion protein. FIG. 15B: Same as FIG. 2B, using a variety of L1210 derivatives as targets. FIG. 15C: Expression of mSLAMF7 (empty curves) on SLAMF7 KO activated CD4+T cells transduced with variants of mSLAMF7 constructs. Filled curves, Ctrl IgG. FIG. 15D: Same as FIG. 15B, but using activated CD4+T cells expressing variants of mSLAMF7 as targets. FIGs. 15E-F: Schematic representation (FIG. 15E) and timecourse of normalized dye 505 fluorescence intensity (FIG. 15F) of LUV-based FRET assay of mSLAMF7 (donor) or hPD-L1 (donor) with mCD47 (acceptor) or hPD-1 (acceptor) monitored by a real time plate reader. FIG. 15G: Time-course of normalized dye 505 fluorescence intensity of LUV- based FRET assay of mSLAMF7 (donor) with mCD47 (acceptor), pretreated with Ctrl IgG MOPC21 , mSLAMF7 mAb 4G2 and mCD47 mAb Miap301 , monitored by a real time plate reader. FIG. 15H: Binding of soluble hCD47-Fc fusion protein to WT or CD47 Raji cells assessed by flow cytometry. All data are means ± s.e.m. ns, not significant; **p < 0.01 and ***p < 0.001. Flow cytometry profiles are representative of 3 (FIGs. 15A,C) or 2 (FIG. 15H) independent experiments. Results are pooled from 4 (FIG. 15B) or 4 (FIG. 15D; for all variants, except 3 for SLAMF7R75A) independent experiments. Each symbol represents one mouse.

[0159] FIGs. 16A-D show that MAb Z10 promotes phagocytosis with SIRPa blockade. FIG. 16A-B: Phagocytosis assay of Daudi (FIG. 16A, left), MM.1 S (FIG. 16A, right), and Fc-silent CD3 + CD28 mAbs activated mouse CD8+T cells expressing (FIG. 16B, right) or not expressing (FIG. 16B, left) hSLAMF7 by WT or SirpaABMDMs assayed by fluorescence microscopy. FIG. 16C: Schematic representations of the impact of MAb Z10, depending on whether MAb Z10 binds SLAMF7 on macrophages ortarget cells. FIG. 16D: Tumors from experiment depicted in FIG. 6I were dissected, weighed, measured and analyzed by flow cytometry. One mouse showed no detectable tumor in the ‘SIRPa mAb (#27) + SLAMF7 mAb (Z10)’ group on day 24. All data are means ± s.e.m. ns, not significant; **p < 0.01 , ***p < 0.001 , and ****p < 0.0001. Results are pooled from 3 (FIG. 16A-B) mice studied in 3 (FIG. 16A-B) independent experiments; 8 [except 7 for ‘SLAMF7 mAb (Z10)’ and ‘SIRPa mAb (#27)’ groups] mice from 2 independent experiments (FIG. 16D) Each symbol represents one mouse.

[0160] FIGs. 17A-B show that MAb Z10 does not affect conjugate formation. FIG. 17A: Formation of conjugates (boxed) between SIRPa KO BMDMs and Raji, in presence of Ctrl IgG or anti- hSLAMF7 MAb Z10, was detected by flow cytometry. Left, representative experiment. The percentages of conjugate formation are indicated below the boxes. Right, quantification of multiple independent experiments. FIG. 17B: Same as FIG. 17A, except that conjugates were assessed by confocal microscopy, after 20 min of co-incubation of SIRPa KO BMDMs and Raji. All data are means ± s.e.m. ns, not significant. Flow cytometry profiles are representative of 3 independent experiments (FIG. 17A). Results are pooled from a total of 3 mice studied in independent experiments. Each symbol represents one mouse.

[0161] FIGs. 18A-B show that MAb Z10 is a non-blocking Mab. FIGs. 18A,B: Same as FIG. 6A, except that BMDMs from WT or SIRPa KO mice and the indicated target cells, expressing or not expressing hCD47 [Raji (FIG. 18A), Daudi (FIG. 18B)] were studied. All data are means ± s.e.m. ns, not significant; **p < 0.01 and ****p < 0.0001 . Results are pooled from 3 mice studied in 3 independent experiments. Each symbol represents one mouse. FIGs. 19A-B show that MAb Z10 binds to the second Ig-like domain of SLAMF7. FIGs. 19A,B: Binding of anti-hSLAMF7 MAbs Z10, elotuzumab (Elo) and Z8 to CD47 KO 293T cells, expressing various chimeras between hSLAMF7 and mSLAMF7 (FIG. 19A) or various mutants of hSLAMF7 (FIG. 19B), was determined by flow cytometry. For the chimeras, the residues in hSLAMF7 (numbered) were replaced by the equivalent residues in mSLAMF7. For the point mutants, residues were replaced by alanines. +, binding; + / -, partial binding; -, no binding. Asterisks indicate the site of the mutation. Flow cytometry profiles are representative of 3 (except 2 for N177, P178, V179, S180) independent experiments.

[0162] FIGs. 20A-B show the impact of CD47 blockade, SIRPa blockade and agonistic anti-SLAMF7 MAb on phagocytosis. FIGs. 20A,B: Models of the effects of CD47 blockade, SIRPa blockade and agonistic anti-SLAMF7 Mab Z10 on phagocytosis of SLAMF7-positive (FIG. 20A) and antibody- opsonized (FIG. 20B) tumor cells are depicted. +, phagocytosis; -, no phagocytosis.

[0163] FIG. 21 A depicts the amino acid sequence of the light chain variable region (VL) of antibody Z10 disclosed herein. The residues defining the CDR1 , CDR2 and CDR3 domains (Chothia numbering) are underlined.

[0164] FIG. 21 B depicts the nucleotide sequence encoding the light chain variable region (VL) of antibody Z10 disclosed herein.

[0165] FIG. 21 C depicts the amino acid sequence of the light chain constant region (kappa) of antibody Z10 disclosed herein.

[0166] FIG. 21 D depicts the nucleotide sequence encoding the light chain constant region (kappa) of antibody Z10 disclosed herein.

[0167] FIG. 22A depicts the amino acid sequence of the heavy chain variable region (VH) of antibody Z10 disclosed herein. The residues defining the CDR1 , CDR2 and CDR3 domains (Chothia numbering) are underlined.

[0168] FIG. 22B depicts the nucleotide sequence encoding the heavy chain variable region (VH) of antibody Z10 disclosed herein.

[0169] FIG. 22C depicts the amino acid sequence of the heavy chain constant region (lgG1) of antibody Z10 disclosed herein.

[0170] FIG. 22D depicts the nucleotide sequence encoding the heavy chain constant region (lgG1) of antibody Z10 disclosed herein.

[0171] FIG. 23 depicts the amino acid sequence of human SLAMF7 (UniProt accession No. Q9NQ25).

[0172] DETAILED DISCLOSURE

[0173] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the technology (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising", "having", "including", and "containing" are to be construed as open- ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

[0174] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context.

[0175] The use of any and all examples, or exemplary language (“e.g.”, "such as") provided herein, is intended merely to better illustrate embodiments of the claimed technology and does not pose a limitation on the scope unless otherwise claimed.

[0176] No language in the specification should be construed as indicating any non-claimed element as essential to the practice of embodiments of the claimed technology.

[0177] Herein, the term "about" has its ordinary meaning. The term “about” is used to indicate that a value includes an inherent variation of error for the device or the method being employed to determine the value, or encompass values close to the recited values, for example within 10% of the recited values (or range of values).

[0178] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All subsets of values within the ranges are also incorporated into the specification as if they were individually recited herein.

[0179] Where features or aspects of the disclosure are described in terms of Markush groups or list of alternatives, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member, or subgroup of members, of the Markush group or list of alternatives.

[0180] Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in biology, cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0181] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present disclosure are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T. A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D. M. Glover and B. D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1- 4, IRL Press (1995 and 1996), and F. M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-lnterscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J. E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

[0182] In the studies described herein, the present inventors have shown that CD47 suppresses phagocytosis by a mechanism that is independent from SIRPa, and that involves direct cis interaction to and sequestration of the cell-intrinsic pro-phagocytic ligand SLAMF7 on the tumor cells, thereby preventing phagocytosis initiated by SLAMF7. Disruption of the CD47-SLAMF7 interaction on tumor cells using an anti-SLAMF7 antibody, in combination with SIRPa inhibition, was shown to restore tumor cell phagocytosis and anti-tumor immunity.

[0183] Accordingly, the present disclosure provides a SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on cells such as tumor cells. The SLAMF7- binding molecule may be used for increasing the susceptibility of cells (e.g., tumor cells) expressing SLAMF7 and CD47 to phagocytosis (e.g., for inducing the phagocytosis of cells expressing SLAMF7 and CD47), and / or for the treatment of a cancer comprising tumor cells expressing SLAMF7 and CD47 in a subject (e.g., a human subject). The present disclosure also provides a method for increasing the susceptibility of cells (e.g., tumor cells) expressing SLAMF7 and CD47 to phagocytosis (e.g., for inducing the phagocytosis of cells expressing SLAMF7 and CD47), the comprising contacting the cells with a SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on the cells. The present disclosure also provides the use of a SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on cells (e.g., tumor cells) for increasing the susceptibility of cells expressing SLAMF7 and CD47 to phagocytosis (e.g., for inducing the phagocytosis of cells expressing SLAMF7 and CD47). The present disclosure also provides the use of a SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on cells (e.g., tumor cells) for the manufacture of a medicament for increasing the susceptibility of cells expressing SLAMF7 and CD47 to phagocytosis (e.g., for inducing the phagocytosis of cells expressing SLAMF7 and CD47).

[0184] The expression “increasing the susceptibility to phagocytosis” means that cells are more likely or prone to be recognized and ingested by phagocytic immune cells such as neutrophils and monocytes / macrophages (relative to untreated cells). The SLAMF7-binding molecule described herein may be useful for eliminating any undesired cells that express or overexpress SLAMF7 and CD47, for example infected cells, defective cells, apoptotic cells or tumor cells. In an embodiment, the SLAMF7-binding molecule described herein is for increasing the susceptibility to phagocytosis of tumor cells.

[0185] The present disclosure also provides a method for treating a cancer comprising tumor cells expressing SLAMF7 and CD47 in a subject (e.g., a human subject), comprising administering an effective amount of a SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 to the subject. The present disclosure also provides the use of a SLAMF7- binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on tumor cells for treating a cancer comprising tumor cells expressing SLAMF7 and CD47 in a subject (e.g., a human subject). The present disclosure also provides the use of a SLAMF7-binding molecule that reduces or inhibits the interaction between SLAMF7 and CD47 on the tumor cells for the manufacture of a medicament for treating a cancer comprising tumor cells expressing SLAMF7 and CD47 in a subject (e.g., a human subject).

[0186] It would be understood by the skilled person that the SLAMF7-binding molecule does not inhibit the SLAMF7-SLAMF7 homotypic trans interaction, which is involved in the induction or stimulation of phagocytosis by the immune cells (e.g., macrophages).

[0187] As used herein, the term “SLAMF7-binding molecule” refers to any molecule capable of binding to SLAMF7 and reducing / inhibiting the interaction of SLAMF7 with CD47. The term “binding molecule” encompasses antibodies, antibody fragments and non-antibody binding agents, for example antibody mimetics such as those described in Yu et al. (2017) Annu Rev Anal Chem 10(1):293-320. Thus, the SLAMF7-binding molecules defined herein may be antibodies, antibody fragments, antibody mimetics, peptides, small molecules (e.g., compounds with molecular weights of less than 1000 Daltons), adnectins, affibodies, affilins, affimers, affitins, alphabodies, anticalins, aptamers, armadillo repeat protein-based scaffolds, atrimers, avimers, DARPins, fynomers, knottins, Kunitz domain peptides, monobodies, and nanofitins. The ability of the SLAMF7-binding molecule to reduce / inhibit the interaction of SLAMF7 with CD47 may be assessed using methods / assays known in the art, for example using a FRET-based assay as described in the Examples below.

[0188] In an embodiment, the SLAMF7-binding molecule is an antibody or an antigen-binding fragment thereof. The term “antibody or antigen-binding fragment thereof’ as used herein refers to any type of antibody / antibody fragment including monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), humanized antibodies, CDR-grafted antibodies, chimeric antibodies and antibody fragments so long as they exhibit the desired antigenic specificity / binding activity. Antibody fragments comprise a portion of a full-length antibody, generally an antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, linear antibodies, single-chain antibody molecules (e.g., single-chain FV, scFV), single domain antibodies (e.g., from camelids), shark NAR single domain antibodies, and multispecific antibodies formed from antibody fragments. Antibody fragments can also refer to binding moieties comprising CDRs or antigen binding domains including, but not limited to, VHregions (VH, VH-VH), anticalins, PepBodies, antibody-T-cell epitope fusions (Troybodies) or Peptibodies.

[0189] The term "monoclonal antibody" as used herein refers to an antibody from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are substantially similar and bind the same epitope(s), except for possible variants that may arise during production of the monoclonal antibody, such variants generally being present in minor amounts. Such monoclonal antibody typically includes an antibody comprising a variable region that binds a target, wherein the antibody was obtained by a process that includes the selection of the antibody from a plurality of antibodies. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones or recombinant DNA clones. It should be understood that the selected antibody can be further altered, for example, to improve affinity for the target, to humanize the antibody, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered variable region sequence is also a monoclonal antibody of this invention. In addition to their specificity, the monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including the hybridoma method (e.g., Kohler et al., Nature, 256:495 (1975); Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 , (Elsevier, N. Y., 1981), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technologies (see, e.g., Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol., 222:581-597 (1991); Sidhu et al., J. Mol. Biol. 338(2) :299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Nat. Acad. Sci. USA 101 (34): 12467-12472 (2004); and Lee et al. J. Immunol. Methods 284(1 -2): 119-132 (2004) and technologies for producing human or human-like antibodies from animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO98 / 24893, WO96 / 34096, WO96 / 33735, and WO91 / 10741 , Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immune, 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591 ,669 (all of GenPharm); 5,545,807; WO 97 / 17852, U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661 ,016, and Marks et al., Bio / Technology, 10: 779-783 (1992); Lonberg et al., Nature, 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al., Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology, 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol., 13: 65-93 (1995). Antibodies capable of specifically binding to the conformational epitope from NTSR1 defined herein can also be produced using phage display technology. Antibody fragments that selectively bind to the conformational epitope from NTSR1 defined herein can then be isolated. Exemplary methods for producing such antibodies via phage display are disclosed, for example, in U.S. Patent No. 6,225,447. The monoclonal antibodies herein specifically include "chimeric" or “recombinant” antibodies in which a portion of the light and / or heavy chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). Chimeric antibodies of interest herein include "humanized" antibodies.

[0190] In an embodiment, the SLAMF7-binding molecule, e.g., antibody or antigen-binding fragment thereof, binds to a domain or epitope comprising residues 172-174, 177 and 181-183 of human SLAMF7. The amino acid sequence of human SLAMF7 is depicted in FIG. 23 (SEQ ID NO:23), with the above-noted residues in bold and underlined. The results of the studies described herein (see, e.g., FIGs. 7E and 19A-B) show that an antibody recognizing the abovenoted domain or epitope is able to dissociate the CD47-SLAMF7 cis interaction on tumor cells, and thus any SLAMF7-binding molecule binding to the same domain or epitope is expected to have the ability to interfere with the CD47-SLAMF7 cis interaction. In an embodiment, the binding of the SLAMF7-binding molecule, e.g., antibody or antigen-binding fragment thereof, is reduced by at least 50% or 60% if the valine residue at position 174 of human SLAMF7 is mutated to alanine. In an embodiment, the binding of the SLAMF7-binding molecule, e.g., antibody or antigen-binding fragment thereof, is reduced by at least 50% if the asparagine residue at position 182 of human SLAMF7 is mutated to alanine.

[0191] In an embodiment, the SLAMF7-binding molecule is an antibody or an antigen-binding fragment thereof, and it comprises the complementary determining regions (CDRs) of antibody Z10 described in the present application. The amino acid sequences of the light and heavy chain variable regions of antibody Z10 are depicted in FIGs. 21A (SEQ ID NO:16) and 22A (SEQ ID NQ:20), respectively, with the residues defining the CDRs (CDR1 , CDR2 and CDR3) indicated. The term "complementarity determining regions" or "CDRs" when used herein refers to parts of immunological receptors that make contact with a specific ligand (SLAMF7) and determine its specificity. The CDRs of immunological receptors are the most variable part of the receptor protein, giving receptors their diversity, and are carried on six loops at the distal end of the receptor's variable domains, three loops coming from each of the two variable domains of the receptor.

[0192] In an embodiment, the antibody or antigen-binding fragment thereof comprises the following CDRs: a light chain CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence KASQDVDTAVA, a light chain CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence WASTRHT, a light chain CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence QQYRSYPFT, a heavy chain CDR1 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence GIDFSRY, a heavy chain CDR2 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence NPDSST, and a heavy chain CDR3 comprising or consisting of an amino acid sequence having at least 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence PGDYDAWYFDV.

[0193] In an embodiment, one ortwo residues in one or more of the above-noted CDRs sequences are substituted. In a further embodiment, one residue in one or more of the above-noted CDRs sequences are substituted. Such substitutions may be useful for increasing the affinity of the antibody or antigen-binding fragment thereof for SLAMF7 (e.g., an affinity-matured antibody or antigen-binding fragment thereof) and / or for reducing non-specific binding.

[0194] In an embodiment, the antibody or antigen-binding fragment thereof comprises the following CDRs: a light chain CDR1 comprising or consisting of the amino acid sequence KASQDVDTAVA (SEQ ID NO:1), a light chain CDR2 comprising or consisting of the amino acid sequence WASTRHT (SEQ ID NO:2), a light chain CDR3 comprising or consisting of the amino acid sequence QQYRSYPFT (SEQ ID NO:3), a heavy chain CDR1 comprising or consisting of the amino acid sequence GIDFSRY (SEQ ID NO:4), a heavy chain CDR2 comprising or consisting of the amino acid sequence NPDSST (SEQ ID NO:5), and a heavy chain CDR3 comprising or consisting of the amino acid sequence PGDYDAWYFDV (SEQ ID NO:6).

[0195] In an embodiment, the antibody or antigen-binding fragment thereof comprises the following framework regions (FRs): a light chain FR1 comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence DITMSQSHKFMSTSVGDRVSITC (SEQ ID NO:7), a light chain FR2 comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence WYQQKPGQSPKLLIY (SEQ ID NO:8), a light chain FR3 comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (SEQ ID NO:9), a light chain FR4 comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence WGTGTTVTVSS (SEQ ID NO: 10), a heavy chain FR1 comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence EVKLLQSGGGLVQPGGSLKLSCAAS (SEQ ID NO:11), a heavy chain FR2 comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence WMSWVRRAPGKGLEWIGEI (SEQ ID NO: 12), a heavy chain FR3 comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence INYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR (SEQ ID NO:13), and / or a heavy chain FR4 comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence WGTGTTVTVSS (SEQ ID NO:14).

[0196] In an embodiment, the antibody or antigen-binding fragment thereof comprises the following framework regions (FRs): a light chain FR1 comprising or consisting of the amino acid sequence of SEQ ID NO:7, a light chain FR2 comprising or consisting of the amino acid sequence of SEQ ID NO:8, a light chain FR3 comprising or consisting of the amino acid sequence of SEQ ID NO:9, a light chain FR4 comprising or consisting of the amino acid sequence of SEQ ID NQ:10, a heavy chain FR1 comprising or consisting of the amino acid sequence of SEQ ID NO:11 , a heavy chain FR2 comprising or consisting of the amino acid sequence, a heavy chain FR3 comprising or consisting of the amino acid sequence of SEQ ID NO: 12, and / or a heavy chain FR4 comprising or consisting of the amino acid sequence of SEQ ID NO:13. In a further embodiment, the antibody or antigen-binding fragment thereof all the above-noted FRs.

[0197] In an embodiment, the antibody or antigen-binding fragment thereof comprises a light chain variable region (VL) comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence depicted in FIG. 21A (SEQ ID NO: 16). In an embodiment, the differences relative to the reference variable light chain sequence are within one or more of the FRs. In a further embodiment, the antibody or antigen-binding fragment thereof comprises a variable light chain comprising or consisting of the sequence depicted in FIG. 21A (SEQ ID NO:16).

[0198] In an embodiment, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 50%, 60%, 70%, 75%, 80%, 85%, 90% or 95% identity with the amino acid sequence depicted in FIG. 22A (SEQ ID NQ:20). In an embodiment, the differences relative to the reference variable heavy chain sequence are within one or more of the FRs. In a further embodiment, the antibody or antigenbinding fragment thereof comprises a variable heavy chain comprising or consisting of the sequence depicted in FIG. 22A (SEQ ID NQ:20).

[0199] The sequences of the CDRs and FRs described herein are based on the numbering scheme of Chothia (Al-Lazikani et al., J Mol Biol. 1997 Nov 7;273(4):927-48). However, the skilled person would understand that the amino acids forming the CDRs and FRs regions in the sequences of antibody Z10 may vary depending on the numbering scheme used. Tables 1 and 2 below depict the sequences of the CDRs and FRs regions of antibody Z10 according to commonly used antibody numbering schemes.

[0200] Table 1 : Predicted FR and CDR sequences in antibody Z10 light chain variable region

[0201] Region Definition Sequence Fragment (SEQ ID NO:) Residues Length LFR1 Chothia DITMSQSHKFMSTSVGDRVSITC - (7) 1 -23 23

[0202] AbM DITMSQSHKFMSTSVGDRVSITC - (7) 1 -23 23 Kabat DITMSQSHKFMSTSVGDRVSITC - (7) 1 -23 23

[0203] Contact DITMSQSHKFMSTSVGDRVSITCKASQDV (24) 1 -29 29

[0204] IMGT DITMSQSHKFMSTSVGDRVSITCKAS— (25) 1 -26 26

[0205] CDR-L1 Chothia KASQDVDTAVA- (1) 24-34 11

[0206] AbM KASQDVDTAVA- (1) 24-34 11

[0207] Kabat KASQDVDTAVA- (1) 24-34 11

[0208] Contact - DTAVAWY (26) 30-36 7

[0209] IMGT — QDVDTA— - (27) 27-32 6

[0210] LFR2 Chothia -WYQQKPGQSPKLLIY (8) 35-49 15

[0211] AbM -WYQQKPGQSPKLLIY (8) 35-49 15

[0212] Kabat -WYQQKPGQSPKLLIY (8) 35-49 15

[0213] Contact — QQKPGQSPK — (28) 37-45 9

[0214] IMGT VAWYQQKPGQSPKLLIY (29) 33-49 17

[0215] CDR-L2 Chothia — WASTRHT (2) 50-56 7

[0216] AbM — WASTRHT (2) 50-56 7

[0217] Kabat — WASTRHT (2) 50-56 7

[0218] Contact LLIYWASTRH- (30) 46-55 10 IMGT — WA - 50-51 2

[0219] LFR3 Chothia - GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (9) 57 - 88 32

[0220] AbM - GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (9) 57 - 88 32

[0221] Kabat - GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (9) 57 - 88 32

[0222] Contact — TGVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (31) 56-88 33

[0223] STRHTGVPDRFTGSGSGTDFTLTISNVQSEDLADYFC „oo37

[0224] CDR-L3 Chothia QQYRSYPFT (3) 89 - 97 9

[0225] AbM QQYRSYPFT (3) 89 - 97 9

[0226] Kabat QQYRSYPFT (3) 89 - 97 9

[0227] Contact QQYRSYPF- (33) 89 - 96 8

[0228] IMGT QQYRSYPFT (3) 89-97 9

[0229] LFR4 Chothia -FGSGTKLEIK (10) 98- 107 10

[0230] AbM -FGSGTKLEIK (10) 98- 107 10

[0231] Kabat -FGSGTKLEIK (10) 98- 107 10

[0232] Contact TFGSGTKLEIK (34) 97- 107 11

[0233] IMGT -FGSGTKLEIK (10) 98- 107 10

[0234] Table 2: Predicted FR and CDR sequences in antibody Z10 heavy chain variable region

[0235] Region Definition Sequence Fragment Residues Length

[0236] HFR1 Chothia EVKLLQSGGGLVQPGGSLKLSCAAS (11) 1 -25 25

[0237] AbM EVKLLQSGGGLVQPGGSLKLSCAAS (11) 1 -25 25

[0238] Kabat EVKLLQSGGGLVQPGGSLKLSCAASGIDFS (35) 1 -30 30

[0239] Contact EVKLLQSGGGLVQPGGSLKLSCAASGIDF- (36) 1 -29 29

[0240] IMGT EVKLLQSGGGLVQPGGSLKLSCAAS - (11) 1 -25 25

[0241] CDR-H1 Chothia GIDFSRY— (4) 26-32 7

[0242] AbM GIDFSRYWMS (37) 26-35 10

[0243] Kabat - RYWMS (38) 31 -35 5 Contact - SRYWMS (39) 30-35 6

[0244] IMGT GIDFSRYW- (40) 26-33 8

[0245] HFR2 Chothia WMSWVRRAPGKGLEWIGEI (12) 33-51 19 AbM — WVRRAPGKGLEWIG- (41 ) 36-49 14

[0246] Kabat —WVRRAPGKGLEWIG- (41) 36-49 14

[0247] Contact — WVRRAPGKGLE - (42) 36-46 11

[0248] IMGT -MSWVRRAPGKGLEWIGE- (43) 34-50 17

[0249] CDR-H2 Chothia - NPDSST - (5) 52-57 6

[0250] AbM — EINPDSSTIN - (44) 50-59 10

[0251] Kabat — EINPDSSTINYAPSLKD (45) 50-66 17

[0252] Contact WIGEINPDSSTIN - (46) 47-59 13

[0253] IMGT — INPDSSTI - (47) 51 -58 8

[0254] HFR3 Chothia INYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR 58-98 41 (13)

[0255] AbM -YAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR 60-98 39

[0256] (48)

[0257] Kabat - KFIISRDNAKNTLYLQMSKVRSEDTALYYCAR (49) 67-98 32

[0258] Contact -YAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYC- 60-96 37

[0259] (50)

[0260] IMGT -NYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYC- 59-96 38 (51)

[0261] CDR-H3 Chothia -PGDYDAWYFDV (6) 99-109 11

[0262] AbM -PGDYDAWYFDV (6) 99-109 11

[0263] Kabat -PGDYDAWYFDV (6) 99-109 11

[0264] Contact ARPGDYDAWYFD- (52) 97- 108 12

[0265] IMGT ARPGDYDAWYFDV (53) 97-109 13

[0266] HFR4 Chothia -WGTGTTVTVSS (14) 110-120 11

[0267] AbM -WGTGTTVTVSS (14) 110-120 11

[0268] Kabat -WGTGTTVTVSS (14) 110-120 11

[0269] Contact VWGTGTTVTVSS (54) 109-120 12

[0270] IMGT -WGTGTTVTVSS (14) 110-120 11

[0271] Variations in the antibodies or antigen-binding fragments thereof described herein, can be made, for example, using any of the techniques and guidelines for conservative and nonconservative mutations set forth, for instance, in U.S. Patent No.5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the antibody that results in a change in the amino acid sequence as compared with the native sequence antibody. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the anti-SLAMF7 antibody or antigen-binding fragment thereof. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the antibody or antigenbinding fragment thereof with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence. In embodiment, the variant exhibit at least 50%, 55% or 60%, preferably at least 65, 70, 75, 80, 90, 95, 96, 97, 98 or 99% sequence identity with the sequence of the antibody or antigen-binding fragment thereof described herein, and maintain the ability to specifically bind to SLAMF7.

[0272] "Identity" refers to sequence identity between two polypeptides. Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0273] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0274] In an embodiment, the anti-SLAMF7 antibody or antigen-binding fragment thereof comprises at least one constant domain, e.g., a constant domain of a light and / or heavy chain, or a fragment thereof. In a further embodiment, the anti-SLAMF7 antibody or antigen-binding fragment thereof comprises a Fragment crystallizable (Fc) fragment of the constant heavy chain of an antibody. The Fc fragment may comprise two or three constant domains, e.g., a CH2domain and CH3domain. The Fc region may be obtained from a human IgG 1 , a human lgG4, or a variant of a human lgG1 or lgG4 having up to ten amino acid modifications, for example. In an embodiment, the Fc fragment comprises or consists of the CH2domain and CH3domain of a human antibody, preferably a human IgG such as lgG1. In an embodiment, the anti-SLAMF7 antibody or antigen-binding fragment thereof comprises a light chain constant domain comprising at least 50%, 55% or 60%, preferably at least 65, 70, 75, 80, 90, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence depicted in FIG. 21C (SEQ ID NO:18). In an embodiment, the anti-SLAMF7 antibody or antigen-binding fragment thereof comprises a light chain constant domain comprising the amino acid sequence depicted in FIG. 21C (SEQ ID NO:18). In an embodiment, the anti-SLAMF7 antibody or antigen-binding fragment thereof comprises a heavy chain constant domain comprising at least 50%, 55% or 60%, preferably at least 65, 70, 75, 80, 90, 95, 96, 97, 98 or 99% sequence identity with the amino acid sequence depicted in FIG. 22C (SEQ ID NO:22). In an embodiment, the anti-SLAMF7 antibody or antigen-binding fragment thereof comprises the amino acid sequence depicted in FIG. 22C (SEQ ID NO:22).

[0275] Covalent modifications of antibodies or antigen-binding fragments thereof are included within the scope of this disclosure. Covalent modifications include reacting targeted amino acid residues of the antibody or antigen-binding fragment thereof with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the antibody or antigen-binding fragment thereof. Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0276] Other types of covalent modification of the antibody or antigen-binding fragment thereof included within the scope of this disclosure include altering the native glycosylation pattern of the antibody or antigen-binding fragment thereof (Beck et al., Curr. Pharm. Biotechnol. 9: 482-501 , 2008; Walsh, Drug Discov. Today 15: 773-780, 2010), and linking the antibody or antigen-binding fragment thereof to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG), polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301 ,144; 4,670,417; 4,791 ,192 or 4,179,337.

[0277] The SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) may further comprise one or more modifications that confer additional biological properties to antibody or antigen-binding fragment thereof such as protease resistance, plasma protein binding, increased plasma half-life, intracellular penetration, etc. Such modifications include, for example, covalent attachment of molecules / moiety to the antibody or antigen-binding fragment thereof such as fatty acids (e.g., C6-Ci8), attachment of proteins such as albumin (see, e.g., U.S. Patent No. 7,268,113); sugars / polysaccharides (glycosylation), biotinylation or PEGylation (see, e.g., U.S. Patent Nos. 7,256,258 and 6,528,485). The above description of modification of the antibody or antigen-binding fragment thereof does not limit the scope of the approaches nor the possible modifications that can be engineered. Thus, in another aspect, the present disclosure provides a conjugate comprising the antibody or antigen-binding fragment thereof described herein and one or more additional molecules or agents (hereinafter secondary molecules or agents). The antibody or antigen-binding fragment thereof may be conjugated to any type of synthetic or natural secondary molecules or agents, such as peptides, proteins, saccharides / polysaccharides, lipids, naturally-occurring or synthetic polymers / co-polymers, etc. to modify one or more properties of the antibody or antigen-binding fragment thereof.

[0278] In an embodiment, the conjugate comprises a covalent link or bond between the antibody or antigen-binding fragment thereof and the molecule conjugated thereto. The molecule may be conjugated directly to the antibody or antigen-binding fragment thereof, or indirectly via a linker. The linker may be a polypeptide linker comprising one or more amino acids or another type of chemical linker (e.g., a carbohydrate linker, a lipid linker, a fatty acid linker, a polyether linker, PEG, etc.

[0279] In an embodiment, the SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) is labelled or conjugated with one or more moieties. The SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) may be labeled with one or more labels such as a biotin label, a fluorescent label, an enzyme label, a coenzyme label, a chemiluminescent label, or a radioactive isotope label. In an embodiment, the antibody or antigenbinding fragment thereof is labelled with a detectable label, for example a fluorescent moiety (fluorophore). Useful detectable labels include fluorescent compounds (e.g., fluorescein isothiocyanate, Texas red, rhodamine, fluorescein, Alexa Fluor® dyes, and the like), radiolabels, enzymes (e.g., horseradish peroxidase, alkaline phosphatase and others commonly used in a protein detection assays), streptavidin / biotin, and colorimetric labels such as colloidal gold, colored glass or plastic beads (e.g., polystyrene, polypropylene, latex, etc.). Chemiluminescent compounds may also be used. Such labelled antibodies or antigen-binding fragments thereof may be useful, for example, for the detection of SLAMF7 and / or SLAMF7-expressing cells in vivo or in vitro, e.g., by flow cytometry, immunohistochemistry, etc. The antibody or antigen-binding fragment thereof can also be conjugated to detectable or affinity tags that facilitate detection and / or purification of the antibody or antigen-binding fragment thereof. Such tags are well known in the art. Examples of detectable or affinity tags include polyhistidine tags (His-tags), polyarginine tags, polyaspartate tags, polycysteine tags, polyphenylalanine tags, glutathione S-transferase (GST) tags, Maltose binding protein (MBP) tags, calmodulin binding peptide (CBP) tags, Streptavidin / Biotin-based tags, HaloTag®, Profinity eXact® tags, epitope tags (such as FLAG, hemagglutinin (HA), HSV, S / S1 , c-myc, KT3, T7, V5, E2, and Glu-Glu epitope tags), reporter tags such as p-galactosidase (P-gal), alkaline phosphatase (AP), chloramphenicol acetyl transferase (CAT), and horseradish peroxidase (HRP) tags (see, e.g., Kimple et al., Curr Protoc Protein Sci. 2013; 73: Unit-9.9).

[0280] The SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) may alternatively or further be conjugated to an antitumor agent (chemotherapeutic agent, toxin, radioisotope, etc.) so as to deliver the antitumor agent to the tumor cells that expressed SLAMF7, i.e., using the SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) as a targeting agent for tumor cells. Such conjugates are commonly referred to as protein / peptide- drug conjugates (PDCs) or antibody-drug conjugates (ADCs).

[0281] The antitumor agent may be any compound that has the ability to inhibit the growth and / or kill tumor cells and includes, for example, small molecules, peptides, proteins, oligonucleotides (e.g., siRNA, shRNA), radionuclide agents (e.g.,177Lu,18F,68Ga,90Y,99mTc,111ln,218Bi,221At,225Ac,227Th), as well as drug delivery systems including nanoparticles (e.g., lipid nanoparticles), liposomes, nanotubes, etc., loaded with a therapeutic antitumor agent.

[0282] In an embodiment, the antitumor agent is a chemotherapeutic agent. The term “chemotherapeutic agent” refers to agents that kill tumor cells and / or inhibit their proliferation / growth. Examples in chemotherapeutic agents include alkylating agents (e.g., Cyclophosphamide, Ifosfamide, Mechlorethamine, Chlorambucil, Melphalan, Dacarbazine, Nitrosoureas, Temozolomide, Carmustine, Lomustine, Streptozocin, Busulfan, Procarbazine), anthracyclines (e.g., Daunorubicin, Doxorubicin, Epirubicin, Idarubicin, Mitoxantrone, Valrubicin), Monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), cytoskeletal disruptors (e.g., taxanes such as Paclitaxel, Docetaxel, Abraxane, Taxotere, cabazitaxel), histone deacetylase inhibitors (e.g., Vorinostat, Romidepsin), topoisomerase I inhibitors (e.g., camptothecin analogs such as Irinotecan, Topotecan, SN-38, Silatecan, Exatecan, Lurtotecan, Deruxtecan), topoisomerase II inhibitors (e.g., Etoposide, Teniposide, Tafluposide), kinase inhibitors (e.g., Bortezomib, Erlotinib, Gefitinib, Imatinib, Vemurafenib, Vismodegib, Dasatinib, Nilotinib, Osimertinib, Crizotinib, Dabrafenib, Vemurafenib, Trametinib, Ibrutinib), nucleotide analogs and precursor analogs (e.g., Azacitidine, Azathioprine, Capecitabine, Cytarabine, Doxifluridine, Fluorouracil (5-FU), Gemcitabine, Hydroxyurea, Mercaptopurine, Methotrexate, Tioguanine (Thioguanine)), peptide antibiotics (e.g., Bleomycin, Actinomycin), platinum-based agents (e.g., Carboplatin, Cisplatin, Oxaliplatin), retinoids (Tretinoin, Alitretinoin, Bexarotene), SERCA inhibitors (e.g., Thapsigargin), mitotic inhibitors such as vinca alkaloids and derivative (e.g., Vinblastine, Vincristine, Vindesine, Vinorelbine), tubulin inhibitors such as Maytansinoids, maytansinoid derivatives such as Mertansine (DM1) or Ravtansine (DM4), Auristatins, Calicheamicins, Tubulysins, Amatoxin orAmanitin, as well as natural phytochemicals having antitumor properties such as curcumin, Alkaloids (e.g., Chlorogenic acid, Theobromine, Theophylline), Anthocyanins (e.g., Cyanidin, Malvidin, Carotenoids (Beta-Carotene, Lutein, Lycopene), Coumestans, Flavan-3-Ols, Flavonoids (e.g., Epicatechin, Hesperidin, Isorhamnetin, Kaempferol, Myricetin, Naringin, Nobiletin, Proanthocyanidins, Quercetin, Rutin, Tangeretin), Hydroxycinnamic Acids (e.g., Chicoric acid, Coumarin, Ferulic acid, Scopoletin), Isoflavones (e.g., Daidzein, Genistein), Lignans (e.g., Silymarin), Monoterpenes (e.g., Geraniol, Limonene), Organosulfides (e.g., Allicin, Glutathione, lndole-3-Carbinol, Isothiocyanates, Sulforaphane), Damnacanthal, Digoxin, Phytic acid, Phenolic Acids (e.g., Capsaicin, Ellagic Acid, Gallic acid, Rosmarinic acid, Tannic Acid), Phytosterols (e.g., Beta-Sitosterol), Saponins, Stylbenes (e.g., Pterostilbene, Resveratrol), Triterpenoids (e.g., Ursolic acid), Xanthophylls (e.g., Astaxanthin, Beta-Cryptoxanthin), and Monophenols (e.g., Hydroxytyrosol).

[0283] In another embodiment, the molecule may be conjugated / attached to the side chain of one the amino acids of the SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof). Methods for conjugating moieties to side-chains of amino acids are well known in the art. For example, chemical groups that react with primary amines (-NH2) present in the side-chain of lysine residues such as isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, imidoesters, carbodiimides, anhydrides, and fluorophenyl esters may be used to conjugate the molecule to the antibody or antigen-binding fragment thereof. Most of these groups conjugate to amines by either acylation or alkylation. Cysteine residues present in the self-assembling domain may also be used to attach the antigen.

[0284] In an embodiment, the SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) is part of a multispecific binding molecule (e.g., a multispecific antibody or antigen-binding fragment thereof). In an embodiment, the SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) is part of a bispecific binding molecule (e.g., a bispecific antibody or antigen-binding fragment thereof).

[0285] Exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED) body, leucine zipper, Duobody, lgG1 / lgG2, dual acting Fab (DAF)-lgG, and Mab2bispecific formats (see, e.g., Klein et al. 2012, mAbs 4:6, 1-11 , and references cited therein, for a review of the foregoing formats). Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody- oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al. (2012) Site-specific DNA-antibody conjugates for specific and sensitive immuno-PCR, PNAS 109 (10) 3731-3736; DOI: 10.1073 / pnas.1120682109, and U.S. Patents Nos. 4,496,689; 4,301 ,144; 4,670,417; 4,791 ,192 or 4,179,337).

[0286] In an embodiment, the bispecific molecule (e.g., bispecific antibody or antigen-binding fragment thereof) is designed to simultaneously bind to SLAMF7 and to a checkpoint inhibitor. The term “immune checkpoint inhibitor” (I C I) or “immune checkpoint blocker” (ICB) as used herein refers to an agent that block or inhibit the activity of a negative regulator of the immune response. In an embodiment, the ICI blocks or inhibits the activity of T cells (e.g., CTLs and / or CD4 helper T cells) and / or of NK cells. Examples of such negative regulators of the immune response (i.e., immune checkpoint) include Adenosine A2A receptor (A2AR), B7-H3 (CD276), B7-H4 (VTCN1), B and T Lymphocyte Attenuator (BTLA or CD272), Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4, CD152), CD47 / SIRPa, Indoleamine 2,3-dioxygenase (IDO), Killer-cell Immunoglobulin- like Receptor (KIR), Lymphocyte Activation Gene-3 (LAG3), nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2), poliovirus receptor-related immunoglobulin (PVRIG), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), Programmed Death 1 (PD-1) receptor, PD-L1 , PD-L2, T-cell Immunoglobulin domain and Mucin domain 3 (TIM- 3), V-domain Ig suppressor of T cell activation (VISTA), and Sialic acid-binding immunoglobulin- type lectin 7 (SIGLEC7 or CD328) and SIGLEC9 (CD329). In an embodiment, the immune checkpoint inhibitor is an inhibitor of CTLA-4, PD-1 or PD-L1. Examples of immune checkpoint inhibitors includes anti-PD-1 antibodies / blockers (e.g., Tislelizumab, Penpulimab, Pidilizumab, Sintilimab, Toripalimab, Retifanlimab, Dostarlimab, Nivolumab, Cemiplimab, Pembrolizumab, Spartalizumab, Camrelizumab, JTX-4014, INCMGA00012 (MGA012), AMP-224, AMP-514), anti- PD-L1 antibodies / blockers (e.g., Durvalumab, Avelumab, Atezolizumab, KN035, CK-301 , AUNP12, CA-170, BMS-986189), anti-CTLA-4 antibodies (e.g., Tremelimumab, Ipilimumab), anti- LAG-3 antibodies (e.g., Relatlimab, LAG525 (IMP701), REGN3767 (R3767), Bl 754,091 , tebotelimab (MGD013), eftilagimod alpha (IMP321), FS118), anti-TIM-3 antibodies (MBG453, Sym023, TSR-022), anti-B7-H3 / H4 antibodies (e.g., MGC018, FPA150), CD73 antagonists / anti- CD73 antibodies (e.g., Mupadolimab (CPI-006), Oleclumab (MEDI9447), Uliledlimab, AB680, BMS-986179, NZV930, AK119, SYM024, INCA00186, ORIC-533, IPH5301 , PSB-1248937), and CD39 antagonists (TTX-030, IPH5201 , SRF617), anti-NKG2A antibodies (Monalizumab), anti- PVRIG (e.g., COM701), anti-CEACAM1 antibodies (e.g., CM24), and CD47 blockers / inhibitors (Evorpacept (ALX148), Hu5F9-G4 (5F9), TTI-662, RRx-001) (see, e.g., Marin-Acevedo et al., Next generation of immune checkpoint inhibitors and beyond, Journal of Hematology & Oncology, volume 14, Article number: 45 (2021); Xia et al., CD39 / CD73 / A2AR pathway and cancer immunotherapy, Molecular Cancer, volume 22, Article number: 44 (2023)). The chemical structures and sequences of the above-noted immune checkpoint inhibitors are incorporated herein by reference.

[0287] In an embodiment, the bispecific molecule (e.g., bispecific antibody or antigen-binding fragment thereof) is designed to simultaneously bind to SLAMF7 and to a tumor antigen, for example a tumor antigen expressed by tumor cells also expressing SLAMF7.

[0288] In another embodiment, the bispecific molecule (e.g., bispecific antibody or antigen-binding fragment thereof) is designed to simultaneously bind to SLAMF7 and to SIRPa. More particularly, it may include anti-SIRPa antibodies or antibody fragments, soluble SIRPa polypeptides or fusion proteins that interfere with the interaction between SIRPa and CD47.

[0289] Examples of anti-SIRPa antibodies include antibody BR105 (Wu et al., Journal for ImmunoTherapy of Cancer 2022; 10: e004054. doi: 10.1136 / jitc-2021 -004054, WO2022 / 121980), SIRP-1 and SIRP-2 (Andrejeva et al., J. Immunol. 2021 Feb 15; 206(4): 712-721. doi: 10.4049 / jimmunol.2001019. Epub 2021 Jan 11), ADU-1805 (Voets et al., J Immunother Cancer. 2019 Dec 4;7(1):340. doi: 10.1186 / S40425-019-0772-0, W02018 / 190719), Bl 765063 (OSE- 172), 1 H9 (Liu et al, JCI Insight, 2020; 5(12): e134728), GS-0189 (Narkhede et al., eJHaem, Volume 4, Issue 2, p. 370-380) and CC-95251 (Behrens etal., Cancers (Basel). 2022 Jul; 14(14): 3366). Several anti-SIRPa antibodies are described in PCT publications Nos. W02018 / 190719, WO2022 / 121980, WO2022 / 254379, W02020 / 180811 , WO2021 / 226591 , WO2021 / 226576, W02023 / 010076, WO2019 / 023347, WO2018 / 107058, WO2013 / 056352, and WO2015 / 138600. The antibodies and antigen-binding fragments described in these documents, including the sequences of the CDRs and variable regions, are incorporated herein by reference. Soluble SIRPa polypeptides and fusion proteins capable of inhibiting SIRPa are described in PCT publications Nos. WO2016 / 023040, W02023 / 003331 , WO2013 / 109752, WO2014 / 094122, and WO2017 / 027422. The soluble SIRPa polypeptides and fusion proteins described in these documents are incorporated herein by reference. Representative soluble SIRPa fusion proteins capable of inhibiting SIRPa include TTI-621 (SIRPa-lgG1 Fc), and its high-affinity variant ALX148 (Evorpacept), TTI-622 (SIRPa-lgG4 Fc), IMM-01 , and SL-172154 (see, e.g., Son et al., Front Immunol. 2022; 13: 1027235). Thus, the bispecific antibody may include any of the above-noted anti-SIRPa antibodies or antibody fragments, soluble SIRPa polypeptides or fusion proteins.

[0290] A further aspect of the present disclosure provides nucleic acids encoding the SLAMF7- binding molecule (e.g., antibody or antigen-binding fragment thereof) described herein. The isolated nucleic acid may be a synthetic DNA, a non-naturally occurring mRNA, or a cDNA, for example. The nucleic acid may be inserted within a plasmid, vector, or transcription or expression cassette. The nucleic acids encoding the antibody or antigen-binding fragment described herein may be made and the expressed antibodies or antigen-binding fragments described may be tested using conventional techniques well known in the art.

[0291] In another aspect, the present disclosure provides a cell, for example a recombinant host cell, expressing the SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) described herein. Methods of preparing antibodies or antigen-binding fragments comprise expressing the encoding nucleic acid(s) in a host cell under conditions to produce the antibodies or antigen-binding fragments, and recovering the antibodies or antigen-binding fragments. The process of recovering the antibodies or antigen-binding fragments may comprise isolation and / or purification of the antibodies or antigen-binding fragments. The method of production may comprise formulating the antibodies or antigen-binding fragments into a composition including at least one additional component, such as a pharmaceutically acceptable excipient. The term "recombinant host cell" (or simply "host cell"), as used herein, is intended to refer to a cell into which exogenous DNA has been introduced. It should be understood that such terms are intended to refer not only to the particular subject cell, but, to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "host cell" as used herein. Preferably host cells include prokaryotic and eukaryotic cells selected from any of the Kingdoms of life. Preferred eukaryotic cells include protist, fungal, plant and animal cells. Most preferably host cells include but are not limited to the prokaryotic cell line E. Coir, mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; the fungal cell Saccharomyces cerevisiae, plant cells, or algae cells.

[0292] In another aspect, the present disclosure provides a composition comprising the SLAMF7- binding molecule (e.g., antibody or antigen-binding fragment thereof) defined herein. In an embodiment, the composition further comprises the above-mentioned SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) and a carrier or excipient, in a further embodiment a pharmaceutically acceptable carrier or excipient. Such compositions may be prepared in a manner well known in the pharmaceutical art by mixing the SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) having a suitable degree of purity with one or more optional pharmaceutically acceptable carriers or excipients (see Remington: The Science and Practice of Pharmacy, by Loyd Allen, Jr, 2012, 22ndedition, Pharmaceutical Press; Handbook of Pharmaceutical Excipients, by Rowe et al., 2012, 7thedition, Pharmaceutical Press). The carrier / excipient can be suitable for administration of the SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) thereof by any conventional administration route, for example, for oral, intravenous, parenteral, subcutaneous, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intracapsular, intraspinal, intrathecal, epidural, intracisternal, intraperitoneal, intranasal or pulmonary (e.g., aerosol) administration. In an embodiment, the carrier / excipient is adapted for administration of the SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) by the intravenous or subcutaneous route. In an embodiment, the carriers / excipients are adapted for administration of the SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) by the intravenous route. In another embodiment, the carriers / excipients are adapted for administration of the SLAMF7-binding molecule (e.g., antibody or antigen-binding fragment thereof) by the subcutaneous route.

[0293] An "excipient" as used herein has its normal meaning in the art and is any ingredient that is not an active ingredient (drug) itself. Excipients include for example binders, lubricants, diluents, fillers, thickening agents, disintegrants, plasticizers, coatings, barrier layer formulations, lubricants, stabilizing agent, release-delaying agents and other components. "Pharmaceutically acceptable excipient" as used herein refers to any excipient that does not interfere with effectiveness of the biological activity of the active ingredients (SLAMF7-binding molecule) and that is not toxic to the subject, i.e., is a type of excipient and / or is for use in an amount which is not toxic to the subject. Excipients are well known in the art, and the present system is not limited in these respects. In certain embodiments, one or more formulations of the dosage form include excipients, including for example and without limitation, one or more binders (binding agents), thickening agents, surfactants, diluents, release-delaying agents, colorants, flavoring agents, fillers, disintegrants / dissolution promoting agents, lubricants, plasticizers, silica flow conditioners, glidants, anti-caking agents, anti-tacking agents, stabilizing agents, anti-static agents, swelling agents and any combinations thereof. As those of skill would recognize, a single excipient can fulfill more than two functions at once, e.g., can act as both a binding agent and a thickening agent. As those of skill will also recognize, these terms are not necessarily mutually exclusive. Examples of commonly used excipient include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols, such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are wetting agents or auxiliary substances, such as emulsifying agents, preservatives, or buffers, which increase the shelf life or effectiveness.

[0294] The composition may also comprise one or more additional active agents for the treatment the targeted disease / condition or for the management of symptom(s) of the targeted disease / condition (e.g., pain killers, anti-nausea agents, etc.), as described in more detail below.

[0295] The SLAMF7-binding molecule described herein may be useful for increasing the susceptibility to phagocytosis of tumor cells expressing SLAMF7 and CD47, thereby inducing the phagocytosis of the tumor cells by phagocytic immune cells (e.g., macrophages). Thus, the SLAMF7-binding molecule described herein may be useful for treating cancers comprising tumor cells expressing SLAMF7 and CD47.

[0296] T umor cells expressing or overexpressing SLAMF7 and CD47 include hematopoietic tumor cells, such as multiple myeloma cells or lymphoma cells, as well as in certain types of solid tumors (e.g., breast tumors, Wang SH et al., Am J Cancer Res. 2022;12(10):4721-4736. Published 2022 Oct 15). A certain percentage of bile duct, breast, colorectal, esophagus, glioma, liver, non-small cell lung, melanoma, ovary, pancreas, soft tissue, stomach, upper aerodigestive and urinary tract tumors are SLAMF7 positive (see, e.g., PCT publication No. WO2019 / 200462). In an embodiment, the SLAMF7-binding molecule described herein may be useful for treating hematological cancers / malignancies (also referred to as “blood cancers”). The term “hematological cancers” or “hematological malignancies” refers to cancers that affect the blood, bone marrow, lymph, and lymphatic system, and includes leukemias such as acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML) and acute monocytic leukemia (AMoL), lymphomas such as Hodgkin's lymphomas and non-Hodgkin's lymphomas, and myelomas such as multiple myeloma (MM).

[0297] In an embodiment, the methods and uses described herein further comprising identifying a subject suffering from a cancer expressing or overexpressing SLAMF7 and CD47.

[0298] As used herein, the term "effective amount" refers to a quantity of SLAMF7-binding molecule sufficient to achieve a desired biological, therapeutic and / or prophylactic effect, e.g., an amount which results in inhibition / reduction of the binding of CD47 to SLAMF7 on a tumor cell, or in the prevention of, or a decrease in, the symptoms associated with cancer. The amount of the SLAMF7-binding molecule used or administered to the subject will depend, for example, on the type and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of disease. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The SLAMF7-binding molecule may also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the SLAMF7-binding molecule may be administered to a subject having one or more signs or symptoms of cancer. For example, a "therapeutically effective amount" of the SLAMF7-binding molecule is meant levels in which the physiological effects of cancer are, at a minimum, ameliorated.

[0299] The SLAMF7-binding molecule or composition comprising same described herein may be used in combination with one or more additional active agents or therapies (radiotherapy, surgery, vaccines, etc.) for the treatment the targeted disease / condition or for the management of one or more symptoms of the targeted disease / condition (e.g., pain killers, anti-nausea agents, etc.). In an embodiment, the SLAMF7-binding molecule described herein is used in combination with one or more chemotherapeutic agents, immunotherapies (e.g., with opsonizing antibodies specific for a tumor antigen, such as anti-CD20 antibodies), checkpoint inhibitors (e.g., inhibitors of PD-1 , PD-L1 , CTLA-4, LAG-3), cell-based therapies (e.g., CAR T cells, CAR NK cells), etc. Examples of chemotherapeutic agents suitable for use in combination with the SLAMF7-binding molecule described herein include, but are not limited to, vinca alkaloids, agents that disrupt microtubule formation (such as colchicines and its derivatives), anti-angiogenic agents, therapeutic antibodies, EGFR targeting agents, tyrosine kinase targeting agent (such as tyrosine kinase inhibitors), transitional metal complexes, proteasome inhibitors, antimetabolites (such as nucleoside analogs), alkylating agents, platinum-based agents, anthracycline antibiotics, topoisomerase inhibitors, macrolides, retinoids (such as all-trans retinoic acids or a derivatives thereof); geldanamycin or a derivative thereof (such as 17-AAG), and other cancer therapeutic agents recognized in the art. In some embodiments, chemotherapeutic agents for use in combination with the SLAMF7-binding molecule described herein comprise one or more of adriamycin, colchicine, cyclophosphamide, actinomycin, bleomycin, duanorubicin, doxorubicin, epirubicin, mitomycin, methotrexate, mitoxantrone, fluorouracil, carboplatin, carmustine (BCNU), methyl-CCNU, cisplatin, etoposide, interferons, camptothecin and derivatives thereof, phenesterine, taxanes and derivatives thereof (e.g., taxol, paclitaxel and derivatives thereof, taxotere and derivatives thereof, and the like), topetecan, vinblastine, vincristine, tamoxifen, piposulfan, nab-5404, nab-5800, nab-5801 , Irinotecan, HKP, Ortataxel, gemcitabine, Oxaliplatin, Herceptin®, vinorelbine, Doxil®, capecitabine, Alimta®, Avastin®, Velcade®, Tarceva®, Neulasta®, lapatinib, sorafenib, erlotinib, erbitux, derivatives thereof, and the like. The SLAMF7-binding molecule thereof or composition comprising same described herein may also be used in combination with one or more additional therapeutic antibodies or antibody fragments, e.g., therapeutic antibodies or antibody fragments used for the treatment of tumors.

[0300] The combination of active agents and / or compositions comprising same may be administered or co-administered (e.g., consecutively, simultaneously, at different times) in any conventional dosage form. Co-administration in the context of the present invention refers to the administration of more than one therapeutic in the course of a coordinated treatment to achieve an improved clinical outcome. Such co-administration may also be coextensive, that is, occurring during overlapping periods of time. For example, a first agent (e.g., the SLAMF7-binding molecule thereof described herein) may be administered to a patient before, concomitantly, before and after, or after a second active agent (e.g., a chemotherapeutic agent) is administered. The agents may in an embodiment be combined / formulated in a single composition and thus administered at the same time.

[0301] In an embodiment, the SLAMF7-binding molecule is administered or for use in combination with an agent such as an antibody or antigen-binding fragment thereof capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP), such as an opsonizing antibody.

[0302] In an embodiment, the SLAMF7-binding molecule is administered or for use in combination with a SIRPa inhibitor, such as an agent capable of inhibiting the interaction between CD47 and SIRPa. As used herein, “SIRPa inhibitor” refers to any compound or composition that directly or indirectly inhibits SIRPa expression and / or activity. Without being so limited, candidate compounds modulating the SIRPa expression and / or activity are tested using a variety of methods and assays. It includes molecules such as, without being so limited, siRNA, antisense molecule, protein, peptide, small molecule, antibody, etc. More particularly, it includes anti-SIRPa antibodies or antibody fragments and soluble SIRPa polypeptides and fusion proteins that interfere with the interaction between SIRPa and CD47.

[0303] Examples of anti-SIRPa antibodies include antibody BR105 (Wu et al., Journal for ImmunoTherapy of Cancer 2022; 10:e004054. doi: 10.1136 / j itc-2021 -004054, WO2022 / 121980), SIRP-1 and SIRP-2 (Andrejeva et al., J. Immunol. 2021 Feb 15;206(4):712-721 . doi: 10.4049 / jimmunol.2001019. Epub 2021 Jan 11), ADU-1805 (Voets et al., J Immunother Cancer. 2019 Dec 4;7(1):340. doi: 10.1186 / S40425-019-0772-0, W02018 / 190719), Bl 765063 (OSE- 172), and CC-95251. Several anti-SIRPa antibodies are described in PCT publications Nos. W02018 / 190719, WO2022 / 121980, WO2022 / 254379, W02020 / 180811 , WO2021 / 226591 , WO2021 / 226576, W02023 / 010076, WO2019 / 023347, WO2018 / 107058, WO2013 / 056352, and WO2015 / 138600. The antibodies and antigen-binding fragments described in these documents, including the sequences of the CDRs and variable regions, are incorporated herein by reference. Soluble SIRPa polypeptides and fusion proteins capable of inhibiting SIRPa are described in PCT publications Nos. WO2016 / 023040, W02023 / 003331 , WO2013 / 109752, WO2014 / 094122, and WO2017 / 027422. The soluble SIRPa polypeptides and fusion proteins described in these documents are incorporated herein by reference. Representative soluble SIRPa fusion proteins capable of inhibiting SIRPa include TTI-621 (SIRPa-lgG1 Fc), and its high-affinity variant ALX148 (Evorpacept), TTI-622 (SIRPa-lgG4 Fc), IMM-01 , and SL-172154 (see, e.g., Son et al., Front Immunol. 2022; 13: 1027235).

[0304] In another aspect, the present disclosure provides a combination or combination therapy comprising the SLAMF7-binding molecule described herein and a SIRPa inhibitor. The present disclosure also provides a kit or package comprising the SLAMF7-binding molecule described herein and a SIRPa inhibitor.

[0305] EXAMPLES

[0306] The present disclosure is illustrated in further details by the following non-limiting examples.

[0307] Example 1 : Materials and Methods

[0308] Mice

[0309] Mice lacking SIRPa (Sirpa1) were generated in our laboratory using fertilized C57BL / 6J oocytes and CRISPR-Cas-based genomic editing. The following guide RNAs were used: 5'-AGCAGCGGCCCTAGGCGGCC-3' (SEQ ID NO:55), 5'-GCCCGGCCCCTGGCCGCCTA-3' (SEQ ID NO:56) and 5'-TCCGCGTCCTGTTTCTGTAC-3' (SEQ ID NO:57). After birth, mice were screened by flow cytometry and sequencing of the Sirpa gene. Strains bearing a 593-nucleotide (#46) or 11 -nucleotide (#54) deletion in exon 2 of Sirpa were chosen. These deletions resulted in a frameshift in the second coding exon of the Sirpa gene and caused loss of SIRPa expression by flow cytometry. Heterozygous knock-out (KO) mice were backcrossed to the C57BL / 6J background for more than 10 generations and subsequently bred to homozygosity for experimentation. The #46 mouse strain was used for most experiments, unless specified. Mice lacking all SFRs (SFR KO) or SLAMF7 (Slamf7z, and mice expressing a human (h) SLAMF7 bacterial artificial chromosome transgene (hSLAMF7 BAC Tg mice), in the C57BL / 6J background, were described elsewhere527. CD47 KO Cd47 / '), RAG-1 KO (Ragl ) and C57BL / 6J mice were obtained from the Jackson Laboratory. SIRPa KO mice were bred with SFR KO or SLAMF7 KO mice to create SIRPa-SFR double (D) KO mice and SIRPa-SLAMF7 DKO mice. hSLAMF7 BAC Tg mice were bred with SLAMF7 KO mice to create SLAMF7 KO-hSLAMF7 BAC Tg mice. RAG- 1-SIRPa DKO mice were generated in the same manner as SIRPa KO mice, except that RAG-1 KO oocytes were used for micro-injection of guide RNAs. This approach was needed because the Sirpa and Rag1 genes are located on the same chromosome. A mouse strain bearing a 168- nucleotide deletion in exon 2 of Sirpa was chosen. This deletion resulted in a frameshift in the second coding exon of the Sirpa gene and caused loss of SIRPa expression by flow cytometry. All mice were maintained in the C57BL / 6J background. They were kept in a specific pathogen- free environment. Either males or females were used, between 8 to 12 weeks of age. Littermates were used as control in most experiments, except for some studies where wild-type syngeneic, age and sex-matched mice were used. Animal experimentation was done in accordance with the Canadian Council of Animal Care and approved by the IRCM Animal Care Committee.

[0310] Cells

[0311] Macrophages were generated as described5. Briefly, for mouse macrophages, bone- marrow-derived macrophages (BMDMs) were generated by growing freshly isolated bone marrow cells for 7 days in medium supplemented with 30% (vol / vol) L929 cell-conditioned medium, as a source of colony-stimulating factor-1 (CSF-1). Human macrophages were produced from peripheral blood mononuclear cells, which were isolated from healthy donors using Ficoll- Paque™ PLUS (Cat#36-101-6383, GE Healthcare), according to the manufacturer’s protocol and as approved by the IRCM Human Ethics Board. Peripheral blood mononuclear cells were then seeded onto Petri dishes containing serum-free RPMI medium for 30 min. After gentle washes to remove non-adherent cells, adherent cells (which mostly represent monocytes) were differentiated into macrophages by culture in medium supplemented with 10% human serum and 10 ng ml1CSF-1 (Cat# 300-25, Peprotech) for 7 days. Mouse CD4+or CD8+T cells, depleted of natural killer T (NKT) cells using anti-NK1.1 monoclonal antibody (MAb), were purified from spleen by negative selection using the EasySep™ Purification Kits (Cat# 19852 or 19853, STEMCELL Technologies). Activated CD4+T cells were obtained by stimulating CD4+T cells with 4 pg ml1concanavalin A (Cat# C5275, Sigma-Aldrich) for 2 days, followed by expansion for 3 days in medium containing 50 units ml1interleukin-2 (Cat# 212-12, Peprotech). Activated CD8+T cells were obtained by stimulating CD8+T cells with 3 pg ml’1Fc-silent anti-CD3 (145-2C11 , Absolute Biotech Company) plus 1 pg ml’1Fc-silent anti-CD28 (D665, Absolute Biotech Company) MAbs for 2 days.

[0312] CRISPR-Cas9 knockout and retroviral infection

[0313] CD47 KO L1210 cells and CD47 KO 293T cells were generated by CRISPR-Cas-mediated genome editing, using plasmid PX458 (Cat# 48138, Addgene) and the following guide RNAs: mouse CD47 (mCD47): 5'-CACCGAGCAACAGCGCCGCCGCCAA-3' (SEQ ID NO:58) and 5'- ACCGTTGGCGGCGGCGCTGTTGCT-3' (SEQ ID NO:59); human CD47 (hCD47): 5'- CTGGTAGCGGCGCTGTTGCT-3' (SEQ ID NO:60). CD47 KO Raji cells and CD47 KO Daudi cells were generated using the Neon Transfection System Kit (Cat# MPK10096, Thermo Fisher Scientific), guide RNA 5'-CTACTGAAGTATACGTAAAG-3' (SEQ ID NO:61) or scrambled guide RNA (Cat# 1072544, IDT), and the Cas9 nuclease (Cat# 1081058, IDT). CD47 KO cells were then purified by cell sorting.

[0314] Constructs encoding wild-type mouse (m) CD47, Flag-tagged mCD47 (Flag tag added at the carboxyl terminus), phenylalanine 37-to-aspartate 37 (F37D) mCD47, wild-type mSLAMF7, arginine 75-to-alanine 75 (R75A) mSLAMF7, glutamate 77-to-alanine 77 (E77A) mSLAMF7, wildtype mSIRPa, mSIRPa first Ig-like (variable; V) domain, wild-type hCD47, hSIRPa version 1 (V1) and hSIRPa V2 were generated by PCR or overlap extension PCR. They were then cloned into either the pFB-GFP or the pMIGR-GFP retroviral vector (which used the mouse stem cell virus promoter), which also encode the green fluorescent protein (GFP). For L1210 derivatives overexpressing SLAMF7, SLAMF7 was cloned into a promoter-modified pFB-GFP (which used the EF-1a promoter). After transfection in Phoenix-Eco cells, viral supernatants were recovered and used for spin infection of the indicated cell lines or primary cells. Plasmids expressing GFP alone were used as controls. 48 hours after retroviral infection, GFP-positive cells were sorted, expanded and used for experimentation. L1210, EL-4 and BI-141 were sorted twice. L1210 derivatives expressing the Tac antigen (hCD25) were generated as described5.

[0315] Recombinant proteins

[0316] To produce Fc fusion proteins containing the extracellular domain of mCD47, mSLAMF7, mSIRPa, mSIRP Ha, mSIRP H b, mSIRP Hc, hSLAMF7, hSLAMF7 first Ig-like [variable; lg(V)] domain fused to mSLAMF7 second Ig-like [constant; lg(C)] domain, or mSLAMF7 lg(V) fused to hSLAMF7 lg(C), cDNAs were cloned into pFc-hlgGI (LALAPG), which encodes in frame the Fc portion of human lgG1 (hlgG1) with the “LALAPG” mutation (leucine 234-to-alanine 234; leucine 235-to-alanine 235; proline 329-to-glycine 329) that prevents binding to Fc receptors (FcRs). SIRPa-Fc fusion proteins TTI-62112and its high-affinity variant ALX14813were produced by synthesizing the cDNAs from published sequences, followed by cloning into pFc-hlgGI (LALAPG). For production, cDNAs were transfected in 293T cells. Then, fusion proteins were purified with protein A Sepharose® (Cat# GE17-1279-03, Sigma-Aldrich). All recombinant proteins were quantified by SDS-PAGE and Coomassie blue staining, using bovine serum albumin (BSA) as standard.

[0317] To produce recombinant versions of mCD47, mSLAMF7, hPD-1 and hPD-L1 , cDNAs encoding their extracellular segment, in which a SNAP tag was inserted after the amino-terminal signal peptide and a 10x histidine (His) tag was inserted after the carboxyl terminus of the extracellular domain, were cloned in pPPI4 vector. After transfection in 293T cells, recombinant proteins were purified from cell culture medium using GE Ni Sepharose® Excel (GE17371201 , Sigma-Aldrich) and eluted with 0.5 M imidazole. Proteins were further purified by size exclusion, using a Superdex® 200 Increase 10 / 300 GL column (Cat# GE28990944, Sigma-Aldrich), in 4-(2- hydroxyethyl)-1 -piperazineethanesulfonic acid (HEPES)-buffered saline buffer (50 mM HEPES- NaOH, pH 7.5, 150 mM NaCI, 10% glycerol). Gel-filtered proteins were labeled either with SNAP- Cell-505 (Cat#S9103S, New England Biolabs) or with SNAP-Cell-TMR (Cat# S9105S, New England Biolabs) following the manufacturer’s instructions. Free dye was removed using a PD- 10 desalting column (Cat# 87766, Thermo Fisher Scientific). All proteins were quantified by SDS- PAGE and Coomassie blue staining, using BSA as a standard.

[0318] Monoclonal antibodies

[0319] To generate anti-mSIRPa MAbs, SIRPa KO mice were immunized with recombinant mSIRPa-Fc fusion proteins. Splenocytes from hyperimmune mice were then fused with FO cells using polyethylene glycol (Cat# P7306, Sigma-Aldrich), and cultured in hypoxanthine- aminopterin-thymidine medium for ~8 days. Hybridomas were initially screened by ELISA using the mSIRPa-Fc fusion protein or an irrelevant Fc fusion protein as control. The hybridomas producing anti-mSIRPa antibodies were then subjected to two rounds of sub-cloning. Anti- mSIRPa MAbs were screened for: 1) the capacity to stain EL-4 cells expressing full-length mSIRPa or a variant of mSIRPa containing only the first Ig-like domain, which is implicated in CD47 binding, using flow cytometry; 2) the ability to block the binding of a mCD47-Fc fusion protein to EL-4 cells expressing mSIRPa using flow cytometry; 3) the ability to bind SIRPa-Fc, but not SIRP J-- Fc fusion proteins, using ELISA. Anti-mSIRPa MAbs #23 and #27 exhibited blocking activity towards SIRPa and did not cross-react with any of the SIRP J- isoforms.

[0320] Anti-hSLAMF7 hybridomas were generated in the similar way, except that SLAMF7 KO mice and recombinant hSLAMF7-Fc fusion proteins were utilized. After two rounds of subcloning, anti-hSLAMF7 MAbs were screened for: 1) the ability to bind hSLAMF7-Fc, but not mSLAMF7-Fc. They were also tested for binding to hSLAMF7(V)-mSLAMF7(C)-Fc or mSLAMF7(V)-hSLAMF7(C)-Fc, to determine if they reacted with the first or second Ig-like domain of hSLAMF7; 2) the capacity to stain EL-4 cells expressing or not hSLAMF7, or CD47 KO 293T cells expressing or not hSLAMF7, or the human-mouse SLAMF7 chimeras; 3) the capacity to block the phagocytosis of CD47 KO Raji or CD47 KO Daudi by wild-type BMDMs. Anti- hSLAMF7 MAbs Z1 , Z8, Z9 and Z27 bound specifically to the first IgG-like domain, whereas MAbs Z10, Z26, Z30, Z46 and elotuzumab (Elo) bound specifically to second Ig-like domain. MAb Z8 was a blocking MAbs. MAbs Z10 and elotuzumab were non-blocking MAbs.

[0321] Blocking anti-mSLAMF7 MAb 4G2 was previously described16; blocking anti-mCD47 hybridoma Miap301 was provided by Dr. Timo van den Berg (Sanqin Institute, Amsterdam, The Netherlands); blocking anti-mSIRPa hybridoma MY-1 was provided by Takashi Matozaki (Kobe University, Kobe, Japan); and blocking anti-hSLAMF7 hybridoma 162 was reported elsewhere29. To create recombinant versions of blocking anti-mSIRPa MAb MY-1 , blocking anti-mSIRPa MAbs #23 and #27, blocking anti-mCD47 MAb Miap301 , blocking anti-mSLAMF7 MAb 4G2, blocking anti-hSLAMF7 MAb 162, and novel anti-hSLAMF7 MAbs Z1 , Z8, Z9, Z10, Z26, Z27, Z30 and Z46, cDNA sequences encoding the variable regions of heavy chain (VH) and light chain (VL) were determined using total cellular RNA from the hybridomas, followed by RNA sequencing or reverse transcription-PCR, as described30. To create recombinant versions of control immunoglobulin G (IgG) MAb MOPC21 , blocking anti-hSIRPa MAbs KWAR23, 18D5, 50A and 40A, blocking anti- hCD47 MAbs B6H12 and AO-176, and non-blocking anti-hSLAMF7 Mab elotuzumab, VH and VL sequences were obtained from previous studies14or patents (WO2017178653A2, US10851164B2, US20200095318, US7709610B2, EP2569013A2). cDNAs encoding these sequences were then synthesized commercially. In all cases, the VH- and VL-encoding cDNAs were cloned in frame into one or both of the following expression plasmids30: pAb- mlgG2a(LALAPG), which contains genes encoding Fc-silent mouse lgG2a (mlgG2a) with the LALAPG mutation, and pAb-mlgK; pAb-hlgGI (LALAPG), which contains genes encoding Fc- silent hlgG 1 with the LALAPG mutation, and pAb-hlgK. Recombinant MAbs were then expressed in 293T cells and purified with protein A Sepharose® (Cat# GE17-1279-03, Sigma-Aldrich). All recombinant MAbs were quantified by SDS-PAGE and Coomassie blue staining, using BSA as a standard.

[0322] For opsonization of target cells with IgG, Fc-intact anti-hCD25 MAb 7G7 (mlgG2a; BioXCell) and Fc-intact anti-hCD20 MAb S18015E (mlgG2a; BioLegend) were used. For flow cytometry assays, anti-CD45 MAb 30-F11 , anti-CD11 b MAb M1 / 70, anti-F4 / 80 MAb BM8, anti-Ly6G MAb 1A8, anti-NK1.1 MAb PK136, anti-SIRPa MAb P84, anti-CD64 VX54-5 / 7.1 , anti-CD16 / 32 MAb 93, anti-CD18 MAb M18 / 2, anti-CD11a MAb M17 / 4, anti-CD11c MAb N418, anti-CD200R1 MAb OX-110, anti-Flag MAb L5, and anti-rat IgG Poly4054 were used. All were obtained from Biolegend. For immunoprecipitations and immunoblots, anti-Flag MAb M2 (Sigma-Aldrich), anti- Myc MAb 9B11 (Cell Signaling Technology), anti-CD47 MAb AF1866, (R&D Systems) and anti- SLAMF7 rabbit antiserum (generated in-house) were used. Anti-p-actin AC-74 (Sigma-Aldrich) was used for immunofluorescence.

[0323] Phagocytosis assays

[0324] Phagocytosis was evaluated using a microscopy-based assay or a pHrodo-based assay, as described15. For the microscopy-based assay, 5x104macrophages were seeded in a 24-well tissue culture plate. The next day, target cells were labeled with 2.5 pM of carboxyfluorescein succinimidyl ester (CFSE, Cat# C34554, Life Technologies). After incubating macrophages in serum-free medium for 1 hour, 2x105CFSE-labeled target cells were added to macrophages for 2 hours, in the presence or absence of 10 pg ml1Fc-silent MAbs. Macrophages were then washed and imaged with an inverted microscope (Carl Zeiss Axiovert S100 TV). For the pHrodo- based assay, target cells were pre-labeled with 100 ng ml1of pHrodo™ green STP ester (Cat# P35369, Thermo Fisher Scientific), prior to addition to macrophages. pHrodo dyes are non- fluorescent at neutral pH and become fluorescent in acidic environments such as phagolysosomes. After 2 hours, cells were harvested and stained with anti-F4 / 80 MAb BM8 (Biolegend) to identify macrophages and analyzed by flow cytometry. For phagocytosis of IgG- coated tumor cells, L1210 expressing Tac (hCD25) were opsonized with anti-hCD25 MAb 7G7 (mlgG2a) for 30 min, prior to the phagocytosis assays. Conversely, Raji were opsonized with anti- hCD20 MAb S18015E (mlgG2a). For phagocytosis of C3bi-opsonized tumor cells, L1210 was incubated with C5-deficient serum (Cat# C1163, Sigma-Aldrich) for 30 min, prior to the phagocytosis assay.

[0325] Soluble Fc fusion protein binding assay and flow cytometry

[0326] To test binding of the mCD47-Fc fusion protein to cells, BMDMs were detached from plates by treatment with phosphate-buffered saline (PBS) containing 2 mM ethylenediaminetetraacetic acid and washed. After blocking Fc-receptors with a mix of mlgG2a MAb 7G7 and anti-CD16 / 32 Mab 2.4G2, cells were incubated for 30 min on ice with mCD47-Fc (hlgG1) (Cat# 1866-CD-050, R&D Systems) or mCD47-Fc (hlgG1 LALAPG) (produced by our group). After the incubation period, cells were washed again and incubated for 30 min on ice with Alexa Fluor™ 647-labeled F(ab')2fragments goat anti-human lgG1 , Fc-specific (Cat# 109-606-098, Jackson Immune Research). After additional washes, fluorescence was evaluated by flow cytometry. To examine binding of the mSLAMF7-Fc fusion protein to cells, BI-141 cells expressing or not expressing mSLAMF7 were incubated for 30 min on ice with 10 pg ml’1anti-mCD47 MAb (mlgG2a LALAPG). Subsequently, cells were stained with various concentrations of mSLAMF7-Fc (hlgG 1 LALAPG) plus Alexa Fluor™ 647-labeled F(ab')2 fragments goat anti-human IgG 1 , Fc-specific (Cat# 109- 606-098, Jackson Immune Research) for 5 min on ice. After additional washes, fluorescence was evaluated by flow cytometry. To determine the impact of SLAMF7 mutations on homotypic binding to SLAMF7, BI-141 cells expressing wild-type SLAMF7 or mutated mSLAMF7 (R75A or E77A) were incubated with mSLAMF7-Fc (hlgG 1 LALAPG) on ice for 30 min, followed by flow cytometry. To ascertain the effect of CD47 mutations on binding to SIRPa, CD47 KO 293T cells and CD47 KO L1210 cells expressing wild-type mCD47 or mutated mCD47 (F37D) were incubated with mSIRPa-Fc (h IgG 1 LALAPG) on ice for 30 min. Cells were then washed and incubated with Alexa Fluor™ 647-labeled F(ab')2fragments goat anti-human lgG1 , Fc-specific (Cat# 109-606-098, Jackson Immune Research) on ice for 30 min. After additional washes, fluorescence was evaluated by flow cytometry. To address the FcR-binding capacity of Fc-silent and Fc-intact MAbs, wild-type BMDMs, which express a wide range of FcRs, were incubated with or without Fc-intact (mlgG2a) or Fc-silent (mlgG2a LALAPG) IgG MOPC21 , or Fc-intact (h IgG 1 ) or Fc-silent (hlgG 1 LALAPG) anti-hSLAMF7 MAb Z10, priorto flow cytometry. To map the binding site of anti- SLAMF7 MAbs on hSLAMF7, CD47 KO 293 T cells were transfected with the indicated hSLAMF7 plasmids for 48 hours, before staining with anti-hSLAMF7 MAbs Z10, Z8 or elotuzumab. To determine expression of cell surface markers, Fc receptors were blocked as above, prior to staining with the indicated antibodies.

[0327] Subcutaneous tumor transplantation assay

[0328] L1210 (0.5x106), L1210 derivatives (0.5x106) or Raji (5x106) were injected subcutaneously into the right flank of 8- to 10-week-old RAG-1 KO or RAG-1 -SIRPa DKO mice. When the tumor was about 5x5 mm in diameter, mice were injected intraperitoneally with 200 pg of Fc-silent control IgG MAb MOPC21 , anti-CD47 MAb Miap301 , anti-SIRPa MAb #27 or anti-SLAMF7 MAb Z10, generated by our group, or Fc-intact anti-hCD25 MAb 7G7 and control IgG Mab C1.18.4 (BioXCell). Experiments were terminated when or before tumor volume reached 1 .5 cm3. T umors were then dissected and weighed. Volumes were also assessed. Tumors were sliced into small pieces and digested with 10 pg ml1deoxyribonuclease I (Cat# D4513, Sigma-Aldrich) and 25 pg ml’1liberase (Cat# 5401054001 , Sigma-Aldrich). Then, cells were passed through a strainer using the plunger end of a syringe. After washing, total cell numbers were determined. Immune cells were detected by staining with the relevant antibodies and flow cytometry.

[0329] T cell transfer assay

[0330] To address the impact of loss of CD47 on survival of activated T cells in vivo, concanavalin A-activated wild-type CD4+T cells (labeled with CFSE) and CD47 KO CD4+T cells [labeled with CellTrace™ violet (CTV, Cat# C34557, Life Technologies)] were mixed 1 :1 and injected intravenously into wild-type or SIRPa KO mice. After 24 hours, mice were bled, and blood was depleted of red blood cells using a red blood cell lysis buffer (Cat# R7757, Sigma-Aldrich). The presence of CFSE- or CTV-positive cells was detected by flow cytometry. T o ascertain the impact of CD47 blockade or SIRPa blockade on the survival of activated T cells in vivo, wild-type CD4+T cells were injected intravenously into wild-type or SLAMF7 KO mice, in the presence of Fc- silent control IgG MAb MOPC21 , anti-CD47 MAb Miap301 or anti-SIRPa MAbs (#23 and #27).

[0331] Conjugate formation and actin polarization assays Conjugate formation and actin polarization were tested as described5. In brief, for the microscopy-based conjugate formation assay, BMDMs were labeled with CTV and plated overnight. The next day, target cells were labeled with CFSE. Macrophages and target cells were then mixed at a 1 :4 ratio in serum-free culture medium in the presence of Fc-silent MAbs, and incubated for 20 min at 37°C to allow conjugate formation. Cells were subsequently washed extensively to remove unconjugated cells. Images were obtained by using a LSM710 confocal microscope (Carl Zeiss). Conjugates between BMDMs and target cells were counted. Forthe flow cytometry-based conjugate formation assay, macrophages were labeled on ice with anti-F4 / 80 MAb BM8 (Biolegend), while target cells were labeled with CFSE. After washing, macrophages and targets were mixed with Fc-silent MAbs for various periods of time at 37°C. Conjugates were detected by flow cytometry. To study actin polarization, BMDMs were stained with CTV and seeded onto a confocal tissue dish overnight. The next day, target cells were stained with CFSE. Macrophages and targets were then mixed at a 1 :4 ratio in serum-free culture medium with Fc- silent MAbs for 20 min at 37°C. Cells were subsequently fixed, washed and permeabilized, and non-specific staining was blocked in PBS supplemented with 5% BSA for 30 min. Then, cells were washed and incubated for 1 hour with anti-actin MAb AC-74 (Sigma-Aldrich). After washing, cells were incubated for 1 hour with Alexa Fluor™ 647-conjugated anti-mouse IgG (Cat# 115-605-071 , Jackson Immune Research). Cells were then processed and analyzed using a LSM710 confocal microscope (Carl Zeiss). Conjugates with full polarization of actin at the area of contact between the macrophage and the target cell were quantitated.

[0332] Immunoprecipitations, immunoblots and mass spectrometry

[0333] CD47 KO 293T cells were transiently transfected with plasmids encoding mSLAMF7-Myc, mCD47-Flag or both, or control plasmids, using polyethylenimine (PEI, Cat# 23966, Polysciences). The Myc and Flag tags were located at the carboxyl terminus of the proteins. Immunoprecipitations and immunoblots were performed as previously described31. CD47 KO L1210 cells expressing mCD47-Flag and a Brij99-containing lysis buffer were used for mass spectrometry, as reported elsewhere5. The following criteria were used to select potentially relevant CD47 interactors: 1) presence in Flag immunoprecipitates from CD47 KO L1210 cells expressing CD47-Flag, but not from CD47 KO L1210 cells lacking CD47-Flag; 2) observation in a minimum of two of three independent experiments.

[0334] RNA sequencing

[0335] RNA sequencing was performed as previously described32. In essence, RNA was isolated from wild-type and SIRPa KO BMDMs using the RNeasy™ Plus Mini Kit (Cat# 74134, Qiagen), according to the manufacturer’s instructions. cDNA libraries were prepared using the Illumina TruSeq™ Stranded mRNA Kit, according to the manufacturer’s instructions, and sequenced with the Illumina HiSeq™ 2000 Sequencer. Read quality was confirmed using FastQC v0.11 .8, before alignment using STAR v2.5.0 for the mouse GRCm38 v98 Reference genome. Differential expression analysis was performed with DESeq2 v1.22.2 from the raw alignment counts calculated with Featurecounts v1 .5. Differentially expressed genes were defined as genes with an adjusted p value of <0.05 and log2fold change of >1 .0.

[0336] PLA assay

[0337] A PLA was performed with a commercial kit according to the manufacturer’s recommendation (cat. no. DU092008, Duolink In Situ, Sigma-Aldrich). Briefly, after fixing the cells to stabilize proximity, cells were incubated with Fc-silent human lgG1 SLAMF7 monoclonal antibody and Fc-silent mouse lgG2a CD47 monoclonal antibody, followed by oligonucleotide- linked secondary antibodies specifically recognizing each of the primary antibodies. Then, proximity was detected by PCR with complementary oligonucleotides coupled to fluorochromes, and a LSM710 confocal microscope (Carl Zeiss). In each experiment, five images per condition were randomly chosen. A total of 100 cells from three independent experiments were randomly chosen for quantification.

[0338] FRET assay

[0339] For the fluorescence resonance energy transfer (FRET) assay, constructs encoding SNAP- tagged wild-type mCD47, mutant mCD47 (F37D) or wild-type hCD47 were co-transfected with constructs encoding CLIP-tagged wild-type mSLAMF7, mSLAMF7 mutants (R75A or E77A) or wild-type hSLAMF7, into CD47 KO 293T cells. After 2 days, cells were collected and seeded onto poly-D-lysine (Cat# P6407, Sigma-Aldrich)-treated confocal microscopy plates. The next day, cells were labeled with SNAP-Surface Alexa Fluor™ 647 (Cat# S9136S, New England Biolabs) and CLIP-Surface 547 (Cat# S9233S, New England Biolabs) for 45 min at 37°C, in the presence of 10 pg ml1of Fc-silent MAbs. Cells were then fixed with 4% paraformaldehyde (Cat# 22023, Biotium) and washed, prior to the FRET assay. Images were acquired with an LSM710 confocal microscope (Carl Zeiss) by exciting CLIP-Surface 547 (energy donor) at 543 nm and SNAP- Surface Alexa Fluor™ 647 (energy acceptor) at 635 nm. Images before and after acceptor bleaching were acquired for FRET analysis, using Imaged (Fiji) with the AccPbFRET plugin, as previously described3334.

[0340] LUV reconstitution assay

[0341] Large unilamellar vesicle (LUV) reconstitution followed by FRET assay was performed as described35. For LUV preparation, 80% 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, Cat# 850457C, Avanti Polar Lipids) and 20% 1 ,2-dioleoyl-sn-glycero-3-[(N-(5-amino-1- carboxypentyl)iminodiacetic acid)succinyl] (nickel salt) (DGS-NTA-Ni, Cat# 790404C, Avanti Polar Lipids) were mixed in chloroform, dried under a stream of nitrogen, desiccated for 1 hour in a vacuum container and then resuspended in PBS. LUVs were generated by extrusion 20 times through a pair of polycarbonate filters containing pores of 200 nm diameter. 8.3 nM SNAP-Cell- 505-labeled mSLAMF7-His or hPD-L1-His was mixed with 0.23 nM LUVs harboring DGS-NTA- Ni in PBS containing 1.5 mg ml1BSA and 1 mM tris(2-carboxyethyl)phosphine (TCEP) in a 96- well solid black microplate, during which the SNAP-Cell-505 fluorescence was monitored in real time, using a plate reader with 504-nm excitation and 540-nm emission. After 30 min, the fluorescence reading was paused, and the second protein component, that is 25 nM SNAP-Cell- TMR-labeled mCD47-His or hPD-1-His, was injected and the fluorescence was monitored for another 30 min. For some conditions, SNAP-Cell-505-labeled mCD47-His or SNAP-Cell-TMR- labeled mSLAMF7-His was incubated with Fc-silent MAbs (three-fold molar excess) for 30 min before being added to the reaction. Data were normalized for the mean fluorescence intensity of the last 10 data points, before the addition of SNAP-Cell-TMR-labeled protein and plotted with GraphPad™ Prism.

[0342] Statistical analyses

[0343] Prism 9 software (GraphPad™) was used for paired or unpaired Student’s t tests (two- tailed), and for one-way ANOVA followed by Tukey’s or Dunnett’s multiple comparison tests, when appropriate. The normal distribution of the data was tested using the D’Agostino-Pearson normality test (Prism 9) and parametric tests used for statistical analyses accordingly.

[0344] Example 2: Blockade of CD47, but not of SIRPa, promotes phagocytosis triggered by pro-phagocytic ligand SLAMF7

[0345] To compare the impact of SIRPa and CD47 on phagocytosis, the effects of previously described or newly generated blocking anti-CD47 MAbs, blocking anti-SIRPa MAbs, or blocking SIRPa fusion proteins (the latter act by occupying CD47 on tumor cells) were evaluated side-by- side (FIGs. 8A-G). To avoid concomitant engagement of pro-phagocytic FcRs on macrophages, all agents were engineered to be “Fc-silent”, due to mutations in their Fc portion (“LALAPG” mutations) (FIGs. 8A,B)21. These mutations fully abrogated the ability of the MAbs to bind FcRs on macrophages (FIG. 8B).

[0346] When wild-type mouse bone marrow-derived macrophages (BMDMs) were used as phagocytes, blocking anti-mouse CD47 MAb Miap301 augmented phagocytosis of mouse tumor cell lines L1210 (leukemia), P815 (mastocytoma) and SP2 / 0 (multiple myeloma) in vitro, relative to control MAb MOPC21 (FIGs. 1A-C). However, no increase in phagocytosis was seen with blocking anti-mouse SIRPa MAbs #23 and #27, which were generated for the present study (FIGs. 1B,C and 8C-E). These observations were made either with microscopy-based or with pHrodo-based phagocytosis assays (FIGs. 1A-C and 9A,B).

[0347] Anti-CD47 MAbs, but not anti-SIRPa MAbs, also suppressed the growth of L1210 in an in vivo sub-cutaneous tumor transplantation assay, using RAG- 1 -deficient (knockout; KO) mice that lack T cells and B cells as recipients, relative to control MAbs (FIGs. 1D and 9C). Moreover, anti- 0047 MAbs, but not anti-SIRPa MAbs, promoted phagocytosis and in vivo elimination of normal activated mouse T cells, which can be also phagocytosed during CD47 blockade5(FIGs. 1E,F). The ability of anti-CD47 MAbs to trigger elimination of L1210 and activated T cells was abolished when BMDMs or recipient mice lacked SLAMF7, confirming the involvement of the homotypic receptor SLAMF7 in this process5(FIGs. 10A,B).

[0348] In the human system, blocking anti-human CD47 MAbs B6H12 and AO-176, as well as blocking human SIRPa-Fc fusion protein TTI-621 and high-affinity variant ALX148, promoted the capacity of blood-derived human macrophages to phagocytose human lymphoma cell line Raji, compared to control IgG (FIG. 1G and 10C). The augmented phagocytosis of Raji was also SLAMF7-dependent (FIG. 10D). However, blocking anti-human SIRPa MAbs KWAR23 and 18D5 did not augment phagocytosis (FIG. 1G). A similar lack of effect of blocking anti-human SIRPa MAbs KWAR23, 18D5, 50A and 40Awas seen with mouse BMDMs ectopically expressing human SIRPa as phagocytes (FIG. 10C). Many of these human SIRPa-CD47-targeting agents are being tested in the clinic as anti-cancer therapies3.

[0349] Hence, blockade of CD47, but not of SIRPa, promoted in vitro phagocytosis and in vivo elimination of tumor cells expressing the cell-intrinsic pro-phagocytic ligand SLAMF7.

[0350] Example 3: Genetic deficiency of CD47 or SIRPa phenocopies the impact of pharmacological blockade

[0351] T o confirm the divergence of impact seen with pharmacological blockade of CD47 or SIRPa, the consequences of genetic ablation of CD47 on target cells or SIRPa on macrophages were compared (FIG. 2A). Wild-type BMDMs displayed greater phagocytosis of CD47 KO L1210, CD47 KO activated T cells, and CD47 KO lymphoma cell lines Raji and Daudi, compared to their wildtype CD47-positive counterparts, as described5(FIGs. 2B-E and 11 A). However, relative to wildtype BMDMs, SIRPa KO BMDMs did not exhibit augmented phagocytosis of CD47-positive targets (FIGs. 2B-E and 11 A). Like wild-type BMDMs, SIRPa KO BMDMs only displayed greater phagocytosis of target cells when CD47 was absent from targets. Similar results were obtained in in vivo elimination assays using L1210 or activated T cells, expressing or not expressing CD47, that were injected in mice expressing or not expressing SIRPa (FIGs. 2F-I and 11 B) . The lack of impact of SIRPa deficiency on phagocytosis was unlikely to be due to altered development or differentiation of SIRPa KO BMDMs, as suggested by their unchanged RNA sequencing profiles, compared to wild-type BMDMs (FIG. 12A). Moreover, other than lack of SIRPa, SIRPa KO BMDMs displayed no alteration in expression of various cell surface markers, including SLAMF7 (FIG. 12B). Analogous findings were made with BMDMs from a second SIRPa KO mouse strain (FIGs. 12C-E). Thus, loss of CD47 on tumor cells or of SIRPa on macrophages mimicked the distinct consequences of blockade of CD47 or SIRPa on phagocytosis. Example 4: Inactivation of CD47 or SIRPa promotes phagocytosis of antibody-opsonized tumor cells

[0352] To ascertain if the disparate effects of blockade or loss of CD47 and SIRPa extended to other pro-phagocytic receptors, tumor cells were “opsonized” by antibodies (IgG) or complement (C3bi), which trigger the macrophage pro-phagocytic receptors FcRs and CD11 b (Mac-1), respectively8(FIGs. 3A-E). Unlike with non-opsonized targets (FIGs. 1 and 2), anti-SIRPa MAbs were as equally efficient as anti-CD47 MAbs at augmenting phagocytosis of IgG-opsonized L1210 or Raji, compared to control MAbs (FIGs. 3A-C). In an analogous way, SIRPa KO BMDMs displayed greater phagocytosis of IgG-opsonized targets, compared with wild-type BMDMs (Fig. 3d). In addition, mice lacking SIRPa exhibited slower growth of IgG-opsonized CD47-positive L1210 in an in vivo tumor assay, compared to mice expressing SIRPa (FIGs. 3F and 13). Similar effects were noted with complement-opsonized targets (FIG. 3E). Therefore, blockade or loss of CD47 on target cells and blockade or loss of SIRPa on macrophages were equally efficient at augmenting phagocytosis of targets opsonized by exogenous ligands for FcRs or CD11 b.

[0353] Example 5: CD47 physically interacts in cis with pro-phagocytic ligand SLAMF7 on tumor cells

[0354] One possibility to explain the inability of blockade or loss of SIRPa to augment phagocytosis of non-opsonized targets was that CD47 triggered inhibitory receptors other than SIRPa that compensated for blockade or loss of SIRPa on macrophages. However, a soluble CD47-Fc fusion protein displayed no residual binding to SIRPa KO BMDMs, suggesting that this notion was unlikely (FIG. 4A). Another scenario was that CD47 was interacting in cis, directly or indirectly, with SLAMF7 at the surface of tumor cells, thereby disallowing SLAMF7 on tumor cells from triggering SLAMF7 on macrophages.

[0355] To assess this possibility, CD47 was immunoprecipitated from L1210 tumor cells and potential CD47-associated proteins were detected by mass spectrometry. As the anti-CD47 MAbs target the extracellular domain of CD47, L1210 cells expressing a Flag-tagged variant of CD47 were generated and immunoprecipitation of CD47 was conducted using anti-Flag MAbs (FIGs. 14A,B). CD47 abundantly co-immunoprecipitated with SLAMF7 (FIGs. 4B and 14C). Several other proteins were also detected, although less prominently and none was a known pro- phagocytic ligand. Co-immunoprecipitation of epitope-tagged versions of CD47 and SLAMF7 was also identified in transiently transfected 293T cells (FIG. 4C).

[0356] To confirm that CD47 and SLAMF7 were in physical proximity at the plasma membrane, fluorescence resonance energy transfer (FRET) studies were conducted, using CD47 KO 293T cells expressing a CD47 variant labeled with an energy acceptor and a SLAMF7 variant labeled with an energy donor (FIGs. 4D,E and 14D). Photobleaching of acceptor-labeled CD47 resulted in increased fluorescence of donor-labeled SLAMF7, indicating energy transfer between the two molecules (FIGs. 4D,E). Energy transfer implies close physical proximity. The FRET between CD47 and SLAMF7 was reduced by a blocking anti-CD47 MAb, which interacts with the SIRPa- binding domain of CD47, compared to control IgG (FIGs. 4D,E). It was also diminished by a point mutation in the SIRPa-binding domain of CD47 [phenylalanine 37-to-aspartate 37 (F37D) mutation]. Likewise, it was reduced by mutations of the putative self-ligand-binding sequences of SLAMF7 [arginine 75-to-alanine 75 (R75A) or glutamate 77-to-alanine 77 (E77A) mutations]2223(FIGs. 14E-H). The F37D CD47 mutant also failed to repress phagocytosis when introduced in CD47 KO L1210 and did not bind a SIRPa-Fc fusion protein, compared to wild-type CD47 (FIGs. 15A,B). Furthermore, the R75A and E77A SLAMF7 mutants failed to rescue the pro-phagocytic effect of SLAMF7 when expressed in SLAMF7 KO T cells, and did not bind to a soluble SLAMF7- Fc fusion protein, compared to wild-type SLAMF7, in keeping with the idea that these mutations abolished the SLAMF7-SLAMF7 homotypic interaction (FIGs. 14F and 15C,D).

[0357] To assess if the physical proximity of CD47 and SLAMF7 was due to a direct interaction, FRET assays were performed using cell-free large unilamellar vesicles (LUVs) reconstituted with purified extracellular domains of CD47 and SLAMF7, which were labeled with energy acceptor and donor, respectively (FIG. 4F). Addition of CD47, but not of unrelated receptor PD-1 , quenched the fluorescence of SLAMF7, indicating energy transfer and proximity (FIG. 4G). The FRET was reduced by blocking anti-CD47 MAb or blocking anti-SLAMF7 MAb 4G2 (FIG. 4H).

[0358] Together, these results implied that CD47 directly interacted in cis with SLAMF7 at the surface of tumor cells, and that this interaction was mediated by their canonical ligand-binding domains.

[0359] Example 6: Evidence that freeing of SLAMF7 from CD47 promotes phagocytosis

[0360] To evaluate if release of SLAMF7 from CD47 was a trigger for phagocytosis, two approaches were used. First, using a binding assay with a SLAMF7-positive cell line, it was observed that blocking anti-CD47 MAbs augmented the ability of a soluble SLAMF7-Fc fusion protein to bind to these cells, compared to a control Fc fusion protein (FIGs. 5A,B). No binding was seen when the cell line lacked SLAMF7 (FIG. 5C). Second, it was hypothesized that overexpression of SLAMF7 on tumor cells might create a pool of CD47-free SLAMF7 that could elicit phagocytosis. When SLAMF7 was overexpressed (~30-fold) in L1210 cells, there was a recovery of phagocytosis during SIRPa blockade, to nearly the same extent as that seen with CD47 deficiency (FIGs. 5D,E). This effect involved SLAMF7 on macrophages (FIG. 5F). There was no increase in phagocytosis if tumor cells overexpressed SLAMF7 but lacked CD47 (FIGs. 5D,E).

[0361] Combined with the results showing that a CD47 mutant (F37D) unable to bind SLAMF7 in cis did not suppress phagocytosis by SIRPa KO BMDMs (FIG. 15A,B), this data suggested that the release of SLAMF7 from CD47, by (i) blocking anti-CD47 antibodies, (ii) enforced expression of SLAMF7 or (iii) mutation of CD47, promoted phagocytosis.

[0362] Example 7: Identification of a first-in-class agonistic anti-SLAMF7 MAb

[0363] To obtain more direct evidence that freeing SLAMF7 from CD47 triggered phagocytosis, it was sought to identify pro-phagocytic anti-SLAMF7 MAbs, which act by dissociating SLAMF7 from CD47. To this end, a large collection of novel anti-human SLAMF7 MAbs were generated. The non-blocking anti-SLAMF7 Mab, elotuzumab, which is used in the clinic for the treatment of multiple myeloma24(FIG. 6A), was also studied. All MAbs reacted with human SLAMF7, but not with mouse SLAMF7, and were modified to be Fc-silent (see Example 1).

[0364] One newly generated MAb, MAb Z10, was able to stimulate phagocytosis of Raji (FIGs. 6A,B). This effect was seen with SIRPa KO mouse BMDMs, but not with wild-type BMDMs. The combined impact of MAb Z10 and inactivation of SIRPa was analogous to that of anti-CD47 Mab B6H12. None of the other anti-SLAMF7 MAbs, including elotuzumab, had a pro-phagocytic impact. The combined impact of Z10 and SIRPa deficiency on phagocytosis was analogous to that of the CD47 monoclonal antibody B6H12. In addition, Z10 triggered the ability of Sirpa~7~ BMDMs to phagocytose Daudi cells and the multiple myeloma cell line MM.1S, which express human SLAMF7 endogenously and Fc-silent CD3 + CD28 monoclonal antibody-activated mouse CD8+ T cells transduced with human SLAMF7 (FIGs. 16A-B).

[0365] The agonistic effect of MAb Z10 was also seen with human macrophages, and with mouse macrophages retrovirally transduced by human SLAMF7, in the presence of Raji as targets (FIGs. 6E-G). However, no impact was seen when macrophages expressed human SLAMF7, but targets expressed mouse SLAMF7 (FIG. 6H), indicating that the pro-phagocytic effect of MAb Z10 involved expression of human SLAMF7 on the target cells (FIG. 17A). In an in vivo subcutaneous tumor growth assay using RAG-1-SIRPa double (D) KO mice, MAb Z10 also displayed anti-tumor activity against Raji, compared to control IgG (FIGs. 6I and 17B). In Rag1~~ mice subcutaneously injected with Raji cells, intraperitoneal co-administration of the SIRPa monoclonal antibody no. 27 and Z10 suppressed tumor growth and increased survival more than CD47 monoclonal antibody B6H12 relative to control MOPC21 IgG (FIGs. 6I, 6J and 16D). No antitumor effect was seen with SIRPa monoclonal antibody no. 27 alone, Z10 alone, SIRPa monoclonal antibody no. 27 + elotuzumab or elotuzumab alone (FIGs. 6J and 16D). Thus, Z10 is an agonist monoclonal antibody that stimulated phagocytosis and prevented tumor growth when combined with SIRPa blockade, an impact that involved Z10 binding to SLAMF7 on tumor cells, not on macrophages.

[0366] Example 8: Anti-SLAMF7 MAb Z10 frees SLAMF7 from CD47 and is non-blocking

[0367] FRET studies showed that MAb Z10, but not elotuzumab, dissociated the cis interaction between CD47 and SLAMF7, in a manner analogous to anti-CD47 MAb B6H12 (FIGs. 7A,B). Furthermore, in confocal microscopy analyses, the combination of MAb Z10 and SIRPa blockade enhanced actin polarization in macrophages exposed to tumor cells, without promoting conjugate formation between macrophages and tumor cells, in a manner comparable to CD47 blockade5(FIGs. 7C and 10A,B).

[0368] To examine if MAb Z10 was a blocking MAb, its binding to chimeras between human and mouse SLAMF7 was first analyzed. Like elotuzumab, MAb Z10 interacted with the SLAMF7 second Ig-like domain [lg(C)], which is not involved in ligand-binding (i.e., SLAMF7-SLAMF7 interaction) (FIG. 7D). In contrast, another anti-SLAMF7 MAb, MAb Z8, interacted with the first Ig-like domain [lg(V)j, which is responsible for ligand-binding (FIG. 7D). Next, it was assessed whether MAb Z10 was able to suppress SLAMF7-dependent phagocytosis. MAb Z10 did not interfere with phagocytosis of CD47 KO Raji or Daudi, which are phagocytosed via SLAMF7, in keeping with the idea that MAb Z10 was a non-blocking MAb towards the SLAMF7-SLAMF7 homotypic interaction (FIGs. 18A,B).

[0369] By creating additional chimeras between human and mouse SLAMF7, it was found that replacement of amino acids 169-176 or 181-188 in human SLAMF7 abolished binding of Mab Z10 and elotuzumab, but not of MAb Z8 (FIG. 19A). Furthermore, point mutations (to alanines) of human SLAMF7 showed that arginine 181 (R181), asparagine 182 (N182), phenylalanine 183 (F183) or, to a lesser extent, valine 174 (V174) were involved for binding of MAb Z10 and elotuzumab, but not of MAb Z8 (FIGs. 7E and 19B). V174 and N182 were more involved in the binding of MAb Z10, relative to elotuzumab. The latter finding suggested differences in the binding mechanisms of MAb Z10 and elotuzumab to SLAMF7. Mutation of amino acids 172, 173 or 177 abolished or greatly reduced binding of all MAbs, implying that these mutations compromised expression of SLAMF7 at the cell surface (FIG. 19B).

[0370] Hence, like anti-CD47 MAbs, the first-in-class agonistic anti-SLAMF7 MAb Z10 dissociated the CD47-SLAMF7 cis interaction on tumor cells, without interacting with sequences directly involved in the SLAMF7-SLAMF7 interaction, suggesting that the effect of Z10 was due to conformational modification of SLAMF7 rather than direct blocking of CD47-SLAMF7, which seemingly implicated sequences similar to those involved in the SLAMF7-SLAMF7 interaction.

[0371] Although the present invention has been described hereinabove by way of specific embodiments thereof, it can be modified, without departing from the spirit and nature of the subject invention as defined in the appended claims. In the claims, the word "comprising" is used as an open-ended term, substantially equivalent to the phrase "including, but not limited to". The singular forms "a", "an" and "the" include corresponding plural references unless the context clearly dictates otherwise. REFERENCES

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Claims

WHAT IS CLAIMED IS:

1. A SLAM family member 7 (SLAMF7)-binding molecule that reduces or inhibits the interaction between SLAMF7 and Cluster of Differentiation 47 (CD47) on a cell.

2. The SLAMF7-binding molecule of claim 1 , wherein the SLAMF7-binding molecule binds to a domain or epitope comprising residues 172-174, 177 and 181-183 of human SLAMF7.

3. The SLAMF7-binding molecule of claim 1 or 2, wherein the SLAMF7-binding molecule is an antibody or an antigen-binding fragment thereof.

4. The SLAMF7-binding molecule of claim 3, wherein the antibody or antigen-binding fragment thereof comprises the following complementary determining regions (CDRs): a light chain CDR1 comprising the amino acid sequence KASQDVDTAVA (SEQ ID NO:1), a light chain CDR2 comprising the amino acid sequence WASTRHT (SEQ ID NO:2), a light chain CDR3 comprising the amino acid sequence QQYRSYPFT (SEQ ID NO:3), a heavy chain CDR1 comprising the amino acid sequence GIDFSRY (SEQ ID NO:4), a heavy chain CDR2 comprising the amino acid sequence NPDSST (SEQ ID NO:5), and a heavy chain CDR3 comprising the amino acid sequence PGDYDAWYFDV (SEQ ID NO:6).

5. The SLAMF7-binding molecule of claim 4, wherein the antibody or antigen-binding fragment thereof comprises the following framework regions (FRs): a light chain FR1 comprising the amino acid sequence DITMSQSHKFMSTSVGDRVSITC (SEQ ID NO:7), a light chain FR2 comprising the amino acid sequence WYQQKPGQSPKLLIY (SEQ ID NO:8), a light chain FR3 comprising the amino acid sequence GVPDRFTGSGSGTDFTLTISNVQSEDLADYFC (SEQ ID NO:9), a light chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NO: 10), a heavy chain FR1 comprising the amino acid sequence EVKLLQSGGGLVQPGGSLKLSCAAS (SEQ ID NO:11), a heavy chain FR2 comprising the amino acid sequence WMSWVRRAPGKGLEWIGEI (SEQ ID NO:12), a heavy chain FR3 comprising the amino acid sequence INYAPSLKDKFIISRDNAKNTLYLQMSKVRSEDTALYYCAR (SEQ ID NO:13), and / or a heavy chain FR4 comprising the amino acid sequence WGTGTTVTVSS (SEQ ID NO: 14).

6. The SLAMF7-binding molecule of claim 5, wherein the antibody or antigen-binding fragment thereof comprises all the FRs defined in claim 5.

7. The SLAMF7-binding molecule of any one of claims 3 to 6, wherein the antibody or antigenbinding fragment thereof comprises a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 16.

8. The SLAMF7-binding molecule of any one of claims 3 to 7, wherein the antibody or antigenbinding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO:20.

9. The SLAMF7-binding molecule of any one of claims 3 to 8, wherein the antibody or antigenbinding fragment thereof further comprises at least one constant domain or a fragment thereof.

10. The SLAMF7-binding molecule of claim 9, which comprises a Fragment crystallizable (Fc) fragment of a heavy chain constant region of an antibody.11 . The SLAMF7-binding molecule of any one of claims 3 to 10, wherein the antibody is a fully human antibody or a chimeric antibody.

12. The SLAMF7-binding molecule of claim 11 , wherein the antibody is an IgG 1 antibody.

13. The SLAMF7-binding molecule of any one of claims 1 to 12, wherein the SLAMF7-binding molecule is conjugated to an antitumor agent.

14. The SLAMF7-binding molecule of claim 13, wherein the antitumor agent is a chemotherapeutic agent, a radionuclide, or a checkpoint inhibitor.

15. The SLAMF7-binding molecule of any one of claims 1 to 14, which is a bispecific binding molecule.

16. The SLAMF7-binding molecule of claim 15, wherein the bispecific binding molecule comprising a binding module that binds to signal inhibitory regulatory protein alpha (SIRPa).

17. The SLAMF7-binding molecule of claim 16, wherein the binding module that binds to SIRPa is an anti- SIRPa antibody or an antigen-bonding fragment thereof.

18. The SLAMF7-binding molecule of any one of claims 1 to 17, for use in increasing the susceptibility to phagocytosis of cells expressing SLAMF7 and Cluster of Differentiation 47 (CD47).

19. The SLAMF7-binding molecule for use according to claim 18, wherein the cells are tumor cells.

20. The SLAMF7-binding molecule of any one of claims 1 to 17, for use in the treatment of a cancer comprising tumor cells expressing SLAMF7 and CD47 in a subject.21 . The SLAMF7-binding molecule for use according to claim 19 or 20, wherein the tumor cells are hematopoietic tumor cells.

22. The SLAMF7-binding molecule for use according to claim 21 , wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

23. The SLAMF7-binding molecule for use according to any one of claims 18 to 22, wherein the SLAMF7-binding molecule is for use in combination with a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or with an agent capable of inducing antibody-dependent cell- mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

24. The SLAMF7-binding molecule for use according to claim 23, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigen-binding fragment thereof, or a soluble SIRPa polypeptide.

25. A method for increasing the susceptibility to phagocytosis of cells expressing SLAM family member 7 (SLAMF7) and Cluster of Differentiation 47 (CD47), the comprising contacting the tumor cells with the SLAMF7-binding molecule defined in any one of claims 1 to 17.

26. The method of claim 25, wherein the cells are tumor cells.

27. The method of claim 26, wherein the tumor cells are hematopoietic tumor cells.

28. The method of claim 27, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

29. The method of any one of claims 25 to 28, wherein the method further comprises contacting the cells with a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or an agent capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

30. The method of claim 29, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigen-binding fragment thereof, or a soluble SIRPa polypeptide.

31. Use of the SLAMF7-binding molecule defined in any one of claims 1 to 17 for increasing the susceptibility to phagocytosis of tumor cells expressing SLAMF7 and CD47.

32. Use of the SLAMF7-binding molecule defined in any one of claims 1 to 17 for the manufacture of a medicament for increasing the susceptibility to phagocytosis of tumor cells expressing SLAMF7 and CD47.

33. The use of claim 31 or 32, wherein the tumor cells are hematopoietic tumor cells.

34. The use of claim 33, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

35. The use of any one of claims 31 to 34, wherein the SLAMF7-binding molecule or medicament is for use in combination with a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or with an agent capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

36. The use of claim 35, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigenbinding fragment thereof, or a soluble SIRPa polypeptide.

37. A method for treating a cancer comprising tumor cells expressing SLAMF7 and CD47 in a subject in need thereof, the method comprising administering to the subject an effective amount of the SLAMF7-binding molecule defined in any one of claims 1 to 17.

38. The method of claim 37, wherein the tumor cells are hematopoietic tumor cells.

39. The method of claim 38, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

40. The method of any one of claims 37 to 39, further comprising administering to the subject an effective amount of a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or of an agent capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

41. The method of claim 40, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigen-binding fragment thereof, or a soluble SIRPa polypeptide.

42. Use of the SLAMF7-binding molecule defined in any one of claims 1 to 17 for treating a cancer comprising tumor cells expressing SLAMF7 and CD47 in a subject.

43. Use of the SLAMF7-binding molecule defined in any one of claims 1 to 17 for the manufacture of a medicament for treating a cancer comprising tumor cells expressing SLAMF7 and CD47 in a subject.

44. The use of claim 42 or 43, wherein the tumor cells are hematopoietic tumor cells.

45. The use of claim 44, wherein the hematopoietic tumor cells are multiple myeloma cells or lymphoma cells.

46. The use of any one of claims 42 to 45, wherein the SLAMF7-binding molecule or medicament is for use in combination with a signal inhibitory regulatory protein alpha (SIRPa) inhibitor, and / or with an agent capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP).

47. The use of claim 46, wherein the SIRPa inhibitor is an anti-SIRPa antibody or an antigenbinding fragment thereof, or a soluble SIRPa polypeptide.

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