Combination of a BTN3A activating antibody, a BCL2 inhibitor and a hypomethylating agent for use in the treatment of cancer

Abstract

The present invention relates to therapeutic combinations of BTN3A activating antibodies, Bcl-2 family inhibitors and hypomethylating agents that are particularly useful in the treatment of cancer, particularly hematological malignancies. More particularly, the disclosure relates to the combination of BTN3A activating antibodies that activate the cytolytic function of Vy9V52 T cells, and venetoclax, which selectively inhibits the Bcl2 receptor, and hypomethylating agents, such as azacitidine, to synergistically and specifically promote Vy9V52 T cell anti-cancer activity.

Description

[Technical Field]

[0001] Disclosed below is a therapeutic combination of a BTN3A activating antibody, a Bcl-2 inhibitor and a hypomethylating agent that is particularly useful for the treatment of cancer, particularly hematological malignancies. The disclosure more particularly relates to the combination of a BTN3A activating antibody that activates the cytolytic function of Vy9V52 T cells, and venetoclax, which selectively inhibits the Bcl2 receptor, and a hypomethylating agent, such as azacitidine, to synergistically and specifically promote Vy9V52 T cell anti-cancer activity. [Background technology]

[0002] To date, various therapeutic and vaccine strategies have been proposed for the treatment of cancer that rely on the patient's immune response, more particularly, on mobilizing T lymphocytes against malignant cells. Several immunomodulatory antibodies against CTLA-4, PD-1, and PD-L1 have already been approved for clinical use by multiple regulatory agencies worldwide. While these drugs represent a major advance in cancer therapy, there remains unmet medical need for a large proportion of cancer patient populations who do not respond to currently available treatments.

[0003] Gamma delta (γδ) T cells are a non-conventional T cell subset with characteristics of both innate and adaptive immune responses that play a key role in immune surveillance against malignancies and infections (Holtmeier W et al., eds. Chem Immunol Allergy [Internet]. Basel: KARGER; 2005 [cited 19 August 2022]. pp. 151–183. Available at: https: / / www.karger.com / Article / FullText / 86659 ). In the blood of healthy adult humans, Vγ9Vδ2 T cells constitute the majority (50–90%) of circulating γδ T cells (Kabelitz D et al., Cell Mol Immunol. 2020 Sep;17(9):925–939), accounting for 1–5% of all blood lymphocytes (Pauza CD et al., Gamma Delta T Cell Therapy for Cancer: It Is Good to be Local. Front Immunol [Internet]. 2018 [cited November 19, 2018]). Unlike conventional αβ T cells, which recognize MHC-restricted antigens and constitute a large subset of circulating T cells, γδ T cells are activated in an MHC-independent manner. More specifically, Vγ9Vδ2 T cell activation is triggered by the intracellular accumulation of phosphoantigens (pAg), which are overproduced in response to viral and bacterial infections, metabolic stress, or gene dysregulation during carcinogenesis. Activation of Vγ9Vδ2 T cells under these pathophysiological conditions induces a wide range of functional activities, including production of cytokines and chemokines, cytolysis of infected or transformed target cells, and interactions with other cells, including epithelial cells, monocytes, dendritic cells (DCs), neutrophils, and B cells (Blazquez JL, Benyamine A, Pasero C, Olive D. New Insights Into the Regulation of γδ T Cells by BTN3A and Other BTN / BTNL in Tumor Immunity. Front Immunol [Internet]. 2018 [cited August 6, 2018]).Vγ9Vδ2 T cells have become attractive targets for cancer immunotherapy due to their potent antitumor activity (Pauza CD et al., supra) and the association between infiltration into malignant tissues and favorable prognosis (Gentles AJ et al., Nat Med. 2015 Aug;21(8):938-945; Tosolini M et al., OncoImmunology. 2017 Mar 4;6(3):e1284723. PMID:28405516).

[0004] In recent years, numerous Vγ9Vδ2 T cell-based immuno-oncological therapeutic approaches have been investigated in various tumors. These approaches involve either in vivo activation of Vγ9Vδ2 T cells using aminobisphosphonates (ABPs), such as zoledronate or synthetic phosphoantigens (i.e., BrHPP), in combination with IL-2, or adoptive transfer of autologous or allogeneic Vγ9Vδ2 T cells into patients after in vitro / ex vivo expansion (Kabelitz D et al., 2020, supra). Although these two Vγ9Vδ2 T cell-based therapies appear to be safe, the clinical responses obtained have been variable among patients (Kabelitz D et al., 2020, supra). In particular, in the case of leukemia, only low responses have been observed in clinical trials in which Vγ9Vδ2 T cells were stimulated in vivo with ABPs. In contrast, expansion and transfer of Vy9V52 T cells has a response rate of over 50% (Kunkele KP et al., Cells. 2020 Mar 30;9(4):829; Barros M de S et al., Front Immunol. 2021 Sep 22;12:729085), thereby supporting the utility of Vy9V52 T cells, in particular, in AML therapy, and their use is being further investigated in multiple clinical trials (Saura-Esteller J et al., Front Immunol. 2022 Jun 16;13:915837). Although apparently successful, in vitro / ex vivo expansion and transfer of Vy9V52 T cells is laborious, suggesting that novel strategies are needed to enhance the clinical benefit of Vy9V52 T cell-mediated antitumor immunity using in vivo activation and expansion.

[0005] The patent publications WO2012080351(A1), WO2012080769(A1), WO20200257031(A1) and WO2020136218 refer to various antibodies against BTN3A that are capable of activating cytokine production, proliferation and cytolytic function of Vy9V52 T cells.

[0006] Previous studies have shown that venetoclax and the hypomethylating agent 5-azacytidine have direct anti-leukemia activity. Furthermore, 5-azacytidine has been shown to enhance cancer cell recognition by immune effector cells by inducing stress ligand expression (Gang AO et al., Blood Cancer J. 2014 March;4(3):e197-e197; Lee JB et al., Blood. 2021 July 22;138(3):234-245). Venetoclax has also been shown to enhance T cell- and NK cell-mediated cytotoxicity against AML blasts (Lee et al., 2021, supra; Wu et al., Int Immunopharmacol. 2022 March;104:108-497). However, venetoclax treatment causes lymphopenia in AML patients, which may be clinically harmful given that Vγ9Vδ2 T cells typically represent less than 5% of all T cells in the peripheral blood of adult humans. Summary of the Invention

[0007] The inventors have shown that ICT01-mediated activation of Vy9V52 T cells partially protects Vy9V52 T cells from venetoclax-induced cell death, and that the combination of ICT01 with venetoclax and 5-azacytidine significantly improves AML cell killing.

[0008] The present invention relies on the use of venetoclax and the hypomethylating agent ICT01 in combination, which shows superior AML killing compared to single agents. By inducing Vy9V52 T cell anti-cancer activity, Vy9V52 T cells are protected from cell death and AML cells undergo chemotherapy-induced apoptosis.

[0009] Specific Embodiments E1. A BTN3A activating antibody for use in treating cancer in a subject in need thereof, wherein a therapeutically effective amount of the BTN3A activating antibody is administered to the subject in combination with a therapeutically effective amount of a Bcl2 family inhibitor compound, e.g., a Bcl2 inhibitor, either simultaneously, sequentially, or separately, and optionally further in combination with a hypomethylating agent.

[0010] E2. A BTN3A activating antibody for use according to embodiment E1, wherein said Bcl2 inhibitor is venetoclax.

[0011] E3. The BTN3A activating antibody binds to human BTN3A with a K of 10 nM or less as measured by surface plasmon resonance. D , preferably a K of 5 nM or less D 4. A BTN3A activating antibody for use according to embodiment E1 or E2 which binds at

[0012] E4. The BTN3A-activating antibody, in co-culture with BTN3A-expressing cells, inhibits activation of γδ T cells, typically Vγ9Vδ2 T cells, as measured by a degranulation assay, at an EC of less than 5 μg / ml, preferably 1 μg / ml or less. 50 A BTN3A activating antibody for use according to any one of embodiments E1 to E3, which induces

[0013] E5. The BTN3A activating antibody (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3; comprising HCDRs 1 to 3 of SEQ ID NOs: 12 to 14 and LCDRs 1 to 3 of SEQ ID NOs: 15 to 17; comprising HCDRs 1 to 3 of SEQ ID NOs: 18 to 20 and LCDRs 1 to 3 of SEQ ID NOs: 21 to 23; or A BTN3A activating antibody for use according to any one of embodiments E1 to E4, which competes for binding with an antibody selected from mAb 20.1 produced by the hybridoma deposited at the CNCM under deposit number I-4401, mAb 7.2 produced by the hybridoma deposited at the CNCM under deposit number I-4402, and an antibody having a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6.

[0014] E6. A BTN3A activating antibody for use according to any one of embodiments E1-E5, wherein the BTN3A antibody comprises a mutated or chemically modified IgG1 constant region that abolishes or reduces binding to Fcγ receptors compared to a corresponding antibody having a wild-type IgG1 isotype constant region.

[0015] E7. A BTN3A activating antibody for use according to any one of embodiments E1 to E6, wherein said mutated IgG1 constant region is of the IgG1 triple mutation L247F, L248E and P350S.

[0016] E8. A BTN3A activating antibody for use according to any one of embodiments E1 to E7, wherein said anti-BTN3A antibody is an antibody comprising a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

[0017] E9. A BTN3A activating antibody for use according to any one of embodiments E1 to E8, wherein the BTN3A activating antibody is administered in combination with venetoclax and further in combination with azacitidine or decitabine, either simultaneously, sequentially or separately.

[0018] E10. A BTN3A activating antibody for use according to any one of embodiments E1-E9, wherein said cancer is a hematological malignancy.

[0019] E11. An anti-BTN3A activating antibody for use according to any one of embodiments E1-E10, wherein said cancer is acute myeloid leukemia.

[0020] E12. A BTN3A activating antibody for use according to any one of embodiments E1 to E11, wherein the cancer is acute myeloid leukemia, and the BTN3A activating antibody is an antibody comprising a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and the BTN3A activating antibody is administered in combination with venetoclax, and further in combination with azacitidine or decitabine, either simultaneously, sequentially or separately.

[0021] E13. A BTN3A activating antibody for use according to any one of embodiments E1-E12, wherein said subject is ineligible for intensive chemotherapy.

[0022] E14. A BTN3A activating antibody for use according to any one of embodiments E1-E13, wherein said subject is over 75 years of age.

[0023] E15. A BTN3A activating antibody for use according to any one of embodiments E1 to E14, wherein said BTN3A activating antibody is administered once every three weeks or once every four weeks.

[0024] E16. A BTN3A activating antibody for use according to any one of embodiments E1 to E15, wherein said BTN3A activating antibody is administered intravenously.

[0025] E17. A BTN3A activating antibody for use according to any one of embodiments E1 to E16, wherein the BTN3A activating antibody is administered at a unit dose of about 7 to about 200 mg, e.g., about 75 mg, e.g., once every 21 days, for 1 to 22 cycles.

[0026] E18. A BTN3A activating antibody for use according to any one of embodiments E1 to E17, wherein venetoclax is administered orally once daily.

[0027] E19. A BTN3A activating antibody for use according to any one of embodiments E1 to E18, wherein venetoclax is administered orally in a unit dose of about 50 mg to about 500 mg.

[0028] E20. A BTN3A activating antibody for use according to any one of embodiments E1 to E19, wherein azacitidine or decitabine is administered as the hypomethylating agent.

[0029] E21. A BTN3A activating antibody for use according to any one of embodiments E1 to E20, wherein azacitidine is administered as the hypomethylating agent.

[0030] E22. Hypomethylating agents are administered at approximately 50 mg / m 2 ~about 100mg / m 2 The BTN3A activating antibody for use according to any one of embodiments E1 to E21, wherein the antibody is administered at a dose of

[0031] E23. Hypomethylating agents are administered at approximately 75 mg / m 2 The BTN3A activating antibody for use according to any one of embodiments E1 to E22, wherein the antibody is administered at a dose of

[0032] E24. A BTN3A activating antibody for use according to any one of embodiments E1 to E23, wherein the hypomethylating agent is administered once daily.

[0033] E25. A BTN3A activating antibody for use according to any one of embodiments E1 to E24, wherein the hypomethylating agent is administered for 5 to 7 consecutive days.

[0034] E26. Hypomethylating agents (a) Administered for 7 consecutive days on days 1–7 of a 28-day cycle; or (b) administered for 5 consecutive days on days 1-5 of a 28-day cycle, followed by 2 days off, and then administered for 2 consecutive days on days 8-9; or (c) A BTN3A activating antibody for use according to any one of embodiments E1 to E25, administered for 6 consecutive days on days 1 to 6 of a 28-day cycle, followed by 1 day off, and then optionally administered once on day 8.

[0035] E27. A BTN3A activating antibody for use according to any one of embodiments E1 to E26, wherein the hypomethylating agent is administered subcutaneously or intravenously.

[0036] E28. A BTN3A activating antibody for use according to any one of embodiments E1 to E27, wherein venetoclax is initially administered for at least one cycle, followed by a recovery period, and the BTN3A activating antibody is administered, for example, after a recovery period of at least 10 to 14 days, or together with the administration of cycle 2 of venetoclax.

[0037] E29. A BTN3A activating antibody for use according to any one of embodiments E1 to E27, wherein the first administration of venetoclax is administered after the first cycle of treatment with the BTN2A activating antibody, preferably 21 days after the first administration of the BTN3A activating antibody.

[0038] E30. A method of treating cancer in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of a BTN3A activating antibody in combination with a therapeutically effective amount of a Bcl2 family inhibitor compound, e.g., a Bcle2 inhibitor, either simultaneously, sequentially or separately, and optionally further in combination with a hypomethylating agent.

[0039] E31. The method of embodiment E30, wherein said Bcl2 inhibitor is venetoclax.

[0040] E32. The BTN3A activating antibody binds to human BTN3A with a K of 10 nM or less as measured by surface plasmon resonance. D , preferably a K of 5 nM or less D The method of embodiment E30 or E31, wherein

[0041] E33. The BTN3A-activating antibody inhibits activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with BTN3A-expressing cells, as measured by a degranulation assay at an EC of less than 5 μg / ml, preferably 1 μg / ml or less. 50 The method of any one of embodiments E30-E32, wherein the induction is with

[0042] E34. The BTN3A activating antibody (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1, and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:3; comprising HCDRs 1 to 3 of SEQ ID NOs: 12 to 14 and LCDRs 1 to 3 of SEQ ID NOs: 15 to 17; comprising HCDRs 1 to 3 of SEQ ID NOs: 18 to 20 and LCDRs 1 to 3 of SEQ ID NOs: 21 to 23; or The method of any one of embodiments E30 to E33, wherein the antibody competes for binding with an antibody selected from mAb 20.1 produced by the hybridoma deposited at the CNCM under deposit number I-4401, mAb 7.2 produced by the hybridoma deposited at the CNCM under deposit number I-4402, and an antibody having a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6.

[0043] E35. The method of any one of embodiments E30-E34, wherein said BTN3A antibody comprises a mutated or chemically modified IgG1 constant region that abolishes or reduces binding to Fcγ receptors compared to a corresponding antibody having a wild-type IgG1 isotype constant region.

[0044] E36. The method of any one of embodiments E30-E35, wherein said mutated IgG1 constant region is of the IgG1 triple mutant L247F, L248E and P350S.

[0045] E37. The method of any one of embodiments E30-E36, wherein said anti-BTN3A antibody is an antibody comprising a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

[0046] E38. The method of any one of embodiments E30-E37, wherein said BTN3A activating antibody is administered in combination with venetoclax and further in combination with azacitidine or decitabine, either simultaneously, sequentially or separately.

[0047] E39. The method of any one of embodiments E30-E38, wherein said cancer is a hematological malignancy.

[0048] E40. The method of any one of embodiments E30-E39, wherein said cancer is acute myeloid leukemia.

[0049] E41. The method of any one of embodiments E30-E40, wherein the cancer is acute myeloid leukemia, and the BTN3A activating antibody is an antibody comprising a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and the BTN3A activating antibody is administered in combination with venetoclax, and further in combination with azacitidine or decitabine, either simultaneously, sequentially or separately.

[0050] E42. The method of any one of embodiments E30-E41, wherein said subject is not eligible for intensive chemotherapy.

[0051] E43. The method of any one of embodiments E30-E42, wherein said subject is over 75 years of age.

[0052] E44. The method of any one of embodiments E30-E43, wherein the BTN3A activating antibody is administered once every three weeks or once every four weeks.

[0053] E45. The method of any one of embodiments E30-E44, wherein the BTN3A activating antibody is administered intravenously.

[0054] E46. The method of any one of embodiments E30-E45, wherein the BTN3A activating antibody is administered at a unit dose of about 7 to about 200 mg, e.g., about 75 mg, e.g., once every 21 days, for 1 to 22 cycles.

[0055] E47. The method of any one of embodiments E30-E46, wherein venetoclax is administered orally once daily.

[0056] E48. The method of any one of embodiments E30-E47, wherein venetoclax is administered orally in a unit dose of about 50 mg to about 500 mg.

[0057] E49. The method of any one of embodiments E30-E48, wherein azacitidine or decitabine is administered as the hypomethylating agent.

[0058] E50. The method of any one of embodiments E30-E49, wherein azacitidine is administered as a hypomethylating agent.

[0059] E51. Hypomethylating agents are administered at approximately 50 mg / m 2 ~about 100mg / m 2 The method of any one of embodiments E30 to E50, wherein the dose is

[0060] E52. Hypomethylating agents are approximately 75 mg / m 2 The method of any one of embodiments E30 to E51, wherein the dose is

[0061] E53. The method of any one of embodiments E30-E52, wherein the hypomethylating agent is administered once daily.

[0062] E54. The method of any one of embodiments E30-E53, wherein the hypomethylating agent is administered for 5 to 7 consecutive days. E55. Hypomethylating agents (a) Administered for 7 consecutive days on days 1–7 of a 28-day cycle; or (b) administered for 5 consecutive days on days 1-5 of a 28-day cycle, followed by 2 days off, and then administered for 2 consecutive days on days 8-9; or (c) The method of one of embodiments E30 to E54, wherein the dose is administered for 6 consecutive days on days 1 to 6 of a 28-day cycle, followed by 1 day off, and then optionally once on day 8.

[0063] E56. The method of any one of embodiments E30-E55, wherein the hypomethylating agent is administered subcutaneously or intravenously.

[0064] E57. The method of any one of embodiments E30-E56, wherein venetoclax is initially administered for at least one cycle, followed by a recovery period, and the BTN3A activating antibody is administered, for example, after a recovery period of at least 10-14 days, or together with the administration of cycle 2 of venetoclax.

[0065] E58. The method of any one of embodiments E30-E56, wherein the first administration of venetoclax is administered after the first cycle of treatment with a BTN2A activating antibody, preferably 21 days after the first administration of a BTN3A activating antibody. [Brief explanation of the drawings]

[0066] [Figure 1a]Figure 3: Activation with ICT01 protects Vy9V52 T cells from venetoclax-induced cell death. A) Mean ± SEM frequency of live Vy9V52 T cells (black), apoptotic Vy9V52 T cells (light grey) or dead Vy9V52 T cells (dark grey) in HD-PBMCs after 48 h of treatment with venetoclax (left) or 5-azacytidine (right). N=4 HD. B) Mean frequency of live Vy9V52 T cells in HD-PBMCs after 48 h of treatment with venetoclax, 5-azacytidine or their combination. N=4 HD. C) Mean ± SEM of the frequency of live Vy9V52 T cells (black), apoptotic Vy9V52 T cells (light gray), or dead Vy9V52 T cells (dark gray) in HD-PBMCs 48 h after treatment with venetoclax in the presence of ICT01 (right) or its isotype control (hIgG1S, left). N=4 HDs. D) Mean ± SEM of the frequency of live Vy9V52 T cells in HD-PBMCs 48 h after treatment with venetoclax in the presence of ICT01 (black) or its isotype control (hIgG1S, light gray), normalized to the condition without venetoclax. N=4 HDs, each dot represents one healthy donor. ***p<0.005, 2-way ANOVA. E) Mean ± SEM of median CD69 mean fluorescence intensity (MFI) of Vy9V52 T cells in HD-PBMCs treated for 48 h with ICT01 (black) or its isotype control (hIgG1S, light grey). N=5 HD, each dot represents one healthy donor. [Figure 1b]Figure 3: Activation with ICT01 protects Vy9V52 T cells from venetoclax-induced cell death. A) Mean ± SEM frequency of live Vy9V52 T cells (black), apoptotic Vy9V52 T cells (light grey) or dead Vy9V52 T cells (dark grey) in HD-PBMCs after 48 h of treatment with venetoclax (left) or 5-azacytidine (right). N=4 HD. B) Mean frequency of live Vy9V52 T cells in HD-PBMCs after 48 h of treatment with venetoclax, 5-azacytidine or their combination. N=4 HD. C) Mean ± SEM of the frequency of live Vy9V52 T cells (black), apoptotic Vy9V52 T cells (light gray), or dead Vy9V52 T cells (dark gray) in HD-PBMCs 48 h after treatment with venetoclax in the presence of ICT01 (right) or its isotype control (hIgG1S, left). N=4 HDs. D) Mean ± SEM of the frequency of live Vy9V52 T cells in HD-PBMCs 48 h after treatment with venetoclax in the presence of ICT01 (black) or its isotype control (hIgG1S, light gray), normalized to the condition without venetoclax. N=4 HDs, each dot represents one healthy donor. ***p<0.005, 2-way ANOVA. E) Mean ± SEM of median CD69 mean fluorescence intensity (MFI) of Vy9V52 T cells in HD-PBMCs treated for 48 h with ICT01 (black) or its isotype control (hIgG1S, light grey). N=5 HD, each dot represents one healthy donor. [Figure 1c]Figure 3: Activation with ICT01 protects Vy9V52 T cells from venetoclax-induced cell death. A) Mean ± SEM frequency of live Vy9V52 T cells (black), apoptotic Vy9V52 T cells (light grey) or dead Vy9V52 T cells (dark grey) in HD-PBMCs after 48 h of treatment with venetoclax (left) or 5-azacytidine (right). N=4 HD. B) Mean frequency of live Vy9V52 T cells in HD-PBMCs after 48 h of treatment with venetoclax, 5-azacytidine or their combination. N=4 HD. C) Mean ± SEM of the frequency of live Vy9V52 T cells (black), apoptotic Vy9V52 T cells (light gray), or dead Vy9V52 T cells (dark gray) in HD-PBMCs 48 h after treatment with venetoclax in the presence of ICT01 (right) or its isotype control (hIgG1S, left). N=4 HDs. D) Mean ± SEM of the frequency of live Vy9V52 T cells in HD-PBMCs 48 h after treatment with venetoclax in the presence of ICT01 (black) or its isotype control (hIgG1S, light gray), normalized to the condition without venetoclax. N=4 HDs, each dot represents one healthy donor. ***p<0.005, 2-way ANOVA. E) Mean ± SEM of median CD69 mean fluorescence intensity (MFI) of Vy9V52 T cells in HD-PBMCs treated for 48 h with ICT01 (black) or its isotype control (hIgG1S, light grey). N=5 HD, each dot represents one healthy donor. [Figure 1d]Figure 3: Activation with ICT01 protects Vy9V52 T cells from venetoclax-induced cell death. A) Mean ± SEM frequency of live Vy9V52 T cells (black), apoptotic Vy9V52 T cells (light grey) or dead Vy9V52 T cells (dark grey) in HD-PBMCs after 48 h of treatment with venetoclax (left) or 5-azacytidine (right). N=4 HD. B) Mean frequency of live Vy9V52 T cells in HD-PBMCs after 48 h of treatment with venetoclax, 5-azacytidine or their combination. N=4 HD. C) Mean ± SEM of the frequency of live Vy9V52 T cells (black), apoptotic Vy9V52 T cells (light gray), or dead Vy9V52 T cells (dark gray) in HD-PBMCs 48 h after treatment with venetoclax in the presence of ICT01 (right) or its isotype control (hIgG1S, left). N=4 HDs. D) Mean ± SEM of the frequency of live Vy9V52 T cells in HD-PBMCs 48 h after treatment with venetoclax in the presence of ICT01 (black) or its isotype control (hIgG1S, light gray), normalized to the condition without venetoclax. N=4 HDs, each dot represents one healthy donor. ***p<0.005, 2-way ANOVA. E) Mean ± SEM of median CD69 mean fluorescence intensity (MFI) of Vy9V52 T cells in HD-PBMCs treated for 48 h with ICT01 (black) or its isotype control (hIgG1S, light grey). N=5 HD, each dot represents one healthy donor. [Figure 1e]Figure 3: Activation with ICT01 protects Vy9V52 T cells from venetoclax-induced cell death. A) Mean ± SEM frequency of live Vy9V52 T cells (black), apoptotic Vy9V52 T cells (light grey) or dead Vy9V52 T cells (dark grey) in HD-PBMCs after 48 h of treatment with venetoclax (left) or 5-azacytidine (right). N=4 HD. B) Mean frequency of live Vy9V52 T cells in HD-PBMCs after 48 h of treatment with venetoclax, 5-azacytidine or their combination. N=4 HD. C) Mean ± SEM of the frequency of live Vy9V52 T cells (black), apoptotic Vy9V52 T cells (light gray), or dead Vy9V52 T cells (dark gray) in HD-PBMCs 48 h after treatment with venetoclax in the presence of ICT01 (right) or its isotype control (hIgG1S, left). N=4 HDs. D) Mean ± SEM of the frequency of live Vy9V52 T cells in HD-PBMCs 48 h after treatment with venetoclax in the presence of ICT01 (black) or its isotype control (hIgG1S, light gray), normalized to the condition without venetoclax. N=4 HDs, each dot represents one healthy donor. ***p<0.005, 2-way ANOVA. E) Mean ± SEM of median CD69 mean fluorescence intensity (MFI) of Vy9V52 T cells in HD-PBMCs treated for 48 h with ICT01 (black) or its isotype control (hIgG1S, light grey). N=5 HD, each dot represents one healthy donor. [Figure 2a]Figure 2: Activation of Vy9V52 T cells with ICT01 protects Vy9V52 T cells from cell death induced by Bcl-2 family member inhibitors (navitoclax, ABT-737 and MIK665). A) Heatmap showing the mean frequency of viable Vy9V52 T cells in HD-PBMCs assessed by flow cytometry after 48 h treatment with increasing concentrations of the indicated Bcl-2 family member inhibitors. For ABT-737, N=3 HD, for navitoclax and MIK665, N=5. B-D) Frequency (mean ± SEM) of viable Vγ9Vδ2 T cells in HD-PBMCs assessed by flow cytometry after 48 h treatment with the indicated concentrations of ABT-737 (N = 3 HDs) (B), navitoclax (N = 5 HDs) (C), or MIK665 (N = 5 HDs) (D) in the presence of 1 μg / mL ICT01 (black) or its isotype control hIgG1S (light gray). Data are normalized to no ABT-737, navitoclax, or MIK665 treatment; each dot represents one healthy donor. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, 2-way ANOVA. [Figure 2b]Figure 2: Activation of Vy9V52 T cells with ICT01 protects Vy9V52 T cells from cell death induced by Bcl-2 family member inhibitors (navitoclax, ABT-737 and MIK665). A) Heatmap showing the mean frequency of viable Vy9V52 T cells in HD-PBMCs assessed by flow cytometry after 48 h treatment with increasing concentrations of the indicated Bcl-2 family member inhibitors. For ABT-737, N=3 HD, for navitoclax and MIK665, N=5. B-D) Frequency (mean ± SEM) of viable Vγ9Vδ2 T cells in HD-PBMCs assessed by flow cytometry after 48 h treatment with the indicated concentrations of ABT-737 (N = 3 HDs) (B), navitoclax (N = 5 HDs) (C), or MIK665 (N = 5 HDs) (D) in the presence of 1 μg / mL ICT01 (black) or its isotype control hIgG1S (light gray). Data are normalized to no ABT-737, navitoclax, or MIK665 treatment; each dot represents one healthy donor. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, 2-way ANOVA. [Figure 2c]Figure 2: Activation of Vy9V52 T cells with ICT01 protects Vy9V52 T cells from cell death induced by Bcl-2 family member inhibitors (navitoclax, ABT-737 and MIK665). A) Heatmap showing the mean frequency of viable Vy9V52 T cells in HD-PBMCs assessed by flow cytometry after 48 h treatment with increasing concentrations of the indicated Bcl-2 family member inhibitors. For ABT-737, N=3 HD, for navitoclax and MIK665, N=5. B-D) Frequency (mean ± SEM) of viable Vγ9Vδ2 T cells in HD-PBMCs assessed by flow cytometry after 48 h treatment with the indicated concentrations of ABT-737 (N = 3 HDs) (B), navitoclax (N = 5 HDs) (C), or MIK665 (N = 5 HDs) (D) in the presence of 1 μg / mL ICT01 (black) or its isotype control hIgG1S (light gray). Data are normalized to no ABT-737, navitoclax, or MIK665 treatment; each dot represents one healthy donor. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, 2-way ANOVA. [Figure 2d]Figure 2: Activation of Vy9V52 T cells with ICT01 protects Vy9V52 T cells from cell death induced by Bcl-2 family member inhibitors (navitoclax, ABT-737 and MIK665). A) Heatmap showing the mean frequency of viable Vy9V52 T cells in HD-PBMCs assessed by flow cytometry after 48 h treatment with increasing concentrations of the indicated Bcl-2 family member inhibitors. For ABT-737, N=3 HD, for navitoclax and MIK665, N=5. B-D) Frequency (mean ± SEM) of viable Vγ9Vδ2 T cells in HD-PBMCs assessed by flow cytometry after 48 h treatment with the indicated concentrations of ABT-737 (N = 3 HDs) (B), navitoclax (N = 5 HDs) (C), or MIK665 (N = 5 HDs) (D) in the presence of 1 μg / mL ICT01 (black) or its isotype control hIgG1S (light gray). Data are normalized to no ABT-737, navitoclax, or MIK665 treatment; each dot represents one healthy donor. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, 2-way ANOVA. [Figure 3a] Figure 2: Pre-treatment ICT01-mediated activation reduces venetoclax sensitivity of Vy9V52 T cells. A) Schematic of the treatment sequence. B) Mean ± SEM frequency of CD25+ Vy9V52 T cells in HD-PBMCs on day 3 of treatment with ICT01 (black) or its isotype control (hIgG1S, light grey) and venetoclax. N=6 HD. C) Mean ± SEM relative number of viable Vy9V52 T cells and D) Mean ± SEM percent (%) viable Vy9V52 T cells compared to the no venetoclax condition on day 3 of treatment with ICT01 (black) or its isotype control (hIgG1S, light grey) and venetoclax. N=5 HD. Each dot represents one healthy donor. *p<0.05, 2-way ANOVA. [Figure 3b]Figure 2: Pre-treatment ICT01-mediated activation reduces venetoclax sensitivity of Vy9V52 T cells. A) Schematic of the treatment sequence. B) Mean ± SEM frequency of CD25+ Vy9V52 T cells in HD-PBMCs on day 3 of treatment with ICT01 (black) or its isotype control (hIgG1S, light grey) and venetoclax. N=6 HD. C) Mean ± SEM relative number of viable Vy9V52 T cells and D) Mean ± SEM percent (%) viable Vy9V52 T cells compared to the no venetoclax condition on day 3 of treatment with ICT01 (black) or its isotype control (hIgG1S, light grey) and venetoclax. N=5 HD. Each dot represents one healthy donor. *p<0.05, 2-way ANOVA. [Figure 3c] Figure 2: Pre-treatment ICT01-mediated activation reduces venetoclax sensitivity of Vy9V52 T cells. A) Schematic of the treatment sequence. B) Mean ± SEM frequency of CD25+ Vy9V52 T cells in HD-PBMCs on day 3 of treatment with ICT01 (black) or its isotype control (hIgG1S, light grey) and venetoclax. N=6 HD. C) Mean ± SEM relative number of viable Vy9V52 T cells and D) Mean ± SEM percent (%) viable Vy9V52 T cells compared to the no venetoclax condition on day 3 of treatment with ICT01 (black) or its isotype control (hIgG1S, light grey) and venetoclax. N=5 HD. Each dot represents one healthy donor. *p<0.05, 2-way ANOVA. [Figure 3d]Figure 2: Pre-treatment ICT01-mediated activation reduces venetoclax sensitivity of Vy9V52 T cells. A) Schematic of the treatment sequence. B) Mean ± SEM frequency of CD25+ Vy9V52 T cells in HD-PBMCs on day 3 of treatment with ICT01 (black) or its isotype control (hIgG1S, light grey) and venetoclax. N=6 HD. C) Mean ± SEM relative number of viable Vy9V52 T cells and D) Mean ± SEM percent (%) viable Vy9V52 T cells compared to the no venetoclax condition on day 3 of treatment with ICT01 (black) or its isotype control (hIgG1S, light grey) and venetoclax. N=5 HD. Each dot represents one healthy donor. *p<0.05, 2-way ANOVA. [Figure 4a] Figure 1: Phenotypic activation and proliferation of Vy9V52 T cells in HD-PBMCs stimulated with ICT01 are largely unaffected by treatment with venetoclax and 5-azacytidine: A) Schematic of the treatment sequence (left) and mean ± SEM (right) frequency of CD25-expressing Vy9V52 T cells in HD-PBMCs on day 4 of treatment with venetoclax, 5-azacytidine, or their combination in the presence of ICT01 (black) or its isotype control (hIgG1S, light grey). N=6 HD, each dot represents one healthy donor. B) Schematic of the treatment sequence (left) and mean ± SEM (right) frequency of CD25-expressing Vy9V52 T cells in HD-PBMCs on day 3 of treatment with venetoclax, 5-azacytidine, or their combination, followed by ICT01 (black) or its isotype control (hIgG1S, light grey). N=6 HD, each dot represents one healthy donor. C and D) Mean ± SEM frequency of Cell Trace Violet (CTV) dim Vγ9Vδ2 T cells in HD-PBMCs on day 4 of treatment with venetoclax, 5-azacytidine, or their combination, and ICT01 (black) or its isotype control (hIgG1S, light gray) in the absence (C) or presence (D) of IL2, N=6 HD, each dot represents one healthy donor. *p<0.05, RN 1-way ANOVA. [Figure 4b] Figure 1: Phenotypic activation and proliferation of Vy9V52 T cells in HD-PBMCs stimulated with ICT01 are largely unaffected by treatment with venetoclax and 5-azacytidine: A) Schematic of the treatment sequence (left) and mean ± SEM (right) frequency of CD25-expressing Vy9V52 T cells in HD-PBMCs on day 4 of treatment with venetoclax, 5-azacytidine, or their combination in the presence of ICT01 (black) or its isotype control (hIgG1S, light grey). N=6 HD, each dot represents one healthy donor. B) Schematic of the treatment sequence (left) and mean ± SEM (right) frequency of CD25-expressing Vy9V52 T cells in HD-PBMCs on day 3 of treatment with venetoclax, 5-azacytidine, or their combination, followed by ICT01 (black) or its isotype control (hIgG1S, light grey). N=6 HD, each dot represents one healthy donor. C and D) Mean ± SEM frequency of Cell Trace Violet (CTV) dim Vγ9Vδ2 T cells in HD-PBMCs on day 4 of treatment with venetoclax, 5-azacytidine, or their combination, and ICT01 (black) or its isotype control (hIgG1S, light gray) in the absence (C) or presence (D) of IL2, N=6 HD, each dot represents one healthy donor. *p<0.05, RN 1-way ANOVA. [Figure 4c]Figure 1: Phenotypic activation and proliferation of Vy9V52 T cells in HD-PBMCs stimulated with ICT01 are largely unaffected by treatment with venetoclax and 5-azacytidine: A) Schematic of the treatment sequence (left) and mean ± SEM (right) frequency of CD25-expressing Vy9V52 T cells in HD-PBMCs on day 4 of treatment with venetoclax, 5-azacytidine, or their combination in the presence of ICT01 (black) or its isotype control (hIgG1S, light grey). N=6 HD, each dot represents one healthy donor. B) Schematic of the treatment sequence (left) and mean ± SEM (right) frequency of CD25-expressing Vy9V52 T cells in HD-PBMCs on day 3 of treatment with venetoclax, 5-azacytidine, or their combination, followed by ICT01 (black) or its isotype control (hIgG1S, light grey). N=6 HD, each dot represents one healthy donor. C and D) Mean ± SEM frequency of Cell Trace Violet (CTV) dim Vγ9Vδ2 T cells in HD-PBMCs on day 4 of treatment with venetoclax, 5-azacytidine, or their combination, and ICT01 (black) or its isotype control (hIgG1S, light gray) in the absence (C) or presence (D) of IL2, N=6 HD, each dot represents one healthy donor. *p<0.05, RN 1-way ANOVA. [Figure 4d]Figure 1: Phenotypic activation and proliferation of Vy9V52 T cells in HD-PBMCs stimulated with ICT01 are largely unaffected by treatment with venetoclax and 5-azacytidine: A) Schematic of the treatment sequence (left) and mean ± SEM (right) frequency of CD25-expressing Vy9V52 T cells in HD-PBMCs on day 4 of treatment with venetoclax, 5-azacytidine, or their combination in the presence of ICT01 (black) or its isotype control (hIgG1S, light grey). N=6 HD, each dot represents one healthy donor. B) Schematic of the treatment sequence (left) and mean ± SEM (right) frequency of CD25-expressing Vy9V52 T cells in HD-PBMCs on day 3 of treatment with venetoclax, 5-azacytidine, or their combination, followed by ICT01 (black) or its isotype control (hIgG1S, light grey). N=6 HD, each dot represents one healthy donor. C and D) Mean ± SEM frequency of Cell Trace Violet (CTV) dim Vγ9Vδ2 T cells in HD-PBMCs on day 4 of treatment with venetoclax, 5-azacytidine, or their combination, and ICT01 (black) or its isotype control (hIgG1S, light gray) in the absence (C) or presence (D) of IL2, N=6 HD, each dot represents one healthy donor. *p<0.05, RN 1-way ANOVA. [Figure 5a]Figure 1 shows that sequential treatment with ICT01-activated Vy9V52 T cells followed by venetoclax and 5-azacytidine significantly reduces the number of cells in resistant AML cell lines. A) Sensitivity of the KG1a cell line to venetoclax, 5-azacytidine, or their combination. The figure shows the mean luminescence signal after 48 h of treatment with venetoclax, 5-azacytidine, or their combination relative to the untreated condition, which reflects viable KG1a AML cells. N=2 experiments. B) Mean ± SEM of the relative number of viable KG1a AML cells monitored by flow cytometry after 3 days of coculture with HD-PBMCs (E:T ratio 25:1) treated with 0.1 or 1 μg / mL of ICT01 (black) or its isotype control (hIgG1S, light gray). N=6, each dot represents one healthy donor. C) Schematic of the treatment schedule for killing of KG1a AML cells by HD-PBMCs. D) Relative number (mean ± SEM) of viable KG1a AML cells (left) and frequency (mean ± SEM) of viable KG1a AML cells (right) after 3 days of co-culture with HD-PBMCs with treatment with venetoclax, 5-azacytidine, or their combination versus no venetoclax or 5-azacytidine treatment in the presence of ICT01 (black) or its isotype control (hIgG1S, light gray). N=6, each dot represents one healthy donor. *p<0.05, Wilcoxon test. [Figure 5b]Figure 1 shows that sequential treatment with ICT01-activated Vy9V52 T cells followed by venetoclax and 5-azacytidine significantly reduces the number of cells in resistant AML cell lines. A) Sensitivity of the KG1a cell line to venetoclax, 5-azacytidine, or their combination. The figure shows the mean luminescence signal after 48 h of treatment with venetoclax, 5-azacytidine, or their combination relative to the untreated condition, which reflects viable KG1a AML cells. N=2 experiments. B) Mean ± SEM of the relative number of viable KG1a AML cells monitored by flow cytometry after 3 days of coculture with HD-PBMCs (E:T ratio 25:1) treated with 0.1 or 1 μg / mL of ICT01 (black) or its isotype control (hIgG1S, light gray). N=6, each dot represents one healthy donor. C) Schematic of the treatment schedule for killing of KG1a AML cells by HD-PBMCs. D) Relative number (mean ± SEM) of viable KG1a AML cells (left) and frequency (mean ± SEM) of viable KG1a AML cells (right) after 3 days of co-culture with HD-PBMCs with treatment with venetoclax, 5-azacytidine, or their combination versus no venetoclax or 5-azacytidine treatment in the presence of ICT01 (black) or its isotype control (hIgG1S, light gray). N=6, each dot represents one healthy donor. *p<0.05, Wilcoxon test. [Figure 5c]Figure 1 shows that sequential treatment with ICT01-activated Vy9V52 T cells followed by venetoclax and 5-azacytidine significantly reduces the number of cells in resistant AML cell lines. A) Sensitivity of the KG1a cell line to venetoclax, 5-azacytidine, or their combination. The figure shows the mean luminescence signal after 48 h of treatment with venetoclax, 5-azacytidine, or their combination relative to the untreated condition, which reflects viable KG1a AML cells. N=2 experiments. B) Mean ± SEM of the relative number of viable KG1a AML cells monitored by flow cytometry after 3 days of coculture with HD-PBMCs (E:T ratio 25:1) treated with 0.1 or 1 μg / mL of ICT01 (black) or its isotype control (hIgG1S, light gray). N=6, each dot represents one healthy donor. C) Schematic of the treatment schedule for killing of KG1a AML cells by HD-PBMCs. D) Relative number (mean ± SEM) of viable KG1a AML cells (left) and frequency (mean ± SEM) of viable KG1a AML cells (right) after 3 days of co-culture with HD-PBMCs with treatment with venetoclax, 5-azacytidine, or their combination versus no venetoclax or 5-azacytidine treatment in the presence of ICT01 (black) or its isotype control (hIgG1S, light gray). N=6, each dot represents one healthy donor. *p<0.05, Wilcoxon test. [Figure 5d]Figure 1 shows that sequential treatment with ICT01-activated Vy9V52 T cells followed by venetoclax and 5-azacytidine significantly reduces the number of cells in resistant AML cell lines. A) Sensitivity of the KG1a cell line to venetoclax, 5-azacytidine, or their combination. The figure shows the mean luminescence signal after 48 h of treatment with venetoclax, 5-azacytidine, or their combination relative to the untreated condition, which reflects viable KG1a AML cells. N=2 experiments. B) Mean ± SEM of the relative number of viable KG1a AML cells monitored by flow cytometry after 3 days of coculture with HD-PBMCs (E:T ratio 25:1) treated with 0.1 or 1 μg / mL of ICT01 (black) or its isotype control (hIgG1S, light gray). N=6, each dot represents one healthy donor. C) Schematic of the treatment schedule for killing of KG1a AML cells by HD-PBMCs. D) Relative number (mean ± SEM) of viable KG1a AML cells (left) and frequency (mean ± SEM) of viable KG1a AML cells (right) after 3 days of co-culture with HD-PBMCs with treatment with venetoclax, 5-azacytidine, or their combination versus no venetoclax or 5-azacytidine treatment in the presence of ICT01 (black) or its isotype control (hIgG1S, light gray). N=6, each dot represents one healthy donor. *p<0.05, Wilcoxon test. [Figure 6]This figure shows that treatment with anti-BTN3A m20.1 and venetoclax and 5-azacytidine significantly enhances Vy9V52 T cell-mediated killing of the AML cell line KG1a. KG1a cells were co-cultured with HD-PBMCs at a 5:1 ratio in the presence of m20.1 or isotype control (0.1 μg / mL). After 6 hours, the indicated concentrations of venetoclax, 5-azacytidine, or their combination were added to the culture medium. The relative number of viable KG1a cells was monitored 48 hours later by flow cytometry. The figure shows the relative number (mean ± SEM) of viable KG1a AML cells after 2 days of co-culture with HD-PBMCs with treatment with venetoclax, 5-azacytidine, or their combination in the presence of m20.1 (black) or its isotype control mIgG1 (light gray). N=6, each dot represents one healthy donor, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, 2-way ANOVA. [Figure 7a] Figure 1 shows that ICT01-activated Vy9V52 T cells, followed by treatment with venetoclax and 5-azacytidine, significantly reduced the number of viable Burkitt lymphoma and chronic B-cell leukemia cell lines. Raji cells (Burkitt lymphoma) or JVM2 cells (chronic B-cell leukemia) were co-cultured with HD-PBMCs at a 5:1 ratio in the presence of ICT01 or isotype control (0.1 μg / mL or 1 μg / mL). After 6 hours, the indicated concentrations of venetoclax, 5-azacytidine, or their combination were added to the culture medium. The relative number of viable Raji (A) or JVM2 (B) cells was monitored by flow cytometry 48 hours later. The figure shows the relative numbers (mean ± SEM) of live Raji (A) or JVM2 (B) cells after 2 days of co-culture with HD-PBMCs in the presence of ICT01 (black) or its isotype control hIgG1S (light gray), and the indicated concentrations of venetoclax and 5-azacytidine. N=6, each dot represents one healthy donor. *p<0.05, **p<0.01, 2-way ANOVA. [Figure 7b]Figure 1 shows that ICT01-activated Vy9V52 T cells, followed by treatment with venetoclax and 5-azacytidine, significantly reduced the number of viable Burkitt lymphoma and chronic B-cell leukemia cell lines. Raji cells (Burkitt lymphoma) or JVM2 cells (chronic B-cell leukemia) were co-cultured with HD-PBMCs at a 5:1 ratio in the presence of ICT01 or isotype control (0.1 μg / mL or 1 μg / mL). After 6 hours, the indicated concentrations of venetoclax, 5-azacytidine, or their combination were added to the culture medium. The relative number of viable Raji (A) or JVM2 (B) cells was monitored by flow cytometry 48 hours later. The figure shows the relative numbers (mean ± SEM) of live Raji (A) or JVM2 (B) cells after 2 days of co-culture with HD-PBMCs in the presence of ICT01 (black) or its isotype control hIgG1S (light gray), and the indicated concentrations of venetoclax and 5-azacytidine. N=6, each dot represents one healthy donor. *p<0.05, **p<0.01, 2-way ANOVA. [Figure 8a]Figure 1 shows that ICT01-activated Vy9V52 T cells, followed by treatment with an inhibitor of a Bcl-2 family member, significantly reduces the number of viable AML cell lines. KG1a or MOLM14 AML cell lines were co-cultured with HD-PBMCs at a 5:1 ratio in the presence of ICT01 or an isotype control (0.1 μg / mL). After 6 hours, the indicated concentrations of ABT-737, navitoclax, or MIK665 were added to the culture medium. A) Relative number of viable KG1a cells (mean ± SEM) after 2 days of co-culture with HD-PBMCs with treatment with the indicated concentrations of ABT-737 in the presence of ICT01 (black) or its isotype control hIgG1S (light gray). B) Relative number of viable KG1a cells (mean ± SEM) after 2 days of co-culture with HD-PBMCs and treatment with the indicated concentrations of navitoclax in the presence of ICT01 (black) or its isotype control hIgG1S (light gray). C) Relative number of viable MOLM14 cells (mean ± SEM) after 2 days of co-culture with HD-PBMCs and treatment with the indicated concentrations of MIK665 in the presence of ICT01 (black) or its isotype control hIgG1S (light gray). N=6 HDs, each dot represents one healthy donor. *p<0.05, 2-way ANOVA. [Figure 8b]Figure 1 shows that ICT01-activated Vy9V52 T cells, followed by treatment with an inhibitor of a Bcl-2 family member, significantly reduces the number of viable AML cell lines. KG1a or MOLM14 AML cell lines were co-cultured with HD-PBMCs at a 5:1 ratio in the presence of ICT01 or an isotype control (0.1 μg / mL). After 6 hours, the indicated concentrations of ABT-737, navitoclax, or MIK665 were added to the culture medium. A) Relative number of viable KG1a cells (mean ± SEM) after 2 days of co-culture with HD-PBMCs with treatment with the indicated concentrations of ABT-737 in the presence of ICT01 (black) or its isotype control hIgG1S (light gray). B) Relative number of viable KG1a cells (mean ± SEM) after 2 days of co-culture with HD-PBMCs and treatment with the indicated concentrations of navitoclax in the presence of ICT01 (black) or its isotype control hIgG1S (light gray). C) Relative number of viable MOLM14 cells (mean ± SEM) after 2 days of co-culture with HD-PBMCs and treatment with the indicated concentrations of MIK665 in the presence of ICT01 (black) or its isotype control hIgG1S (light gray). N=6 HDs, each dot represents one healthy donor. *p<0.05, 2-way ANOVA. [Figure 8c]Figure 1 shows that ICT01-activated Vy9V52 T cells, followed by treatment with an inhibitor of a Bcl-2 family member, significantly reduces the number of viable AML cell lines. KG1a or MOLM14 AML cell lines were co-cultured with HD-PBMCs at a 5:1 ratio in the presence of ICT01 or an isotype control (0.1 μg / mL). After 6 hours, the indicated concentrations of ABT-737, navitoclax, or MIK665 were added to the culture medium. A) Relative number of viable KG1a cells (mean ± SEM) after 2 days of co-culture with HD-PBMCs with treatment with the indicated concentrations of ABT-737 in the presence of ICT01 (black) or its isotype control hIgG1S (light gray). B) Relative number of viable KG1a cells (mean ± SEM) after 2 days of co-culture with HD-PBMCs and treatment with the indicated concentrations of navitoclax in the presence of ICT01 (black) or its isotype control hIgG1S (light gray). C) Relative number of viable MOLM14 cells (mean ± SEM) after 2 days of co-culture with HD-PBMCs and treatment with the indicated concentrations of MIK665 in the presence of ICT01 (black) or its isotype control hIgG1S (light gray). N=6 HDs, each dot represents one healthy donor. *p<0.05, 2-way ANOVA. [Figure 9] Figure 1: ICT01 in combination with venetoclax and 5-azacytidine significantly extends survival of MOLM-14-implanted NSG mice. Kaplan-Meier survival curves for each treatment group demonstrate improved efficacy when ICT01 is combined with venetoclax and 5-azacytidine. Statistical analysis was performed using the log-rank (Mantel-Cox) test. **p<0.005, ***p<0.0005. DETAILED DESCRIPTION OF THE INVENTION

[0067] definition In order that this disclosure may be more readily understood, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0068] The terms "polypeptide," "protein," or "peptide," as used herein, refer to any chain of amino acid residues, regardless of chain length or post-translational modification (e.g., glycosylation).

[0069] As used herein, the term "BTN3A" has its general meaning in the art. In certain embodiments, the term refers to a human BTN3A polypeptide, including any of BTN3A1 of SEQ ID NO:24, BTN3A2 of SEQ ID NO:25, or BTN3A3 of SEQ ID NO:26.

[0070] The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site that immunospecifically binds to an antigen. The terms "antibody" and "immunoglobulin" have the same meaning and are used equivalently in this disclosure. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments and antibody variants (including derivatives). The term "antibody," as used herein, also includes bispecific or multispecific molecules. Antibodies can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or receptor ligand), to create bispecific molecules that bind to at least two different binding sites or target molecules. Indeed, antibodies can be derivatized or linked to more than one other functional molecule to create multispecific molecules that bind to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, an antibody of the disclosure can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, etc.) to one or more other binding molecules, such as another antibody, antibody fragment, peptide, or binding mimetic, to yield a bispecific molecule. Moreover, in embodiments where the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity in addition to the first and second target epitopes.

[0071] In natural antibodies from rodents and primates, two heavy chains are linked to each other by disulfide bonds, and each heavy chain is linked to a light chain by a disulfide bond. There are two types of light chains: lambda (λ) and kappa (κ). There are five main heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each chain contains domains with different sequences. In a typical IgG antibody, the light chain contains two domains: a variable domain (VL) and a constant domain (CL). The heavy chain contains four domains: a variable domain (VH) and three constant domains (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light chain (VL) and the heavy chain (VH) determine binding recognition and specificity to the antigen. The light chain constant region domain (CL) and the heavy chain constant region domain (CH) confer important biological properties, such as antibody chain assembly, secretion, transplacental movement, complement fixation, and binding to Fc receptors (FcR).

[0072] An Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment and consists of the variable portions of one light chain and one heavy chain. Antibody specificity resides in the structural complementarity between the antibody binding site and an antigenic determinant. The antibody binding site is primarily composed of residues from the hypervariable or complementarity-determining regions (CDRs). In some cases, residues from non-hypervariable or framework regions (FRs) may also participate in the antibody binding site or influence the overall domain structure and ultimately the binding site. Complementarity-determining regions (CDRs) refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of a native immunoglobulin binding site. The light and heavy chains of an immunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, and L-CDR3, and H-CDR1, H-CDR2, and H-CDR3, respectively. Thus, an antigen-binding site typically contains six CDRs, including a set of CDRs from each of the heavy and light chain V regions. The framework region (FR) refers to the amino acid sequence inserted between the CDRs. Thus, the variable regions of the light and heavy chains typically contain four framework regions and three CDRs in the following order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0073] Residues in antibody variable domains are conventionally numbered according to the system devised by Kabat et al. This system is described in Kabat et al., 1987, Sequences of Proteins of Immunological Interest, US Department of Health and Human Services, NIH, USA (Kabat et al., 1992, hereinafter "Kabat et al."). This numbering system is used herein. The Kabat residue designations do not necessarily correspond directly to the linear numbering of amino acid residues in the sequence of SEQ ID NO: 1. The actual linear amino acid sequence, whether in the framework of the basic variable domain structure or in the complementarity-determining regions (CDRs), may contain fewer or additional amino acids relative to the strict Kabat-numbered sequence, corresponding to truncations of or insertions into structural elements. The correct Kabat numbering of residues can be determined for a given antibody by aligning the homologous residues in the antibody sequence against the "standard" Kabat-numbered sequence. The CDRs of the heavy chain variable domain are located at residues 31 to 35 (H-CDR1), 50 to 65 (H-CDR2), and 95 to 102 (H-CDR3) according to the Kabat numbering system. The CDRs of the light chain variable domain are located at residues 24 to 34 (L-CDR1), 50 to 56 (L-CDR2), and 89 to 97 (L-CDR3) according to the Kabat numbering system.

[0074] It is now well established in the art that the non-CDR regions of a mammalian antibody can be replaced with similar regions from allo- or heterospecific antibodies while retaining the epitope specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies, in which non-human CDRs are covalently linked to human FR and / or Fc / pFc regions to generate a functional antibody.

[0075] As used herein, "humanized" refers to an antibody in which some, most, or all amino acids outside the CDR regions have been replaced with corresponding amino acids derived from a human immunoglobulin molecule. Humanization methods include, but are not limited to, those described in U.S. Patent Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205. U.S. Patent Nos. 5,585,089 and 5,693,761, as well as International Publication WO 90 / 07861, also propose four possible criteria for designing humanized antibodies. The first proposal was to use, as an acceptor, a framework from a specific human immunoglobulin that is exceptionally similar to the donor immunoglobulin being humanized, or to use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of a human immunoglobulin is unusual and the donor amino acid at this position is typical in human sequences, then the donor amino acid can be selected rather than the acceptor amino acid. The third proposal was that the donor amino acid can be selected rather than the acceptor amino acid at positions immediately adjacent to the three CDRs in a humanized immunoglobulin chain. The fourth proposal was that donor amino acid residues be used at framework positions where, in a three-dimensional model of the antibody, the amino acid is predicted to have a side chain atom within 3A of the CDR and thus be capable of interacting with the CDR. The above methods merely illustrate some of the methods that one skilled in the art can employ to create humanized antibodies. Those skilled in the art will be familiar with other methods for antibody humanization. In some humanized forms of antibodies, some, most, or all of the amino acids outside the CDR regions may be replaced with amino acids from a human immunoglobulin molecule, but some, most, or all of the amino acids within one or more CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permissible as long as they do not impair the ability of the antibody to bind a given antigen.Suitable human immunoglobulin molecules can include IgG1, IgG2, IgG3, IgG4, IgA, and IgM molecules. A "humanized" antibody retains the same antigen specificity as the original antibody. However, when using certain humanization methods, the binding affinity and / or specificity of the antibody can be increased using the method of "directed evolution" as described in Wu et al., Mol. Biol. 294:151, 1999.

[0076] Fully human monoclonal antibodies can also be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Patent Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, and 6,150,584, the contents of which are incorporated herein by reference, and the references cited therein. These animals have been genetically modified to lack endogenous (e.g., murine) antibody production function. The animals have been further modified to contain all or part of human germline immunoglobulin gene loci, such that immunization of these animals results in the production of fully human antibodies against the antigen of interest. After immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex / GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke a human anti-mouse antibody (KAMA) response when administered to humans.

[0077] In vitro methods for generating human antibodies also exist. These methods include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference.

[0078] The term "antigen-binding fragment" of an antibody (or simply "antibody fragment"), as used herein, refers to a full-length antibody or one or more fragments thereof that retain the ability to specifically bind to an antigen (e.g., a BTN3A protein as defined above). In certain embodiments, the antibodies provided herein are antibody fragments, more particularly, any protein that comprises the antigen-binding domain of an antibody disclosed herein. Well-known antibody fragments include: Fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL, and CH1 domains; F(ab)2 fragments, i.e., bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single antibody arm; dAb fragments consisting of the VH domain (Ward et al., 1989 Nature 341:544-546), or any fusion protein containing such an antigen-binding fragment; and diabodies (small antibody fragments having two antigen-binding sites; these fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) (VH-VL) on the same polypeptide chain). The two domains on the same chain are forced to pair with complementary domains on another chain, creating two antigen-binding sites, using a linker that is too short to allow inter-domain pairing. Furthermore, although the two domains of an Fv fragment, i.e., VL and VH, are encoded by separate genes, they can be joined using recombinant techniques with a synthetic linker that allows the VL and VH domains to pair into a single-chain protein to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antigen-binding fragment" of an antibody (also referred to herein for short as antibody fragment). More generally, antibody fragments as intended herein also encompass single-domain antibodies, which are antibody fragments comprising all or a portion of an antibody heavy-chain variable domain or all or a portion of an antibody light-chain variable domain.In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 (B1)). These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Suitable antibody fragments include, but are not limited to, Fv, Fab, F(ab')2, Fab', dsFv, scFv, sc(Fv)2, and diabodies. Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells as described herein.

[0079] The term "monoclonal antibody," as used herein, refers to a single-specificity preparation of antibody molecules. A monoclonal antibody displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity that has variable and constant regions derived from or based on human germline immunoglobulin sequences, or from entirely synthetic sequences. The method of preparation of the monoclonal antibody is not related to the binding specificity.

[0080] A "recombinant antibody" is an antibody that is produced, expressed, created, or isolated by recombinant means, e.g., an antibody expressed using a recombinant expression vector transfected into a host cell; an antibody isolated from a recombinant combinatorial antibody library; an antibody isolated from an animal (e.g., a mouse) that is transgenic with human immunoglobulin genes; or an antibody that is produced, expressed, created, or isolated by any other method that combines particular immunoglobulin gene sequences (e.g., human immunoglobulin gene sequences) with other DNA sequences. Recombinant antibodies include, for example, chimeric and humanized antibodies. In some embodiments, recombinant human antibodies of the present disclosure have the same amino acid sequence as a corresponding naturally occurring human antibody, but are structurally different from said naturally occurring human antibody. For example, in some embodiments, recombinant human antibodies have different glycosylation patterns as a result of recombinant production. In some embodiments, recombinant human antibodies are chemically modified by adding or subtracting at least one covalent chemical bond from the structure of a human antibody naturally occurring in humans.

[0081] An "isolated antibody," as used herein, refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to BTN3A is substantially free of antibodies that specifically bind to antigens other than BTN3A). However, an isolated antibody that specifically binds to BTN3A may have cross-reactivity with other antigens, for example, related BTN3A molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0082] The phrases "antibody that recognizes an antigen" and "antibody having specificity for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen." Additionally, the term "anti-BTN3A antibody" or "BTN3A antibody" is used herein simply to mean "an antibody that recognizes BTN3A."

[0083] As used herein, the term "activating antibody" refers to an antibody that can directly or indirectly induce immune function of effector cells. In particular, as used herein, an activating anti-BTN3A antibody has at least the ability to induce activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with BTN3-expressing cells, with an EC50 of less than 5 μg / ml, preferably 1 μg / ml or less, as measured in a degranulation assay (see WO2020 / 025703 for detailed assays).

[0084] As used herein, the term "binding" in the context of antibody binding to a given antigen or epitope, particularly BTN3A, typically refers to a binding activity of about 10 -7 M or less, e.g., about 10 -8 M or less, e.g., about 10 -9 M or less, about 10 -10 M or less, or about 10 -11 K to M and below D where affinity is as determined, for example, by surface plasmon resonance (SPR) technology on a BIAcore 3000 instrument, typically using a soluble form of the antigen as the ligand and the antibody as the analyte. BIACORE® (GE Healthcare, Piscaataway, NJ) is one of a variety of surface plasmon resonance assay formats commonly used for epitope bin panels of monoclonal antibodies. Typically, an antibody binds to a given antigen with an affinity corresponding to a K of 0.05 for binding to a nonspecific antigen (e.g., BSA, casein) that is neither identical nor closely related to the given antigen. D K of 1 / 10 or less, for example, 1 / 100 or less, for example, 1 / 1,000 or less, for example, 1 / 10,000 or less, for example, 1 / 100,000 or less D The antibody binds with an affinity corresponding to K D If the K is very low (i.e., the antibody has high affinity), the K D However, typically, K D It is less than 1 / 10,000 of that.

[0085] The term "affinity," as used herein in the context of antibodies, refers to the strength of the binding of an antibody to an epitope.

[0086] "K on " or "Kass" (K a The term "K"), as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, while "K dis " (Kd) or "K off The term " ", as used herein, is intended to refer to the off-rate of a particular antibody-antigen interaction.

[0087] The term “K D ", as used herein, refers to k off k on the ratio to (i.e., k off / k on ) is intended to refer to the equilibrium dissociation constant, expressed as a molar concentration (M), obtained from D The value is related to the antibody concentration (the amount of antibody needed for a particular experiment) and therefore the K D The lower the value (the lower the concentration), the higher the affinity of the antibody. D The K value can be determined using methods well established in the art. D Preferred methods for determining K values ​​can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988), Coligan et al. (eds.), Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, NY, (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), which are incorporated herein by reference in their entireties. DMethods for determining affinity are those using surface plasmon resonance or those using biosensor systems, examples of which are the Biacore® system (for detailed information on affinity evaluation, see also Rich RL, Day YS, Morton TA, Myszka DG. High-resolution and high-throughput protocols for measuring drug / human serum albumin interactions using BIACORE®. Anal Biochem. 2001 Sep. 15;296(2):197-207) or the Octet® system. The Octet® platform is based on biolayer interferometry (BLI) technology. The principle of BLI technology is based on the optical interference pattern of white light reflected from two surfaces: a layer of immobilized protein and an internal reference layer. Binding between a ligand immobilized on the surface of the biosensor tip and an analyte in solution increases the optical thickness at the biosensor tip, resulting in a shift in the interference pattern measured in nanometers. The wavelength shift (Δλ) is a direct measure of the change in optical thickness of the biological layer; measuring this shift over a period of time and plotting its magnitude as a function of time yields a classical association / dissociation curve. This interaction is measured in real time, allowing the monitoring of binding specificity, association and dissociation rates, and concentration (see Abdiche et al., 2008, but also see results for details). Affinity measurements are typically performed at 25°C.

[0088] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope present on an antigen, e.g., BTN3A. In some embodiments, this term refers to an antibody that detectably binds to human BTN3A expressed on peripheral blood mononuclear cells (PBMCs), preferably with an EC of less than 50 μg / ml, more preferably less than 10 μg / ml, as determined in the Examples. 50(Assays and protocols are typically disclosed in WO 2020 / 025703, see in particular Table 4.) In other embodiments, the antibody binds to the antigenic recombinant polypeptide with a K of 100 nM or less, 10 nM or less, 1 nM or less, 100 pM or less, or 10 pM or less, as measured by SPR measurements as described above. D (However, see also Table 4 of WO 2020 / 025703 for details).

[0089] An antibody that "cross-reacts with an antigen other than BTN3A" has a K of 10 nM or less, 1 nM or less, or 100 pM or less for this antigen other than BTN3A. D An antibody that "does not cross-react with a particular antigen" is intended to refer to an antibody that binds to that antigen with a K of 1 μM or greater. D , or a K of 10 μM or more D The term "antibody" is intended to refer to an antibody that binds to the antigen. In certain embodiments, such antibodies that do not cross-react with the antigen have essentially undetectable binding to these proteins in standard binding assays. In certain embodiments, a humanized antibody of the present disclosure, e.g., mAb1, cross-reacts with cynomolgus monkey BTN3A1, BTN3A2, and BTN3A3 of SEQ ID NOs: 27, 28, and 29, respectively, as measured, for example, by Biacore assay (see, in particular, the relevant assays exemplified in WO 2020 / 025703, with reference to Table 26).

[0090] Specificity may be further demonstrated by an affinity / avidity ratio for binding to a specific antigen versus non-specific binding to other unrelated molecules, e.g., about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or greater (where the specific antigen is a BTN3A polypeptide).

[0091] As used herein, the term "avidity" refers to an informative measure of the overall stability or strength of an antibody-antigen complex. It depends on three major factors: antibody epitope affinity; the valency of both the antigen and the antibody; and the structural arrangement of the interacting moieties. Ultimately, these factors define antibody specificity, i.e., the likelihood that a particular antibody will bind to a precise antigen epitope.

[0092] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0093] As used herein, the term "optimized" means that a nucleotide sequence has been altered to encode an amino acid sequence using codons preferred by the production cell or organism, which is generally a eukaryotic cell, such as a Chinese hamster ovary cell (CHO) or a human cell. An optimized nucleotide sequence is one that has been engineered to completely retain, or retain as much as possible, the amino acid sequence originally encoded by the starting nucleotide sequence. An amino acid sequence encoded by an optimized nucleotide sequence is also referred to as optimized.

[0094] The term "identity," as used herein with reference to polypeptide sequences, refers to the identity of amino acid sequences between two molecules. These molecules are identical at a given amino acid position in both molecules if that position is occupied by the same amino acid. Identity between two polypeptides is a direct function of the number of identical positions. Generally, sequences are aligned (including gaps, if necessary) to obtain the highest order match. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity (%) = number of identical positions / total number of positions × 100), taking into account the number of gaps, and the length of each gap, that need to be introduced for optimal alignment of the two sequences. Comparison of sequences and determination of percent identity between two sequences can be accomplished using the mathematical algorithm described below.

[0095] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988) as incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. Alternatively, the percent identity between two amino acid sequences can be determined using published techniques and widely available computer programs, examples of which are BLASTP, FASTA (Atschul et al., J. Molecular Biol. 215:403, 1990), or the algorithm of Needleman and Wunsch (J. Mol. Biol. 48:444-453, 1970) incorporated into the GAP program in the GCG software package (Devereux et al., Nucleic Acids Res. 12:387, 1984, generally available at http: / / www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0096] The percent identity between two nucleotide or amino acid sequences can also be determined using an algorithm such as the BLASTN program for nucleic acid sequences, which uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=4, and a comparison of both strands.

[0097] Additional antibodies can be identified based on their ability to cross-compete with other antibodies of the present disclosure (e.g., competitively inhibit the binding of other antibodies of the present disclosure in a statistically significant manner) in a standard antigen-binding assay, such as an ELISA binding assay. The ability of a test antibody to inhibit the binding of an antibody of the present disclosure to its target indicates that the antibody and the test antibody can compete for binding to the target; such an antibody may, according to non-limiting theory, bind to the same or related (e.g., structurally similar or spatially proximate) epitope on the target as the competing antibody. Thus, another aspect of the present disclosure provides antibodies that bind to the same antigen as the antibodies disclosed herein and compete with them. As used herein, an antibody "competes" for binding when the competing antibody inhibits binding to a target by an antibody or antigen-binding fragment of the disclosure by more than 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% in the presence of an equimolar concentration of the competing antibody.

[0098] "Combination therapy," "co-administration," "co-administration," or "concurrent administration" refers to the combined administration of at least two therapeutic agents, where a first agent, typically a BTN3A-activating compound, is administered in the same subject in need thereof, either simultaneously or separated by a time interval, in combination with a second agent, e.g., a Bcl2 family inhibitor, and, optionally, in combination with a third agent, e.g., a hypomethylating agent, where the time interval allows the combined partners to exert a cooperative or synergistic effect to treat a disorder, e.g., cancer, and more particularly, a hematological malignancy. While these delivery methods are within the scope described herein, they are not intended to imply that the therapeutic agents must be administered simultaneously and / or formulated for delivery together. A BTN3A-activating antibody disclosed herein, e.g., ICT01, can be administered concurrently with, before, or after one or more other additional therapies or therapeutic agents. These terms are also intended to encompass therapeutic regimens in which the agents are not necessarily administered by the same route of administration.

[0099] As used herein, the terms synergy or synergistic effect, when used in connection with describing the effectiveness of a drug combination, means that the measured effect of the combination is greater than that expected from the sum of the effects of the individual drugs (i.e., greater than an additive effect). In some embodiments, tumor growth rate or tumor size (e.g., rate of change in tumor size (e.g., volume, mass)) is used to determine whether a drug combination is synergistic (e.g., a drug combination is synergistic if the tumor growth rate is slower than expected if the drug combination results in an additive effect). In some embodiments, median overall survival (e.g., less than 12 months) is used to determine whether a drug combination is synergistic (e.g., a drug combination is synergistic if the median overall survival of a subject or population of subjects is longer than expected if the drug combination results in an additive effect). In some embodiments, complete remission (CR) and complete remission with incomplete count recovery (CRi) rates are used to determine whether a drug combination is synergistic (compared to monotherapy). In some embodiments, T cell expansion may also be used to determine whether a drug combination is synergistic (e.g., a drug combination is synergistic if the rate of expansion (determined as a percentage increase in the population compared to baseline values ​​or an increase in absolute cell numbers) of a particular T cell subset is greater than would be expected if the drug combination resulted in an additive effect).

[0100] Activated BTN3A antibody Activating anti-BTN3A antibodies according to the present disclosure typically exhibit one or more of the following properties: (i) the antibody has a K of 10 nM or less, as measured, for example, by SPR as described in the Examples of Patent Application WO 2020025703; D , preferably a K of 1 nM or less D binds to BTN3A at ; (ii) the antibody has a K of 100 nM or less, as measured, for example, by SPR as described in the Examples of Patent Application WO 2020025703. D , preferably a K of 10 nM or less D cross-reacts with cynomolgus monkey BTN3A; (iii) the antibody has an EC of 50 μg / ml or less, preferably 10 μg / ml or less, as measured by the flow cytometry assay described in the Examples of Patent Application WO 2020025703. 50 binds to human PBMCs; (iv) This antibody has an EC value of less than 5 μg / ml, preferably 1 μg / ml or less, in co-culture with BTN3A-expressing cells, as described in the Examples of Patent Application WO 2020025703. 50 induces activation of γδ T cells, typically Vγ9Vδ2 T cells; (v) the antibody has an EC of less than 0.1 μg / mL, preferably 0.01 μg / mL or less, e.g., 100 pg / mL to 0.1 μg / mL, as measured by surface expression of the activation marker CD69. 50 induces in vitro activation of Vγ9Vδ2 T cells in human PBMCs.

[0101] Examples of BTN3A activating antibodies are described in the following paragraphs. In some embodiments, the BTN3A activating antibody is selected from the group consisting of BTN3A antibodies such as those described in International Patent Application Publication Nos. WO 2012080769, WO 2012080351, and WO 2020025703. In some specific embodiments, the BTN3A activating antibody is selected from the humanized antibodies described in WO 2020025703, or is a humanized version of the BTN3A activating antibodies described in WO 2012080769 and WO 2012080351. In some embodiments, the BTN3A activating antibody may be selected from mAb 20.1 and mAb 7.2, or humanized versions thereof, obtainable from one of the hybridomas accessible under CNCM deposit numbers I-4401 and I-4402, such as those described in WO2012080769 and WO2012080351, and may be selected from humanized mAbs 1-6 described in WO2020025703.

[0102] In some embodiments, a BTN3A activating antibody comprises the six CDRs (CDR1 (also referred to as HCDR1), VH CDR2 (also referred to as HCDR2), VH CDR3 (also referred to as HCDR1), VL CDR1 (also referred to as LCDR1), VL CDR2s (also referred to as LCDR2), VL CDR3s (also referred to as HCDR3)) of antibody 20.1 or 7.2 described in WO2012080769 and WO2012080351, or mAb 1-6 described in WO2020025703. In specific embodiments, an anti-BTN3A activating antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and HCDR3 as shown in Table 1 below:

[0103] [Table 1]

[0104] In some embodiments of the antibodies for use disclosed herein, the six CDR regions are 100% identical to the six CDR regions of antibody 20.1 or 7.2 described in WO2012080769 and WO2012080351, or mAbs 1-6 described in WO2020025703; particularly, in some embodiments, the six CDR regions of the antibodies disclosed herein are 100% identical to the six CDR regions of Table 1, particularly mAbs 7.2; 1; 2; 4; and 5.

[0105] Other antibodies for use disclosed herein include antibodies with mutated amino acids by amino acid deletion, insertion or substitution, but which still have at least 60, 70, 80, 90, 95, 96, 97, 98, 99 or 100 percent identity in the CDR regions to the six CDR regions of antibodies 20.1 or 7.2 described in WO2012080769 and WO2012080351, or to the six CDR regions of mAbs 1-6 described in WO2020025703, particularly to the six CDR regions defined in Table 1. Typically, according to the present disclosure, the antibody may have 1, 2, 3 or 4 amino acid differences (including deletions, insertions or substitutions) in one or more CDRs relative to the CDR sequences of antibodies 20.1 or 7.2 described in WO2012080769 and WO2012080351, or relative to the CDR sequences of mAbs 1 to 6 described in WO2020025703, particularly relative to the CDR sequences in Table 1, and more particularly relative to the CDR sequences of mAbs 7.2;1;2;4; and 5.

[0106] In certain embodiments, antibodies for use according to the present disclosure comprise a variant CDRH2 of mAb 20.1 with the substitutions N5S and K10N (also referred to as N53S, K58N according to Kabat numbering).

[0107] In other specific embodiments, antibodies for use according to the present disclosure comprise a variant CDRL1 of mAb 20.1 with the substitution L8V (also referred to as L31V according to Kabat numbering).

[0108] In a more specific embodiment, an antibody for use according to the present disclosure comprises a variant CDRH2 of mAb 20.1 with substitutions N5S and K10N (also referred to as N53S and K58N according to Kabat numbering), and a variant CDRL1 of mAb20.1 with substitution L8V (also referred to as L31V according to Kabat numbering).

[0109] In certain embodiments, a BTN3A activating antibody for use according to the present disclosure comprises the heavy chain CDR1-3 sequences of SEQ ID NO: 34, SEQ ID NO: 35, and SEQ ID NO: 36, and the light chain CDR1-3 sequences of SEQ ID NO: 37, SEQ ID NO: 38, and SEQ ID NO: 39, respectively.

[0110] In certain embodiments, a BTN3A activating antibody for use according to the present disclosure comprises a variable heavy chain VH of SEQ ID NO:40, and a variable light chain of SEQ ID NO:41.

[0111] In certain embodiments, the BTN3A-activating antibody is a bispecific antibody characterized by comprising a first binding moiety that specifically binds to human BTN3A and a second binding moiety that specifically binds to a tumor antigen, typically a malignant cell-targeted tumor antigen of the targeted hematological malignancy. For example, the first binding moiety is a full-length bivalent antibody having the heavy chain CDR sequences of CDRH1 of SEQ ID NO: 34, CDRH2 of SEQ ID NO: 35, and CDRH3 of SEQ ID NO: 36, and the light chain CDR sequences of CDRL1 of SEQ ID NO: 37, CDRL2 of SEQ ID NO: 38, and CDRL3 of SEQ ID NO: 39.

[0112] Other anti-BTN3A activating antibodies, particularly variants of the mAb20.1 activating antibody, are disclosed in WO 2023 / 161457 (Evobright GmbH), the contents of which are incorporated herein in their entirety. Antibodies for use in the present disclosure also include antibodies having at least 90%, particularly at least 95, 96, 97, 98, 99, or 100%, identity to the VH and VL regions defined in Table 2. More particularly, antibodies of the present disclosure include selected humanized recombinant antibodies mAb1, mAb2, mAb4, and mAb5, which are structurally characterized by variable heavy and light chain amino acid sequences and human constant regions (isotypes), as set forth in Table 2 below:

[0113] [Table 2]

[0114] mAb3 and mAb6 are humanized versions of the parent murine BTN3A activating antibody called mAb 20.1, which is described in WO 2012 / 080351.

[0115] The corresponding amino acid and nucleotide coding sequences of the constant isotype regions of IgG1, IgG4, and their mutant versions IgG1 L247F / L248E / P350S and IgG4 S241P / L248E, used to generate mAb1 to mAb6, are well known in the art (Oganesyan et al., 2008; Reddy et al., 2000). The C-terminal lysine found in IgG can be naturally cleaved, and this modification does not affect the properties of the antibody; therefore, this residue can be further deleted in the constructs of mAb1 to mAb6.

[0116] The full-length light and heavy chains and corresponding coding sequences of mAb1, mAb2, mAb4 and mAb5 are shown in Table 3 below.

[0117] [Table 3]

[0118] In certain embodiments that can be combined with the preceding embodiments, the antibody provided herein is an antibody fragment of the antibody defined above. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab'), Fv, unibody, and scFv fragments, diabodies, single domains, or nanobodies, and other fragments. Preferably, the antibody fragment is a monovalent antibody, e.g., a Fab of an scFv fragment.

[0119] In some embodiments, antibodies of the present disclosure compete for binding with the BTN3A antibodies described above, and in particular, antibodies of the present disclosure compete for binding with an antibody selected from mAb 20.1 and mAb 7.2, obtainable from one of the hybridomas accessible under CNCM deposit numbers I-4401 and I-4402, such as those described in WO 2012080769 and WO 2012080351, and also compete for binding with an antibody selected from mAbs 1-6 described in WO 2020025703. In more specific embodiments, antibodies of the present disclosure compete for binding with an antibody selected from mAb 7.2 produced by the hybridoma deposited at the CNCM under deposit number I-4402, and an antibody having a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

[0120] In some embodiments, the antibodies of the present disclosure are chimeric, humanized, or human antibodies. In a preferred embodiment of the present disclosure, the BTN3A antibody is a humanized antibody. Typically, non-human antibodies are humanized to reduce immunogenicity in humans, while retaining at least the same affinity (or better affinity) as the parent non-human antibody. More specifically, the BTN3A antibody is a humanized form of antibody 20.1 or 7.2 disclosed in WO2012080351. In a preferred embodiment, the antibody of the present disclosure is a humanized version of the parent antibody mAb 7.2 disclosed in WO2012080351. Generally, a humanized antibody comprises one or more variable domains, with the CDRs (or portions thereof) derived from a non-human antibody, e.g., murine mAb 7.2, and the FRs (or portions thereof) derived from murine antibody sequences with mutations that reduce immunogenicity. The humanized antibody also optionally comprises at least a portion of a human constant region. Preferably, the recombinant antibody according to the present disclosure is a humanized silenced antibody, typically a humanized silenced IgG1 or IgG4 antibody. Suitable humanized anti-BTN3A antibodies according to the present disclosure are typically described in WO2020025703 and include mAbs having the VH / VL polypeptide sequences in Table 2 and the light / heavy chains in Table 3.

[0121] As used herein, the term "silent" antibody refers to an antibody that has no or low FcγR binding and / or C1q binding, as measured in binding assays such as those described in WO2020025703. In one embodiment, the term "no or low FcγR binding and / or C1q binding" means that the silent antibody exhibits at least 50% less, e.g., less than 80%, of the FcγR binding and / or C1q binding observed with a corresponding antibody having a wild-type human IgG1 or IgG4 isotype.

[0122] Framework or Fc manipulation The antibodies of the present disclosure may include modifications made to framework residues in VH and VL to reduce immunogenicity.

[0123] In some specific embodiments, the antibody of the present disclosure is a humanized monoclonal antibody of the parent murine antibody mAb 7.2, which contains at least the following amino acid mutations in the VH framework region: V5Q; V11L; K12V; R66K; S74F; I75S; E81Q; S82AR; R82BS; R83T; D85E; T87S; L108S; and at least the following amino acid mutations in the Vκ framework region: T5N; V15L; R18T; V19I; K42N; A43I; D70G; F73L; Q100G.

[0124] In other specific embodiments, the antibody of the present disclosure is a humanized monoclonal antibody of the parent murine antibody mAb 7.2, which contains at least the following amino acid mutations in the VH framework region relative to mAb 7.2: V5Q; V11L; K12V; R66K; S74F; I75S; E81Q; S82AR; R82BS; R83T; D85E; T87S; L108S; and at least the following amino acid mutations in the Vκ framework region: T5N; V15L; R18T; V19I; K42N; A43I; S63T; D70G; F73L; Q100G.

[0125] In addition to modifications made to the framework regions, the antibodies of the disclosure typically may be engineered to contain modifications in the Fc region to alter one or more functional properties of the antibody, e.g., serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0126] Furthermore, the antibodies of the present disclosure can be chemically modified (e.g., one or more chemical moieties can be attached to the antibody) or modified to alter glycosylation to alter one or more functional properties of the antibody, each of these embodiments being described in further detail below.

[0127] As used herein, the terms "isotype constant region" or "Fc region" are used interchangeably to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions. The human IgG heavy chain Fc region is generally defined to include amino acid residues from position C226 or P230 to the carboxyl terminus of an IgG antibody, where numbering is according to the EU numbering system. The C-terminal lysine (K447 residue) of the Fc region can be removed, for example, during antibody production or purification, or the corresponding codon can be deleted in a recombinant construct. Thus, antibody compositions of the present disclosure can include antibody populations in which all K447 residues have been removed, antibody populations in which the K447 residue has not been removed, and antibody populations having a mixture of antibodies with and without the K447 residue.

[0128] In certain embodiments, the hinge region of CH1 is modified so that the number of cysteine ​​residues in the hinge region is altered, e.g., increased or decreased. This approach is further described in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine ​​residues in the hinge region of CH1 is altered, for example, to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

[0129] In other embodiments, the Fc-hinge region of the antibody is mutated to decrease the biological half-life of the antibody. More specifically, the antibody has one or more amino acid mutations introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment to reduce Staphylococcus aureus protein A (SpA) binding compared to the SpA binding of the native Fc-hinge domain. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0130] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, an antibody can have its affinity for an effector ligand altered by replacing one or more amino acids with a different amino acid residue, but retain the antigen-binding ability of the parent antibody. The effector ligand for which affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter et al.

[0131] In another embodiment, the antibody can have one or more selected amino acid residues replaced with different amino acid residues to alter C1q binding and / or reduce or eliminate complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 by Idusogie et al.

[0132] In another embodiment, one or more amino acid residues are altered to alter the ability of the antibody to fix complement. This approach is further described in PCT Publication No. WO 94 / 29351 by Bodmer et al.

[0133] In other embodiments, the Fc region is modified by modifying one or more amino acids to reduce the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to reduce the affinity of the antibody for Fcγ receptors. Such antibodies with reduced effector function, particularly reduced ADCC, include silenced antibodies.

[0134] In certain embodiments, an Fc domain of the IgG1 isotype is used, in some particular embodiments, a mutant variant of the IgG1 Fc fragment is used, for example, a silenced IgG1 Fc that reduces or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or bind to Fcγ receptors.

[0135] In certain embodiments, an Fc domain of the IgG4 isotype is used, in some specific embodiments, a mutant variant of an IgG4 Fc fragment is used, for example, a silenced IgG4 Fc that reduces or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or bind to Fcγ receptors.

[0136] Silenced effector function can be obtained by mutation in the Fc constant part of the antibody, and has been described in the art (Baudino et al., J. Immunol. 2008; Strohl, CO Biotechnology 20 2009). An example of a silent IgG1 antibody is the triple mutation variant IgG1 L247F L248E P350S. An example of a silent IgG4 antibody is the double mutation variant IgG4 S241P L248E.

[0137] In certain embodiments, the Fc domain is a silent Fc mutant that prevents glycosylation of the Fc domain at position 314. For example, the Fc domain comprises an amino acid substitution of asparagine at position 314. Examples of such amino acid substitutions are substitutions of N314 with glycine or alanine.

[0138] In yet other embodiments, the glycosylation of the antibody is modified. For example, an aglycosylated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites in the antibody sequence. For example, one or more variable region framework glycosylation sites can be eliminated by making one or more amino acid substitutions, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for antigen. Such approaches are described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861 by Co et al.

[0139] Another modification of the antibodies herein contemplated by the present disclosure is pegylation or hesylation or related techniques. Antibodies can be pegylated, for example, to increase the biological (e.g., serum) half-life of the antibody. To pegylate an antibody, the antibody or a fragment thereof is typically reacted with polyethylene glycol (PEG), such as a reactive ester or aldehyde derivative of PEG, under conditions that result in attachment of one or more PEG groups to the antibody or antibody fragment. Pegylation can be carried out by an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any form of PEG that has been used to derivatize other proteins, such as mono(C1-C10)alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certain embodiments, the antibody to be pegylated is a non-glycosylated antibody. Methods for pegylation of proteins are known in the art and can be applied to the antibodies of the present disclosure. See, for example, European Patent No. 0154316 to Nishimura et al. and European Patent No. 0401384 to Ishikawa et al.

[0140] Another possibility is to fuse at least the antigen-binding region of an antibody of the present disclosure to a protein capable of binding to a serum protein, such as human serum albumin, to increase the half-life of the resulting molecule. Such an approach is described, for example, in Nygren et al., EP 0486525.

[0141] In certain embodiments, the C-terminal lysine commonly present in human IgG heavy chain constant domains is engineered and removed to reduce heterogeneity due to cleavage of this lysine commonly observed during manufacturing or storage. Such modifications do not appreciably alter the desired function of these antibodies, and confer stability advantages to these molecules.

[0142] Nucleic acid molecules encoding antibodies of the present disclosure Also disclosed herein are nucleic acid molecules encoding the anti-BTN3A antibodies of the present disclosure. Exemplary variable light and heavy chain nucleotide sequences are those encoding the variable light and heavy chain amino acid sequences of any one of mAb1, mAb2, mAb4, and mAb5, the latter sequences being readily derived from Tables 1 and 2 using the genetic code and, optionally, taking into account codon bias depending on the host cell type.

[0143] The present disclosure also relates to nucleic acid molecules derived from the latter sequence that have been optimized for protein expression in mammalian cells, such as CHO cell lines.

[0144] Nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. Nucleic acids are "isolated" or "substantially purified" when purified by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art (Ausubel et al., 1988, Current Protocols in Molecular Biology (John Wiley & Sons)), to separate them from other cellular components or contaminants, such as other cellular nucleic acids or cellular proteins. Nucleic acids of the present disclosure can be, for example, DNA or RNA, and may or may not contain intronic sequences. In certain embodiments, nucleic acids can be present in a vector, such as a phage display vector or a recombinant plasmid vector.

[0145] The nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. For example, once DNA fragments encoding VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques to, for example, convert the variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, a VL- or VH-encoding DNA fragment (e.g., the VL and VH segments defined in Table 1) is operably linked to another DNA molecule or to a fragment encoding another protein, such as an antibody constant region or a flexible linker. The term "operably linked," as used in this context, is intended to mean that the two DNA fragments are operably joined, for example, so that the amino acid sequences encoded by the two DNA fragments are in frame or so that a protein is expressed under the control of a desired promoter.

[0146] The isolated DNA encoding the VH region can be converted into a full-length heavy chain gene by operatively linking the VH-encoding DNA to another DNA molecule encoding the heavy chain constant region (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (Kabat et al., KS (1992). Sequences of Proteins of Immunological Interest (DIANE Publishing)), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The heavy chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. In some embodiments, the heavy chain constant region is selected from an IgG1 isotype, e.g., a human IgG1 isotype. In other embodiments, the heavy chain constant region is selected from an IgG4 isotype, e.g., a human IgG4 isotype. For a Fab fragment heavy chain gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only the heavy chain constant region CH1.

[0147] Isolated DNA encoding the VL region can be converted into a full-length light chain gene (e.g., into a Fab light chain gene) by operatively linking the VL-encoding DNA to another DNA molecule encoding the light chain constant region, CL. The sequences of human light chain constant region genes are known in the art (Kabat et al., 1992, supra), and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a kappa or lambda constant region.

[0148] To generate an scFv gene, the VH- and VL-encoding DNA fragments are operably linked to another fragment encoding a flexible linker, for example, another fragment encoding the amino acid sequence (Gly4-Ser)3, thereby enabling expression of the VH and VL sequences as a contiguous single-chain protein in which the VL and VH domains are connected by the flexible linker (Bird et al., 1988, supra; Huston et al., 1988, supra; McCafferty, J. et al., 1990. Nature 348, 552-554).

[0149] Generation of transfectomas producing monoclonal antibodies The antibodies of the present disclosure can be produced in host cell transfectomas, for example, using a combination of recombinant DNA techniques and gene transfection methods, as are well known in the art (Morrison, 1985; Science 229, 1202-1207).

[0150] For example, to express an antibody, or antibody fragment thereof, DNA encoding partial or full-length light and heavy chains can be obtained by standard molecular biological or biochemical techniques (e.g., DNA chemical synthesis, PCR amplification, or cDNA cloning using a hybridoma expressing the antibody of interest), and this DNA can be inserted into an expression vector such that the gene is operably linked to transcriptional and translational control sequences. In this context, the term "operably linked" is intended to mean that the antibody gene is ligated into a vector such that the transcriptional and translational control sequences in the vector perform their intended function of controlling the transcription and translation of the antibody gene. The expression vector and expression control sequences are selected to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or, more typically, both genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and vector, or, if no restriction sites are present, blunt-end ligation). The light and heavy chain variable regions of the antibodies described herein can be used to generate full-length antibody genes of any antibody isotype by inserting them into an expression vector already encoding heavy and light chain constant regions of the desired isotype, such that the VH segment is operably linked to a CH segment(s) in the vector and the VL segment is operably linked to a CL segment in the vector. Additionally or alternatively, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from a host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is linked in-frame to the amino terminus of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide from a non-immunoglobulin protein).

[0151] In addition to the antibody chain genes, the recombinant expression vectors disclosed herein carry control sequences that regulate the expression of the antibody chain genes in a host cell. The term "control sequence" is intended to include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. Such control sequences are described, for example, in Goeddel (Goeddel, DV (1990). [1] Systems for heterologous gene expression., in Methods in Enzymology (Academic Press), pp. 3-7). Those skilled in the art will understand that the design of the expression vector, including the selection of control sequences, can depend on factors such as the choice of host cell to be transformed and the desired expression level of protein. Control sequences for expression in mammalian host cells include promoters and / or enhancers derived from viral elements that direct high levels of protein expression in mammalian cells, such as promoters and / or enhancers derived from cytomegalovirus (CMV), simian virus 40 (SV40), adenovirus (e.g., the adenovirus major late promoter (AdMLP)), and polyoma. Alternatively, non-viral control sequences, such as the ubiquitin promoter or P-globin promoter, can be used. Still further, control elements composed of sequences from different sources, such as the SRa promoter system, which contains sequences from the SV40 early promoter and the long terminal repeat of human T-cell leukemia virus type 1 (Takebe et al., 1988, Mol. Cell. Biol. 8, 466-472).

[0152] In addition to the antibody chain genes and control sequences, the recombinant expression vectors of the disclosure may carry additional sequences, such as sequences that control replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced (see, e.g., U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all by Axel et al.). For example, typically the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on the host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in dhfr-host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0153] For expression of the light and heavy chains, expression vector(s) encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to encompass a wide range of techniques commonly used to introduce foreign DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, and DEAE-dextran transfection. The antibodies of the present disclosure can theoretically be expressed in either prokaryotic or eukaryotic host cells. Expression of antibodies in eukaryotic cells, such as mammalian host cells, yeast, or filamentous fungi, is contemplated because such eukaryotic cells, particularly mammalian cells, are more likely than prokaryotic cells to assemble and secrete properly folded and immunologically active antibodies.

[0154] In one specific embodiment, a cloning or expression vector according to the present disclosure comprises one of the coding sequences for the heavy and light chains of any of mAb1, mAb2, mAb4, and mAb5 operably linked to a suitable promoter sequence.

[0155] Mammalian host cells for expressing recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO) cells, including dhfr- CHO cells (described in Urlaub and Chasin, 1980) used with a DHFR selectable marker (described in Kaufman and Sharp, 1982), the CHOK1 dhfr+ cell line, NSO myeloma cells, COS cells, and SP2 cells, e.g., the GS CHO cell line used with the GS Xceed™ Gene Expression System (Lonza). When a recombinant expression vector encoding an antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient for expression of the antibody within the host cells and, optionally, for secretion of the antibody into the culture medium in which the host cells are grown. The antibody can be recovered and purified from the culture medium, for example, after secretion, using standard protein purification methods (Shukla et al., 2007, J. Chromatogr. B 848, 28-39).

[0156] In one particular embodiment, the host cell of the present disclosure is a host cell transfected with an expression vector comprising a coding sequence suitable for expression of each of mAb1, mAb2, mAb4, and mAb5, operably linked to a suitable promoter sequence.

[0157] For example, the present disclosure relates to a host cell comprising at least the nucleic acids of SEQ ID NO:8 and SEQ ID NO:10 encoding the heavy and light chains of mAb1, respectively.

[0158] The latter host cells can then be further cultured under conditions suitable for the expression and production of an antibody of the present disclosure selected from the group consisting of mAb1, mAb2, mAb3, mAb4, and mAb5, respectively.

[0159] Alternatively, cell-free expression systems can be used to produce any of mAb1, mAb2, mAb3, mAb4, and mAb5. Typically, methods for cell-free expression of proteins or antibodies have been previously described (Stech et al., 2017, Sci. Rep. 7, 12030).

[0160] Anti-BTN3A immunoconjugate In another aspect, the present disclosure features that the anti-BTN3A antibodies, or fragments thereof, disclosed herein are conjugated to a therapeutic moiety. Such conjugates are referred to herein as "immunoconjugates." Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells.

[0161] Cytotoxins can be conjugated to the antibodies of the present disclosure using linker technology available in the art. Examples of linker types that have been used to conjugate cytotoxins to antibodies include, but are not limited to, hydrazones, thioethers, esters, disulfides, and peptide-containing linkers, such as valine-citrulline linkers. For example, linkers can be selected that are susceptible to cleavage by the low pH in lysosomal compartments or by proteases preferentially expressed in tumor tissues, such as cathepsins (e.g., cathepsins B, C, and D).

[0162] For further discussion regarding types of cytotoxins, linkers, and methods for conjugating therapeutic agents to antibodies, see also Panowski et al., 2013 for a review of antibody drug conjugates.

[0163] The antibodies of the present disclosure can also be conjugated to radioisotopes to create cytotoxic radiopharmaceuticals, also called radioimmunoconjugates. Examples of radioisotopes that can be conjugated to antibodies for diagnostic or therapeutic use include iodine. 131 ,indium 111 ,yttrium 90 , and lutetium 177 Methods for preparing radioimmunoconjugates are established in the art.

[0164] Bispecific or multispecific anti-BTN3A antibodies In another aspect, bispecific or multispecific molecules comprising the anti-BTN3A antibodies of the present disclosure are also disclosed herein. The antibodies can be derivatized or linked to another functional molecule, e.g., another peptide or protein (e.g., another antibody or receptor ligand), to create a bispecific molecule that binds to at least two different binding sites or target molecules. Indeed, the antibodies can be derivatized or linked to more than one other functional molecule to create a multispecific molecule that binds to more than two different binding sites and / or target molecules; such multispecific molecules are also intended to be encompassed by the term "bispecific molecule" as used herein. To create a bispecific molecule, the antibodies of the present disclosure can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, etc.) to one or more other binding molecules, e.g., another antibody, antibody fragment, peptide, or binding mimic, to obtain a bispecific molecule.

[0165] Thus, the present disclosure includes bispecific molecules comprising at least one first binding specificity for BTN3A, e.g., one antigen-binding portion of any one of mAb1, mAb2, mAb3, mAb4, mAb5, and mAb6 (or a functional variant thereof), and a second binding specificity for a second target epitope.

[0166] Furthermore, in embodiments in which the bispecific molecule is multispecific, the molecule can further comprise a third binding specificity in addition to the first and second target epitopes.

[0167] In one embodiment, the bispecific molecules disclosed herein comprise at least one antibody or antibody fragment thereof, including, for example, a Fab, Fab', F(ab')2, Fv, unibody, or single-chain Fv, as a binding specificity. The antibody may also be a light or heavy chain dimer, or any minimal fragment thereof, such as an Fv, or a single-chain construct as described in Ladner et al., U.S. Patent No. 4,946,778.

[0168] Other antibodies that can be employed in the bispecific molecules disclosed herein are murine, chimeric, and humanized monoclonal antibodies.

[0169] Bispecific molecules of the present disclosure can be prepared by conjugating the component binding specificities using methods known in the art. For example, each binding specificity of the bispecific molecule can be generated separately and then conjugated to each other. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate (sulfo-SMCC) (Karpovsky et al., 1984 J. Exp. Med. 160, 1686-1701; Liu et al., 1985 Proc. Natl. Acad. Sci. 82, 8648-8652). Other methods include those described in Brennan et al., 1985, Science 229, 81-83; Glennie et al., 1987, J. Immunol. 139, 2367-2375; and Paulus, 1985, Behring Inst. Mitt. 118-132.

[0170] Alternatively, both binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly useful when the bispecific molecule is a mAb x mAb, mAb x scFv, mAb x Fab, Fab x F(ab')2, or ligand x Fab fusion protein. In certain embodiments, the bispecific molecule can comprise a fusion of an anti-BTN3A antibody comprising full-length heavy and light chains that binds to the target epitope with an scFv. In a related, more specific embodiment, the scFv is fused at the C-terminal portions of the antibody heavy and light chains (bivalent binding specificity for the target epitope). Bispecific molecules of the present disclosure can be single-chain molecules comprising one single-chain antibody and one binding determinant, or single-chain bispecific molecules comprising two binding determinants.

[0171] Binding of a bispecific molecule to a specific target can be confirmed, for example, by enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (REA), FACS analysis, bioassays (e.g., growth inhibition and apoptosis), or Western blot assays. Each of these assays generally detects the presence of a particular protein-antibody complex of interest by employing a labeled reagent (e.g., an antibody) specific for the complex of interest. Bcl2 family inhibitors The present disclosure relates to the use of activating BTN3A antibodies in combination with at least one Bcl2 family inhibitor.

[0172] Apoptosis is induced in cells by a caspase activation cascade that can be triggered by the extrinsic and intrinsic pathways. The intrinsic pathway is triggered by the disruption of mitochondrial integrity, which is regulated by BCL-2 family proteins. BCL-2 (B-cell lymphoma 2) was the first protein identified in this family and is the family's namesake. Members are characterized by the expression of four BH (B cell homology) domains. In humans, six anti-apoptotic family members (BCL-2, BCL-XL, BCL-w, BCL-2-related protein A1 (Bfl-1 / A1), MCL-1 (myeloid cell leukemia 1), and BCL-B / Boo) and ten pro-apoptotic family members (including BAX and BAK) have been identified. Under homeostatic conditions, anti-apoptotic proteins ensure cell survival by binding to the key pro-apoptotic proteins BAX and BAK. Stress, such as loss of survival signals, DNA damage, chemicals, or therapeutic agents, disrupts this balance, leading to loss of mitochondrial membrane integrity and the initiation of apoptosis (Perini et al., 2018 J Hematol Oncol. 11(1):65; Roberts, Hematology Am Soc Hematol Educ Program. 2020 Dec 4;2020(1):1-9).

[0173] As used herein, the term Bcl2 family inhibitor refers to (i) BCL-2 and BCL-XL inhibitors, (ii) selective BCL-2 inhibitors, and (iii) inhibitors of MCL-1.

[0174] In certain embodiments, the Bcl2 family inhibitor is a Bcl2 inhibitor selected from (i) a BCL-2 and BCL-XL inhibitor, and (ii) a selective BCL-2 inhibitor.

[0175] In certain embodiments, inhibitors of BCL-2 family proteins include inhibitors of MCL-1, such as AZD5991 (e.g., CAS No. 2143010-83-5) and S64315 (MIK665) (e.g., CAS No. 1799631-75-6).

[0176] In certain embodiments, the Bcl2 inhibitor is selected from BCL-2 and BCL-XL inhibitors, including: ABT-737 (e.g., CAS No. 852808-04-9), navitoclax (e.g., CAS No. 923564-51-6), and AZD4320 (e.g., CAS No. 1357576-48-7).

[0177] The Bcl-2 inhibitor is preferably selected from the group consisting of venetoclax, navitoclax, obatoclax, and even more preferably venetoclax.

[0178] Venetoclax (DCI), also known as ABT-199, selectively binds to and inhibits the BCL-2 (B-cell lymphoma-2) protein. In some hematologic cancers, BCL-2 prevents cancer cells from undergoing apoptosis. The IUPAC name for venetoclax is 4-[4-[[2-(4-chlorophenyl)-4,4-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[3-nitro-4-(oxan-4-ylmethylamino)phenyl]sulfonyl-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide. Venetoclax has the following formula:

[0179] [ka] Venetoclax is marketed as VENCLEXTA™ and is in tablet form. In the United States, venetoclax is indicated for the treatment of (i) adult patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL); and (ii) newly diagnosed acute myeloid leukemia (AML), in combination with injectable 5-azacytidine or decitabine or low-dose cytarabine, in adults who are 75 years of age or older or who have comorbidities that prevent the use of intensive induction chemotherapy. In some embodiments, patients receive 20 mg / m 2 of low-dose cytarabine. 2 of cytarabine is given subcutaneously once daily for 10 consecutive days every four weeks.

[0180] Navitoclax, also known as ABT-263, selectively binds to the anti-apoptotic proteins Bcl-2, Bcl-XL, and Bcl-w, preventing them from binding to the apoptotic effector proteins Bax and Bak. The IUPAC name for navoticlax is 4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohexen-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide.

[0181] Obatoclax, also known as GX15-070, is a pan-inhibitor of Bcl-2 family proteins with proapoptotic activity. Obatoclax is a selective antagonist of the BH3-binding groove of Bcl-2 family proteins, which are overexpressed in several cancers. The IUPAC name for obatoclax is (2Z)-2-[(5Z)-5-[(3,5-dimethyl-1H-pyrrol-2-yl)methylidene]-4-methoxypyrrol-2-ylidene]indole.

[0182] S64315, also known as MIK665, is a highly potent and selective inhibitor of MCL-1 with proapoptotic and antineoplastic activity due to its high expression in various human cancers, including those of hematopoietic and lymphoid origin. The IUPAC name for S64315 is (R)-2-((5-(3-chloro-2-methyl-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-6-(4-fluorophenyl)thieno[2,3-d]pyrimidin-4-yl)oxy)-3-(2-((2-(2-methoxyphenyl)pyrimidin-4-yl)methoxy)phenyl)propanoic acid).

[0183] In a preferred embodiment, the Bcl-2 inhibitor for use in the combinations of the present disclosure is venetoclax. hypomethylating agents In certain embodiments, the combination therapies described herein include a hypomethylating agent. Hypomethylating agents, also known as HMAs or demethylating agents, inhibit DNA methylation. In certain embodiments, hypomethylating agents block the activity of DNA methyltransferase. In certain embodiments, hypomethylating agents include, but are not limited to, azacitidine and decitabine.

[0184] Azacitidine is also known as 5-AC, 5-azacytidine, azacitidine, ladakamycin, 5-AZC, AZA-CR, U-18496, 4-amino-1-β-D-ribofuranosyl-1,3,5-triazin-2(1H)-one, 4-amino-1-[(2R,3R,4S,5R)3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1,3,5-triazin-2-one, or VIDAZA®.

[0185] Azacitidine is a pyrimidine nucleoside analog of cytidine with anti-neoplastic activity. Azacitidine is incorporated into DNA and blocks DNA methylation by reversibly inhibiting DNA methyltransferase. DNA hypomethylation by azacytidine can activate tumor suppressor genes that are silenced by hypermethylation, resulting in anti-tumor effects. Azacitidine can also be incorporated into RNA, disrupting normal RNA function and reducing tRNA cytosine-5-methyltransferase activity.

[0186] In some embodiments, azacitidine is administered at a dose of about 25 mg / m 2 ~about 150mg / m 2 , for example, about 50 mg / m 2 ~about 100mg / m 2 , about 70mg / m 2 ~about 80mg / m 2 , about 50mg / m 2 ~about 75mg / m 2 , about 75mg / m 2 ~about 125mg / m 2 , about 50mg / m 2 , about 75mg / m 2 , about 100mg / m 2 , about 125mg / m 2 , or about 150 mg / m 2 In some embodiments, azacitidine is administered at a dose of about 50 mg / m. In some embodiments, azacitidine is administered once daily. In some embodiments, azacitidine is administered intravenously. In other embodiments, azacitidine is administered subcutaneously. In some embodiments, azacitidine is administered at a dose of about 50 mg / m. 2 ~about 100mg / m 2 (e.g., about 75 mg / m 2 ) for, for example, about 5 to 7 consecutive days in a 28-day cycle. For example, azacitidine is administered at a dose of about 75 mg / m 2 For seven consecutive days on days 1-7 of a 28-day cycle, azacitidine may be administered at a dose of about 75 mg / m 2For five consecutive days on days 1-5 of a 28-day cycle, followed by two days off, and then for two consecutive days on days 8-9. As yet another example, azacitidine may be administered at a dose of about 75 mg / m 2 It is recognized that a dose of 100 mg ...

[0187] Combination kits and compositions Combination Kit A combination of the present disclosure, comprising an anti-BTN3A antibody and a Bcl2 family inhibitor, e.g., a Bcl2 inhibitor, as defined above, can be presented as a combination kit. The term "combination kit" or "kit of parts," as used herein, refers to a pharmaceutical composition or compositions used to administer a BTN3A-activating antibody (e.g., mAb1) according to the present disclosure and a Bcl2 family inhibitor, e.g., a Bcl2 inhibitor (e.g., venetoclax). When both compounds are administered simultaneously, the combination kit can, for example, contain the components, suitably the anti-BTN3A antibody and the Bcl2 family inhibitor, in separate pharmaceutical compositions. When the components, suitably the BTN3A-activating antibody and the Bcl2 inhibitor, are not administered simultaneously, the combination kit can contain the active ingredients in separate pharmaceutical compositions in a single package or in separate pharmaceutical compositions in separate packages.

[0188] In one aspect, (i) a composition comprising a BTN3A activating antibody, e.g., mAb1, as defined above, typically a BTN3A activating antibody, together with a pharmaceutically acceptable excipient, diluent or carrier; (ii) a composition comprising a Bcl2 inhibitor as defined above, e.g., venetoclax, typically a Bcl2 inhibitor, together with a pharmaceutically acceptable excipient, diluent and / or carrier; and (iii) Optionally, a kit-of-parts is provided comprising a hypomethylating agent, such as azacitidine or decitabine.

[0189] In one embodiment of the present disclosure, the kit of parts comprises: (i) a composition comprising a BTN3A activating antibody, e.g., mAb1, as defined above, typically a BTN3A activating antibody, together with a pharmaceutically acceptable excipient, diluent or carrier; (ii) a composition comprising a Bcl2 inhibitor as defined above, e.g., venetoclax, typically a Bcl2 inhibitor, together with a pharmaceutically acceptable excipient, diluent and / or carrier; and (iii) a hypomethylating agent, e.g., azacitidine or decitabine, optionally together with a pharmaceutically acceptable excipient, diluent and / or carrier; The components may be provided in a form suitable for sequential, separate and / or simultaneous administration.

[0190] In one embodiment, the kit of parts comprises: (i) a first container containing a composition comprising a BTN3A activating antibody as defined above, typically a BTN3A activating antibody, together with a pharmaceutically acceptable excipient, diluent and / or carrier; (ii) a second container containing a composition comprising a Bcl2 family inhibitor, e.g., a Bcl2 inhibitor, as defined above, together with a pharmaceutically acceptable excipient, diluent and / or carrier; and (iii) Optionally, a third container containing a hypomethylating agent as defined above, e.g., azacitidine or decitabine.

[0191] The combination kit may also be provided with instructions, such as dosage and administration instructions, which may be of the type provided to a physician or by a physician, such as instructions to a patient.

[0192] composition Thus, in accordance with the present disclosure, a BTN3A activating antibody as defined herein and a Bcl2 family inhibitor as defined herein can be formulated separately into separate compositions, e.g., pharmaceutical compositions, using, e.g., pharmaceutically acceptable carriers.

[0193] As used herein, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, or excipient, and includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. In addition to the active compound (i.e., a BTN3A-activating antibody and / or a Bcl2 family inhibitor), the composition may further include one or more of the following compounds:

[0194] Sterile phosphate-buffered saline is one example of a pharmaceutically acceptable carrier. Other suitable carriers are well known to those skilled in the art (Remington and Gennaro, 1995). The formulation may further include one or more excipients, preservatives, solubilizers, buffers, albumin to prevent protein loss on the vial surface, etc.

[0195] The form of the pharmaceutical composition, the route of administration, the dosage and the regimen will naturally vary depending on the condition to be treated, the severity of the disease, the age, weight and sex of the patient, etc.

[0196] The pharmaceutical compositions of the present disclosure can be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, and the like.

[0197] Preferably, the pharmaceutical composition comprises a vehicle that is pharmaceutically acceptable for injectable formulations, in particular isotonic sterile saline (monosodium phosphate or disodium phosphate and sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc., or mixtures of such salts), or a dry composition, in particular a lyophilized composition, which, upon addition of sterile water or physiological saline, as the case may be, can be made into a solution for injection.

[0198] The dosage used for administration can vary depending on various parameters, in particular depending on the mode of administration used, the symptoms involved, or also on the desired duration of treatment.

[0199] Compositions Comprising BTN3A-Activating Antibodies and Dosing Regimen In some embodiments, the BTN3A-activating antibodies defined herein can thus be formulated into a composition, e.g., a pharmaceutical composition, as defined above, comprising one or a combination of the antibodies disclosed herein, such as one of the antibodies selected from the group consisting of mAb1, mAb2, mAb3, mAb4, and mAb5, or antigen-binding portions thereof, formulated together with a pharmaceutically acceptable carrier.

[0200] The antibodies of the present disclosure can be formulated into the compositions defined above in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the protein) formed with inorganic acids such as, for example, hydrochloric or phosphoric acid, or organic acids such as, for example, acetic, oxalic, tartaric, and mandelic acids. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as, for example, isopropylamine, trimethylamine, histidine, and procaine. Formulations of solutions suitable for infusion or subcutaneous injection of antibodies are described in the art and are reviewed, for example, in Cui et al. (Drug Dev Ind Pharm 2017, 43(4):519-530). In a preferred embodiment, the anti-BTN3A antibody is formulated for intravenous infusion as defined above.

[0201] Pharmaceutical compositions containing BTN3A-activating antibodies can be formulated at various concentrations. For example, the formulations may contain BTN3A-activating antibodies at concentrations of 0.1 μM to 1 mM, more preferably 1 μM to 500 μM, 500 μM to 1 mM, 300 μM to 700 μM, 1 μM to 200 μM, 100 μM to 200 μM, 200 μM to 300 μM, 300 μM to 400 μM, 400 μM to 500 μM, 500 μM to 600 μM, 600 μM to 700 μM, 800 μM to 900 μM, or 900 μM to 1 mM. Typically, the formulations contain BTN3A-activating antibodies at a concentration of 300 μM to 700 μM.

[0202] Typically, therapeutic doses of BTN3A-activating antibodies for human patients range from 100 pg to 700 mg per administration (based on a 70 kg body weight). For example, a maximum therapeutic dose may range from 0.1 to 10 mg / kg per administration, e.g., 0.1 to 5 mg / kg, 1 to 5 mg / kg, or 0.1 to 2 mg / kg. It will be understood that such doses may be administered at different intervals as determined by the oncologist / physician; for example, doses may be administered daily, twice weekly, weekly, every other week, every three weeks, or monthly.

[0203] Typically, in certain embodiments, the BTN3A activating antibody is administered intravenously at doses containing 20 μg to 200 mg, particularly 1 mg to 200 mg or 7 to 200 mg, typically every 21 days.

[0204] In certain embodiments, a suitable dose for intravenous administration of an activating anti-BTN3A antibody may be selected from 1, 7, 10, 20, 50, 75, 100, 125, 150, 175, and 200 mg.

[0205] In certain embodiments, an activating BTN3A antibody (preferably mAb1, described below) for use in the methods of the present disclosure is administered intravenously in doses containing 20 μg to 200 mg in each dose, preferably with a second dose administered at least 15 days, typically about 21 days, after the first dose.

[0206] Compositions Comprising Bcl2 Family Inhibitors and Dosing Regimen Therapy with a Bcl2 family inhibitor can be initiated according to a schedule of weekly dose escalation over a specified period (days or weeks) until the recommended daily dose is reached.

[0207] When treating chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL), venetoclax is currently administered at a daily dose of 20 mg in week 1, 50 mg in week 2, 100 mg in week 3, 200 mg in week 4, and 400 mg in weeks 5 and beyond. When treating AML in combination with another agent, e.g., injectable 5-azacytidine, venetoclax is currently administered at a daily dose of 100 mg on day 1, 200 mg on day 2, and 400 mg on days 3 and beyond. Vidaza® (injectable 5-azacytidine) is administered at a dose of 75 mg / m in a 28-day cycle beginning on day 1 of venetoclax treatment. 2 It is administered intravenously or subcutaneously on days 1 to 7 of each cycle at a dosage of

[0208] In some embodiments, venetoclax is administered orally. In some embodiments, venetoclax is administered in tablet form. In some embodiments, venetoclax is administered daily. In some embodiments, venetoclax is administered at a dose of about 20 mg to about 400 mg, e.g., about 20 mg, about 50 mg, about 100 mg, about 200 mg, or about 400 mg. In some embodiments, venetoclax is administered at a dose of about 400 mg.

[0209] In some embodiments, 5-azacytidine and venetoclax are administered concurrently. In some embodiments, 5-azacytidine and venetoclax are administered sequentially. In some embodiments, when 5-azacytidine and venetoclax are administered sequentially, 5-azacytidine is administered first. In some embodiments, 5-azacytidine and venetoclax are administered as separate dosage forms, e.g., an injectable solution suitable for intravenous or subcutaneous use and / or a tablet or capsule for oral use. In some embodiments, 5-azacytidine and venetoclax are co-formulated as a single unit dosage form, e.g., an injectable solution suitable for intravenous or subcutaneous use, or a tablet or capsule for oral use.

[0210] Other specific dosing regimens with other Bcl2 family inhibitors can be determined by one of skill in the art, particularly with regard to the regimen prescribed for the therapeutic indication being treated by the combination therapy of the present disclosure. Combination Uses and Methods of the Present Disclosure The present disclosure provides a therapeutic combination for use in the treatment of cancer, particularly hematological malignancies, more preferably acute myeloid leukemia, comprising a BTN3A activating antibody, e.g., mAb1, a Bcl2 inhibitor such as a Bcl2 family inhibitor, e.g., venetoclax, as defined above, and optionally a hypomethylating agent, e.g., azacitidine.

[0211] The present disclosure also provides a method of treating cancer in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a BTN3A activating antibody, e.g., mAb1, in combination with a therapeutically effective amount of a Bcl2 family inhibitor, e.g., a Bcl2 inhibitor such as venetoclax, simultaneously, sequentially, or separately, and optionally in combination with a therapeutically effective amount of a hypomethylating agent, e.g., azacitidine or decitabine.

[0212] As used herein, the terms "treat," "treating," or "treatment" refer to one or more of the following: (1) inhibiting the disease; e.g., inhibiting the disease, condition, or disorder (i.e., arresting further progression of the symptoms and / or symptomology) in an individual experiencing or exhibiting the symptoms or symptomology of the disease, condition, or disorder; and (2) ameliorating the disease; e.g., ameliorating the disease, condition, or disorder (i.e., ameliorating the symptoms and / or symptomology) in an individual experiencing or exhibiting the symptoms or symptomology of the disease, condition, or disorder, e.g., reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease. In particular, with respect to the treatment of acute myeloid leukemia, the term "treatment" can refer to inhibiting the proliferation of AML blasts or reducing the number of AML blasts.

[0213] The antibodies of the present disclosure are BTN3A activating antibodies and can activate the cytolytic function, cytokine production and / or proliferation of Vy9V52 T cells, thereby being able to overcome the immunosuppressive mechanisms observed in cancer patients (see, inter alia, WO2012 / 080769, WO2012 / 080351 and WO2020 / 025703) and during chronic infections. The results of the present disclosure now show that the present combination further enhances synergistic and specific killing of AML blasts by Vy9V52 T cells in human PBMCs, highlighting the therapeutic importance of this combination, in particular, for the treatment of hematological malignancies.

[0214] As used herein, the terms "cancer," "hyperproliferative," and "neoplastic" refer to cells capable of autonomous growth, i.e., an abnormal state or pathology characterized by rapidly proliferating cell proliferation. Hyperproliferative and neoplastic disease states may be classified as pathological, i.e., characterizing or constituting a disease state, or non-pathological, i.e., deviating from the norm but not associated with a disease state. The terms are meant to include all types of cancerous growths or tumorigenic processes, metastatic tissues, or malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness.

[0215] Examples of cancer include, but are not limited to, hematological malignancies such as leukemia (e.g., acute myeloid leukemia (AML) or chronic lymphocytic leukemia (CLL), including chronic B-cell leukemia), lymphoma (e.g., small lymphocytic lymphoma (SLL)), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, or myeloma (e.g., multiple myeloma (MM)). In some embodiments, the hematological cancer is myelodysplastic syndrome (MDS) (e.g., a lower-risk MDS, e.g., very-low-risk MDS, low-risk MDS, or intermediate-risk MDS, or a higher-risk myelodysplastic syndrome, e.g., high-risk MDS or very-high-risk MDS).

[0216] In some embodiments, the subject in need of such treatment is a subject suffering from a hematological malignancy, e.g., acute myeloid leukemia, and the subject is ineligible for the use of intensive induction chemotherapy.

[0217] In some embodiments, the subject in need of such treatment is 75 years of age or older and / or has a co-morbidity.

[0218] In some embodiments, the subject in need of such treatment is a subject who is eligible for treatment with venetoclax in combination with a hypomethylating agent according to standard of care.

[0219] Each therapeutic agent of the combination therapy of the present disclosure (i.e., the BTN3A-activating antibody defined above, the Bcl2 inhibitor, and optionally, the hypomethylating agent) can be administered as separate pharmaceuticals administered sequentially in any order. The anti-BTN3A antibody, e.g., mAb1, the Bcl2 inhibitor such as a Bcl2 family inhibitor, e.g., venetoclax, and the hypomethylating agent, e.g., azacitidine, are typically formulated into separate compositions as described above.

[0220] Sequential administration (e.g., in separate pharmaceutical compositions) is particularly useful when the therapeutic agents of the combination therapy are in different dosage forms (e.g., one agent is a tablet or capsule and the other is a sterile liquid) and / or when the therapeutic agents of the combination therapy are administered on different dosing schedules, e.g., a chemotherapeutic agent that is administered at least daily and a biologic therapeutic agent that is administered less frequently, e.g., once per week, once every two weeks, or once every three weeks.

[0221] In some embodiments, the BTN3A activating antibody (e.g., mAb1) is administered before the first administration of a Bcl2 family inhibitor, e.g., a Bcl2 inhibitor (e.g., venetoclax), while in other embodiments, the BTN3A activating antibody (e.g., mAb1) is administered after administration of a Bcl2 family inhibitor, e.g., a Bcl2 inhibitor (e.g., venetoclax).

[0222] The selection of a dosing regimen (also referred to herein as an administration regimen) for the combination therapy of the present disclosure depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissues, or organs in the individual being treated. Preferably, the dosing regimen maximizes the amount of each therapeutic agent delivered to the patient, consistent with an acceptable level of side effects. Thus, the dosage and frequency of each biological and chemotherapeutic agent in the combination will depend, in part, on the specific therapeutic agent, the severity of the cancer being treated, and the characteristics of the patient. Guidance is available for selecting appropriate doses of antibodies, cytokines, and small molecules. See, for example, Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al. (2003) New Engl. J. Med. 348:601-608; Milgrom et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz et al. (2000) New See Engl. J. Med. 342:613-619; Ghosh et al. (2003) New Engl. J. Med. 348:24-32; Lipsky et al. (2000) New Engl. J. Med. 343:1594-1602; Physicians' Desk Reference 2003 (Physicians' Desk Reference, 57th Edition); Medical Economics Company; ISBN: 1563634457; 57th Edition (November 2002).The determination of an appropriate dosing regimen can be made by the clinician using, for example, parameters or factors known or suspected in the art to affect treatment, or predicted to affect treatment, and will depend, for example, on the patient's clinical history (e.g., previous therapies), the type and stage of the cancer being treated, and response biomarkers to one or more of the therapeutic agents of the combination therapy.

[0223] Any appropriate dosage range can be used as determined by the attending healthcare professional. Dosing regimens can be adjusted to obtain the optimum desired response (e.g., a therapeutic or prophylactic response). Dosage amounts are disclosed in the previous section regarding Bcl2 family inhibitors. In some embodiments of combination therapy, a suitable dosage range for a Bcl2 family inhibitor as defined herein, particularly venetoclax, can be, for example, 10 mg to 600 mg. In some embodiments, an exemplary dose is about 100 mg to 400 mg. In some embodiments, the dosing regimen includes providing a Bcl2 inhibitor, such as a Bcl2 family inhibitor, e.g., venetoclax, daily.

[0224] In some embodiments, at least one of the therapeutic agents of the combination therapy is administered using the same dosing regimen (dose, frequency of treatment, and duration of treatment) that would typically be employed to treat the same cancer when the therapeutic agent is used as a monotherapy.

[0225] For example, venetoclax is administered in the same dosing regimen recommended for treating patients with newly diagnosed acute myeloid leukemia in combination with a hypomethylating agent, such as azacitidine (see "Practical Dosing Considerations for Venetoclax," Cancernetwork.com, Oncology, Vol. 33, No. 9).

[0226] When a Bcl2 inhibitor, such as venetoclax, is administered in combination with a hypomethylating agent (e.g., azacitidine), the hypomethylating agent may be administered subcutaneously once daily at 75 mg / m² on days 1-5 and 8-9 of each cycle. In the event of lymphopenia, a maximum of 14 days of recovery between cycles is recommended, with growth factors administered to at least treat neutropenia (Jonas and Pollyea, 2019; Leukemia 33:2795-2804). The next cycle will be determined by the patient's response but will generally be similar to cycle 1.

[0227] In other embodiments, the patient is administered a lower total amount, e.g., a lower dose, less frequent administration, and / or a shorter duration of treatment, of at least one of the therapeutic agents of the combination therapy than when the therapeutic agent is used as a monotherapy.

[0228] With regard to the dosing regimen of BTN3A activating antibodies for use in the present therapeutic combination, any suitable dosage range can be used as determined by the attending medical practitioner.

[0229] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response). The antibodies of the present disclosure can be formulated to be present in a therapeutic mixture at about 1 to 200.0 milligrams. It will be understood that such doses can be administered at different intervals as determined by the oncologist / physician; for example, doses can be administered daily, twice weekly, weekly, biweekly, every three weeks, or monthly.

[0230] Typically, in certain embodiments, the BTN3A activating antibody, e.g., mAb1, is administered intravenously at doses containing 1 mg to 200 mg, e.g., 20 to 100 mg, typically every 21 days. In certain embodiments, suitable doses for intravenous administration of the activating BTN3A antibody, e.g., mAb1, can be selected from 1, 7, 10, 20, 50, 75, 100, 125, 150, 175, and 200 mg.

[0231] In certain embodiments, an activating BTN3A antibody (preferably mAb1, described below) for use in the combination methods of the present disclosure is administered intravenously in doses containing 7 mg to 200 mg in each dose, preferably with a second dose administered at least 15 days after the first dose, typically once every 21 days for 1 to 22 cycles.

[0232] In some embodiments, a Bcl-2 family inhibitor, for example, a Bcl-2 inhibitor such as venetoclax, is administered separately, sequentially, or simultaneously in combination, at a dose of about 10 mg to about 500 mg, for example, about 20 mg to about 400 mg, about 50 mg to about 350 mg, about 100 mg to about 300 mg, about 150 mg to about 250 mg, 50 mg to about 500 mg, about 100 mg to about 500 mg, about 150 mg to about 500 mg, about 200 mg to about 500 mg, about 250 mg to about 500 mg, or about 300 mg to about 500 mg, about 350 mg to about 500 mg, about 400 mg to about 500 mg, about 450 mg to about 500 mg, about 10 mg to about 400 mg, about 10 mg to about 350 mg, about 10 mg to 300 mg, about 10 mg to about 250 mg, about 10 mg to about 200 mg, about 10 mg to about 150 mg, about 10 mg to about 100 mg, about 10 mg to about 50 mg, about 50 mg to about 150 mg, about 150 mg to about 250 mg, about 250 mg to about 350 mg, or about 350 mg to about 400 mg. In some embodiments, the Bcl-2 inhibitor, e.g., venetoclax, is administered at a dose of about 20 mg, 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, or 500 mg. In some embodiments, the Bcl-2 inhibitor, e.g., venetoclax, is administered once daily. In some embodiments, the Bcl-2 family inhibitor, e.g., a Bcl-2 inhibitor, is administered orally.

[0233] In some embodiments, a Bcl-2 inhibitor, such as a Bcl-2 family inhibitor, e.g., venetoclax, is orally administered at a dose of about 350 mg to about 450 mg (e.g., about 400 mg) once daily, e.g., on each day of a 21-day cycle. In some embodiments, the dose of the Bcl-2 inhibitor is escalated over four days in the first cycle to achieve a dose of about 400 mg per day. For example, the doses on days 1, 2, 3, and 4 and thereafter of cycle 1 are about 100 mg, about 200 mg, about 300 mg, and about 400 mg, respectively.

[0234] In some embodiments, a Bcl-2 inhibitor such as a Bcl-2 family inhibitor, e.g., venetoclax, is administered in dose escalation cycles, e.g., for about 5 weeks, followed by a fixed dose, e.g., for at least about 24 months. In some embodiments, the Bcl-2 inhibitor is administered once daily, e.g., for about one week, at a dose of about 10 mg to about 30 mg (e.g., about 20 mg), followed by a dose of about 40 mg to about 60 mg (e.g., about 50 mg), once daily, e.g., for about one week, then a dose of about 80 mg to about 120 mg (e.g., about 100 mg), once daily, e.g., for about one week, then a dose of about 150 mg to about 250 mg (e.g., about 200 mg), once daily, e.g., for about one week, then a dose of about 350 mg to about 450 mg (e.g., about 400 mg), once daily, e.g., for about one week, then a fixed dose, e.g., a dose of about 350 mg to about 450 mg (e.g., about 400 mg), once daily, e.g., for at least about 24 months.

[0235] In certain embodiments, a Bcl2 family inhibitor, e.g., a Bcl-2 inhibitor such as venetoclax, is administered once daily, e.g., in a unit dose of 10 mg to 600 mg, optionally in combination with azacitidine or decitabine, for at least 5 to 9 days, followed by a recovery period of at least 14 days.

[0236] In certain embodiments, a Bcl2 family inhibitor, e.g., a Bcl-2 inhibitor such as venetoclax, and a hypomethylating agent, e.g., azacitidine, are initially administered for at least one cycle, followed by a recovery period of at least 10-14 days, and the BTN3A activating antibody is administered, e.g., after the recovery period of at least 10-14 days, or together with cycle 2 administration of the Bcl2 inhibitor and hypomethylating agent.

[0237] In another specific embodiment, the first administration of a Bcl2 family inhibitor, e.g., a Bcl-2 inhibitor such as venetoclax, and a hypomethylating agent, e.g., azacitidine, occurs after the first cycle of treatment with a BTN3A activating antibody, e.g., mAb1, preferably 21 days after the first administration of the BTN3A activating antibody.

[0238] In certain embodiments, an activating BTN3A antibody (preferably mAb1, described below) for use in the methods of the present disclosure is administered intravenously at doses containing 7 mg to 200 mg per dose, preferably with the second dose administered at least 15 days after the first dose, typically once every 21 days, for 1 to 22 cycles; a Bcl-2 inhibitor, such as venetoclax, is administered, for example, at a unit dose of 10 mg to 600 mg once daily, optionally in combination with azacitidine or decitabine, for at least 5 to 9 days, followed by a recovery period of at least 14 days, wherein venetoclax and a hypomethylating agent, such as azacitidine, are administered initially for at least one cycle, followed by a recovery period of at least 10 to 14 days, and the BTN3A activating antibody is administered, for example, at least 10 to 14 days after the recovery period.

[0239] In certain embodiments, the combination therapy disclosed herein (typically the mAb1 described above and a Bcl2 family inhibitor, e.g., a Bcl-2 inhibitor, particularly venetoclax) may be administered in combination with other anti-neoplastic agents.

[0240] In other particular embodiments, the combination therapy disclosed herein (typically the mAb1 described above and said Bcl2 family inhibitor, e.g., a Bcl-2 inhibitor, particularly venetoclax) may be administered in combination with a cell therapy (particularly a γδ T cell therapy).

[0241] Accordingly, the present disclosure relates to a combination as defined herein for in vivo use to enhance tumor cell efficacy in γδ T cell therapy in a subject in need thereof, typically a subject suffering from cancer, wherein a BTN3A activating antibody and a Bcl2 family inhibitor, e.g., a Bcl-2 inhibitor, may be administered to the subject concurrently, simultaneously, in parallel or sequentially.

[0242] As used herein, the term γδ T cell therapy refers to a therapy comprising administering at least an effective amount of γδ T cells to a subject in need thereof. Such γδ T cells can be allogeneic or autologous. In certain embodiments, γδ T cells can be genetically engineered by deletion or knockout or insertion or knockin of specific genes. In certain embodiments, the γδ T cells include γδ T cells expressing a chimeric antigen receptor. γδ T cells can be ex vivo expanded and / or purified. Alternatively, γδ T cells can also be included in a cell composition containing other blood cells, e.g., other cells of the immune system. For references regarding γδ T cell therapy, see Pauza CD. et al., Front Immunol. 2018 Jun. 8;9:1305.doi:10.3389; Saudemont A. et al., Front Immunol. 2018 Feb. 5;9:153.doi:10.3389. Accordingly, the present disclosure provides a method of treating a subject having tumor cells, e.g., hematological tumor cells, suffering from cancer, including solid tumors or hematological malignancies, particularly leukemias such as acute myeloid leukemia, comprising: (i) administering to the subject an effective amount of an anti-BTN3A activating antibody disclosed herein, typically mAb1, mAb2, mAb3, mAb4, or mAb5, in combination with an effective amount of a Bcl2 inhibitor, e.g., venetoclax, and optionally, in combination with a hypomethylating agent, e.g., azacitidine; and (ii) administering to the subject an effective amount of a γδ T cell composition; The method further relates to a method in which the combination of the effective amount of the anti-BTN3A antibody and the effective amount of the Bcl2 inhibitor can enhance anti-tumor cytolysis of the tumor cells mediated by the γδ T cell composition.

[0243] The present disclosure having thus been fully described, will now be further described by the following examples, which are merely illustrative and are not meant to be further limiting.

[0244] [Table 4] TIFF2025532758000007.tif197149 TIFF2025532758000008.tif210149 TIFF2025532758000009.tif210149 TIFF2025532758000010.tif210149 TIFF2025532758000011.tif210149 TIFF2025532758000012.tif210149 TIFF2025532758000013.tif207149 TIFF2025532758000014.tif170149 TIFF2025532758000015.tif197149 TIFF2025532758000016.tif199149 TIFF2025532758000017.tif172149 [Example]

[0245] 1. Methods and Assays of the Disclosure Methods for characterizing BTN3A activating antibodies for use according to the present disclosure 1.1 Binding affinity assay: multi-cycle kinetic assay (SPR) For anti-BTN3A antibodies, multi-cycle kinetic analysis can be performed using a Biacore T200 (serial number 1909913) instrument (Uppsala, Sweden) running Biacore T200 evaluation software V2.0.1.

[0246] Purified antibodies were diluted to a concentration of 2 μg / ml in 2% BSA / PBS. At the start of each cycle, each antibody was captured onto Protein A at a density of approximately 146.5 RU (RL) (theoretical value for an RMax of approximately 50 RU). After capture, the surface was allowed to stabilize before injecting BTN3A1 antigen (Sino Biological cat. no. 15973-H08H). BTN3A1 was titrated over a 2-fold dilution range from 25 to 0.78 nM in 0.1% BSA / HBS-P+ (running buffer). The association phase was monitored for 400 s, and the dissociation phase was monitored for 35 min (2100 s). Kinetic data were obtained using a flow rate of 50 μl / min to minimize potential mass transfer effects. The Protein A surface was regenerated at the end of each cycle using two injections of 10 mM glycine-HCl pH 1.5. To confirm the stability of the surface and analyte throughout the kinetic cycle, two blank (no BTN3A1) and single-concentration analyte replicates are performed for each antibody tested. The signal from reference channel Fc1 is subtracted from the signals from Fc2, Fc3, and Fc4 to correct for differences in nonspecific binding to the reference surface. Additionally, blank runs are subtracted for each Fc to correct for antigen-independent signal fluctuations, e.g., drift. Sensorgrams are fitted using a one-to-one binding mathematical model with a global RMax parameter and no bulk signal (constant RI = 0 RU).

[0247] 1.2 Flow cytometric binding assay of human PBMCs BTN3A-activating antibodies for use according to the present disclosure can also be characterized for binding to human PBMCs isolated from the blood of healthy donors. PBMCs are isolated from buffy coats using Lymphoprep (Axis-shield, Dundee, UK) density centrifugation. PBMCs are then frozen and stored at -80°C or in liquid nitrogen until needed.

[0248] 1×10 6 100 μl of cells / ml of cells are transferred to each well of a new U-bottom 96-well plate, the plate is then centrifuged, and the supernatant is discarded.

[0249] Serial dilutions of antibody from 0.001 μg / ml to 150 μg / ml are prepared in PBS containing 2 mM EDTA. Human PBMCs are resuspended in 50 μl of the prepared diluted test antibody dose titration series.

[0250] After 30 minutes of incubation at 4°C in the dark, the plates are centrifuged and washed twice with 150 μl / well of PBS+2 mM EDTA, after which the cells are resuspended in 50 μl of a mix consisting of a 1 / 100 dilution of goat anti-human antibody (PE-labeled) and a 1 / 500 dilution of Live / Dead neat IR in PBS+2 mM EDTA.

[0251] After 15 minutes of incubation in the dark at 4°C, the plates are centrifuged and washed once with 150 μl / well of PBS + 2 mM EDTA, after which the cells are resuspended in 200 μl of PBS + 2 mM EDTA. Cells are analyzed on a BD LSR Fortessa Cytometer. Data are analyzed using FlowJo software (Version 10, FlowJo, LLC, Ashland, USA).

[0252] The same protocol can be performed for cynomolgus monkey PBMCs and Daudi-Burkitt lymphoma cell lines.

[0253] 1.3 In vitro functional efficacy: γδ-T cell degranulation assay The assay consists of measuring the activating or inhibitory effect of anti-BTN3A antibodies on γδ T cell degranulation in a Daudi-Burkitt lymphoma cell line (Harly et al., 2012, Blood 120, 11, 2269-2279). γδ T cells are expanded from PBMCs of healthy donors by culturing them with zoledronic acid (1 μM) and IL2 (200 UI / mL) for 11–13 days. IL2 is added on days 5 and 8, and every two days thereafter. The percentage of γδ T cells is determined at the beginning of culture and assessed throughout the culture period by flow cytometry until it reaches at least 80%. Frozen or fresh γδ T cells were then used against the Daudi cell line in a degranulation assay (effector:target (E:T) ratio of 1:1), in which cells were co-cultured for 4 hours at 37°C in the presence of 10 μg / mL of 7.2 and / or 20.1 humanized variants and / or their chimeric versions. For γδ T cell degranulation, activation with PMA (20 ng / mL) plus ionomycin (1 μg / mL) served as a positive control, and medium alone served as a negative control. At the end of the 4-hour co-incubation, cells were analyzed by flow cytometry to assess the percentage of γδ T cells positive for CD107a (lysosomal-associated membrane protein-1 (LAMP-1)) and CD107b (LAMP-2). Because CD107 is recruited to the cell surface following activation-induced granule exocytosis, measurement of surface CD107 is a sensitive marker for identifying recently degranulated cytolytic T cells.

[0254] The same protocol can be performed using AML blasts isolated from patients as target cells instead of Daudi cells.

[0255] 1.4 In vitro functional efficacy: activation of Vγ9Vδ2 T cells in PBMCs The assay consists of measuring the activating effect of BTN3A activating antibodies on Vγ9Vδ2 T cells in PBMCs. Human PBMCs are isolated by Ficoll density gradient centrifugation of peripheral blood (EDTA-buffy coat or heparinized whole blood). When using whole blood, RBCs are removed using 1× RBC lysis buffer (eBioscience) for 10 minutes at room temperature and then washed with PBS + 1% FBS.

[0256] PBMCs or RBC-depleted cells are cultured at 1.5–3 × 10 cells / mL in RPMI 1640 containing 10% FBS and 1% penicillin / streptomycin at 37°C, 5% CO2, in a 200 μL volume in round-bottom 96-well plates with increasing concentrations of BTN3A activating antibodies (dose range 0.00001–100 μg / mL). Activation status is monitored after 2 days of culture by flow cytometry analysis of activation marker surface expression. Cells are washed with PBS + 2% FBS and 2 mM EDTA (FACS buffer). Cells were centrifuged at 1800 rpm for 5 min and then incubated with 10 μL of FcR blocking reagent (Miltenyi Biotec) for 10 min at room temperature (RT). Afterwards, 30–50 μL of the appropriate antibody mix prepared in FACS buffer containing at least fluorescently conjugated anti-CD3, anti-Vγ9 or Vδ2 TCR, and anti-CD69 antibodies was added. To exclude dead cells from the analysis, a viability marker (LIVE / DEAD Fixable Dead Cell Stain) was added in all experiments. Cells were incubated for 30 min at 4°C, washed twice with FACS buffer, and then fixed with Cytofix fixation buffer (BD Bioscience) for flow cytometry analysis. Data were analyzed using flowjo V-10.6 software. Activated Vγ9Vδ2 T cells are defined as CD3+Vδ2+ (or Vγ9+ or Vδ2+Vγ9+)CD69+.

[0257] 1.5 In vitro Vγ9Vδ2 T cell expansion Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation of peripheral blood obtained from the Etablissement Français du Sang (EFS) Provence-Alpes-Côte d'Azur (France). To expand Vγ9Vδ2 T cells, 300 × 10 6 PBMCs were cultured at 1.5 × 10 in the presence of rHuIL-2 (200 UI / mL) and aminobisphosphonate (zoledronate, 1 μM). 6 Cells were resuspended at 1 × 10 cells / ml in a 75 cm flask in RPMI 1640 supplemented with 10% FBS and 1% sodium pyruvate for 10–14 days. From day 5 onwards, rHuIL-2 was refreshed every 2 or 3 days and cells were cultured at 1 × 10 cells / ml. 6 At the end of the expansion phase, the purity of Vy9V52 T cells was assessed by flow cytometry, and once the number of Vy9V52 T cells reached 80 percent viable cells, the cells were frozen in CryoStor CS10 for later use.

[0258] 1.6 Mouse model The mouse strain selected was the highly immunodeficient NSG mouse, which lacks mature T cells, B cells, and natural killer (NK) cells and is also defective in multiple cytokine signaling pathways, making it ideal for human cell transplantation. Six- to eight-week-old female NSG mice were housed in disposable standard cages on ventilated racks at the TrGET platform facility (Cancer Research Center of Marseille, France). Mice were housed under sterile conditions with unlimited access to sterilized food and water and maintained on a 12-h light / dark cycle under temperature and humidity control. Cages contained enriched environments with bedding.

[0259] NSG mice were intravenously (iv) injected with luciferase (luc2)-expressing MOLM-14 (CVCL_7916) cells (0.2 × 106 per mouse) via the tail vein on day 0 in a volume of 100 μl. Bioluminescence analysis was performed on day 0 using a PhotonIMAGER (Biospace Labs) after the addition of endotoxin-free luciferin (30 mg / kg). Mice were randomized into homogenous groups of 6–8 mice based on the intensity of the bioluminescence signal (Table). 3 × 106 human in vitro-expanded Vγ9Vδ2 T cells and rHuIL-15 / IL-15Rα-Fc complexes were injected i.v. on days 1, 8, 15, and 23. ICT01 or hIgG1S was injected i.v. on days 1, 5, 8, 11, 15, 18, 23, and 25.

[0260] The rHuIL-15 / IL-15Rα-Fc complex was pre-complexed for 30 min at room temperature (RT) (0.2 μg rHuIL-15 + 1.2 μg IL-15Rα-Fc per mouse) and mixed with Vγ9Vδ2 T cells and ICT01 or its isotype control (hIgG1S) before injection. The final volume of each injection was 200 μl.

[0261] Venetoclax was administered to mice by oral gavage (og) on ​​days 1-4, then 5 days per week for a total of 3 weeks. 5-azacytidine was administered by intraperitoneal (ip) injection on days 1-4, concurrently with venetoclax treatment. Venetoclax and 5-azacytidine were administered 4-6 hours after Vγ9Vδ2 T cell transfer.

[0262] Bioluminescence signals from MOLM-14 cells were measured on days 0, 7, 14, 21, and 28 after cell injection to track tumor growth. Mice were monitored daily for signs of disease (significant weight loss, ruffled fur, hunched back, weakness, and decreased mobility), and injected animals showing signs of distress were sacrificed. Survival curves were estimated by the Kaplan-Meier method and compared using the log-rank test.

[0263] 2. Results 2.1 ICT01-mediated activation of Vγ9Vδ2 T cells increases resistance to inhibitors of Bcl-2 family members The effect of ICT01 on Vγ9Vδ2 T cell survival upon treatment with Bcl-2 family member inhibitors was assessed in peripheral blood mononuclear cells (HD-PBMCs) from healthy donors using flow cytometry analysis of the apoptotic markers caspase 3 / 7 and dead cell markers.

[0264] Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation of peripheral blood (EDTA-buffy coat) from healthy donors (HD, n = 4-6) and cultured in PBMC medium (RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 1 mM sodium pyruvate) and treated with increasing concentrations of venetoclax (0-0.1 μM), 5-azacytidine (0-0.4 μM), or their combination, or with ICT01 or its isotype control (hIgG1S) used at 1 μg / mL in the presence or absence of ABT-737 (0-1.875 μM), navitoclax (0-1.875 μM), or MIK665 (0-1.875 μM). Cells were harvested on day 2, centrifuged at 1800 rpm for 5 min, and then incubated with 25 μl of FcR blocking reagent (Miltenyi Biotec) for 10 min at room temperature (RT). Then, 25 μL of the following antibody mix prepared in PBS + 2% FBS + 2 mM EDTA (FACS buffer) was added: anti-CD4-BUV395, CD56-BV421, Vd2-PE, CD3-PE-CF594, and CD8-A700 antibodies for immune subset identification; anti-CD25-BV650 and CD69-APC antibodies for monitoring the activation status of Vγ9Vδ2 T cells; and a viability marker (LIVE / DEAD™ Fixable Dead Cell Stain) to distinguish live from dead cells. Cells were incubated for 20 min at room temperature (RT) and washed twice with FACS buffer. Caspase 3 / 7 Green detection reagent (ThermoFisher) was added to determine apoptosis, and cells were incubated at RT for 30 min before flow cytometry analysis.

[0265] While 5-azacytidine had no effect on Vγ9Vδ2 T cell survival, treatment with venetoclax induced apoptosis and death of Vγ9Vδ2 T cells in a concentration-dependent manner (a ∼29% reduction in viable cells with 0.01 μM venetoclax and a maximum ∼49% reduction with 0.1 μM venetoclax compared to the absence of venetoclax) (Fig. 1a, b). In contrast, co-treatment with ICT01 significantly reduced venetoclax-induced cell death (a ∼11% reduction in viability with 0.01 μM venetoclax and a ∼30% reduction with 0.1 μM venetoclax compared to the absence of venetoclax) (Fig. 1c, d). As expected, ICT01 induced specific activation of Vy9V52 T cells, as indicated by increased expression of CD69 and CD25 compared to hIgG1S after 2 days of treatment (Fig. 1e, data not shown), indicating that activation with ICT01 partially protects Vy9V52 T cells from venetoclax-induced cell death.

[0266] As described for venetoclax, treatment with other Bcl-2 family member inhibitors induces apoptosis and death of Vγ9Vδ2 T cells in a concentration-dependent manner, resulting in EC 50 The EC values ​​for ABT-737, navitoclax, and MIK665 were 0.24 μM, 0.16 μM, and 0.24 μM, respectively (Fig. 2a). A significant decrease in Vγ9Vδ2 T cell death induced by Bcl-2 family member inhibitors was observed when PBMCs were simultaneously treated with ICT01. The EC values ​​for Vγ9Vδ2 T cell death were 0.24 μM, 0.16 μM, and 0.24 μM, respectively (Fig. 2b). 50 increased from 0.23 μM in the presence of isotype control to 0.46 μM in the presence of ICT01 for ABT-737 (Fig. 2b), from 0.16 μM in the presence of isotype control to 0.20 μM in the presence of ICT01 for navitoclax (Fig. 2c), and from 0.24 μM in the presence of isotype control to 0.78 μM in the presence of ICT01 for MIK665 (Fig. 2d).

[0267] In a similar experiment, freshly isolated PBMCs were stimulated with ICT01 or its isotype control, hIgG1S, for 1 day and then treated with venetoclax for 2 days (Figure 3a). At the end of the incubation period, the number of viable Vγ9Vδ2 T cells was assessed by flow cytometry. Results showed that venetoclax-induced cell death of Vγ9Vδ2 T cells was significantly reduced by activation with ICT01 (as confirmed by increased surface expression of CD25 (Figure 3b)) (Figures c and d).

[0268] Taken together, these results indicate that in vitro activation of Vy9V52 T cells with ICT01 can protect them from cell death induced by venetoclax and other inhibitors of Bcl-2 family members, providing an advantage over monotherapy as large numbers of Vy9V52 T cells (important for anti-cancer immunity) can act in concert with venetoclax (or other Bcl-2 family member inhibitors) to kill AML blasts.

[0269] 2.2 Venetoclax and 5-azacytidine do not affect ICT01-induced Vγ9Vδ2 T cell activation and proliferation ICT01 has been shown to induce activation and proliferation of resting Vy9V52 T cells. To further evaluate the effects of venetoclax and 5-azacytidine on Vy9V52 T cells, ICT01-induced phenotypic activation, IFNγ production, and proliferation were measured in PBMCs from healthy donors cultured with or without venetoclax, 5-azacytidine, or their combination.

[0270] ICT01-induced phenotypic activation of Vγ9Vδ2 T cells was indicated by upregulation of CD25. Human PBMCs (HD, n=6) from healthy donors were cultured in PBMC medium (RPMI 1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin, and 1 mM sodium pyruvate) with ICT01 or its isotype control (hIgG1S) at 1 μg / mL in the presence or absence of increasing concentrations of venetoclax (0-0.1 μM), 5-azacytidine (0-0.4 μM), or their combination. Cells were harvested on day 4, centrifuged at 1800 rpm for 5 min, and then incubated with 25 μl of FcR blocking reagent (Miltenyi Biotec) for 10 min at room temperature (RT). Then, 25 μL of the following antibody mix prepared in PBS + 2% FBS + 2 mM EDTA (FACS buffer) was added: anti-CD4-BV650, CD56-FITC, Vd2-PE, CD3-PE-CF594, and CD8-A700 antibodies for immune subset identification; anti-CD25-BV650 antibody was added to monitor activation status; and a viability marker (Live / Dead™ Fixable Dead Cell Stain) was added to distinguish live from dead cells. Cells were incubated for 20 min at 4°C, washed twice with FACS buffer, and then analyzed by flow cytometry.

[0271] 5-Azacytidine and venetoclax had no effect on ICT01-induced upregulation of CD25 on Vγ9Vδ2 T cells and therefore had no effect on the phenotypic activation of Vγ9Vδ2 T cells (Figure a).

[0272] In a similar experiment, freshly isolated PBMCs from healthy donors were treated with increasing concentrations of venetoclax (0–0.1 μM), 5-azacytidine (0–0.4 μM), or their combination for 1 day, followed by stimulation with ICT01 or its isotype control, hIgG1S, for 2 days. Again, activation of Vγ9Vδ2 T cells by ICT01 was unaffected by venetoclax and 5-azacytidine treatment, as indicated by similar induction of CD25 expression in all conditions tested (Figure b).

[0273] The function of Vγ9Vδ2 T cells is tightly regulated by activating receptors (NKG2D and DNAM-1) and inhibitory receptors (PD-1, NKG2A, BTLA, and TIM3). To evaluate the effect of venetoclax and 5-azacytidine treatment on the expression of these receptors by Vγ9Vδ2 T cells, frozen human PBMCs (HD, n = 6) from healthy donors were thawed and treated with ICT01 or its isotype control (hIgG1S) at 1 μg / mL in PBMC medium (RPMI 1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin, 1 mM sodium pyruvate, and 50 IU / mL IL-2) in the presence or absence of increasing concentrations of venetoclax (0–0.1 μM), 5-azacytidine (0–0.4 μM), or their combination. Cells were harvested on day 5, centrifuged at 1800 rpm for 5 min, and then incubated with 25 μl of FcR blocking reagent (Miltenyi Biotec) for 10 min at RT. Afterwards, 25 μl of the following antibody mix prepared in PBS + 2% FBS + 2 mM EDTA (FACS buffer) was added: anti-CD4-BUV395, Vδ2-PE, CD3-PE-CF594, and CD8-A700 antibodies for immune subset identification; anti-BTLA-BV421, PD1-BV650, DNAM-1-BV785, TIM3-BB515, NKG2A-PE-Vio770, and NKG2D-APC antibodies for inhibitory and activating receptor detection; and a viability marker (Live / Dead™ Fixable Dead Cell Stain) to distinguish live from dead cells. The cells were incubated at 4°C for 20 min and washed twice with FACS buffer before flow cytometry analysis.

[0274] ICT01 induced an increase in the expression of all analyzed activating and inhibitory receptors on Vy9V52 T cells by day 5 of culture, which was unaffected by treatment with venetoclax and 5-azacytidine (data not shown).

[0275] To evaluate the effects of venetoclax and 5-azacytidine on cytokine production, freshly isolated human PBMCs (HD, n=6) from healthy donors were cultured in PBMC medium (RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 1 mM sodium pyruvate) and treated with ICT01 or its isotype control (hIgG1S) at 1 μg / mL in the presence or absence of increasing concentrations of venetoclax (0-0.1 μM), 5-azacytidine (0-0.4 μM), or their combination. Half of the cells were further treated with 50 IU / mL IL-2. Golgi Stop was added 6–8 h after co-culture. Cells were harvested 16 h later and centrifuged at 1800 rpm for 5 min. Then, cells were incubated with 25 μL of FcR blocking reagent (Miltenyi Biotec) for 10 min at room temperature (RT). Then, 25 μL of the following antibody mix prepared in PBS + 2% FBS + 2 mM EDTA (FACS buffer) was added: anti-CD4-BV650, Vγ9-FITC, CD3-PE-CF594, CD56-PE-Vio770, and CD8-AF700 antibodies for immune subset identification, and a viability marker (Live / Dead™ Fixable Dead Cell Stain) was added to distinguish live from dead cells. After 20 min, 100 μL of CytoFix solution (BD Bioscience) was added, and the cells were incubated for 20 min at 4 °C. 100 μL of Perm / Wash (BD Bioscience) was added, and the cells were centrifuged and washed once with 200 μL Perm / Wash. After washing, the cells were stained with anti-IFNγ-APC antibody for 30 minutes at 4°C, washed twice with Perm / Wash, and analyzed by flow cytometry.

[0276] Treatment with venetoclax or 5-azacytidine alone had no effect on ICT01-induced, IL2-independent IFNγ production by Vγ9Vδ2 T cells (data not shown). When activated with ICT01 alone, treatment with both venetoclax and 5-azacytidine in combination reduced IFNγ production by Vγ9Vδ2 T cells by 30.4–25.2%, but this was not observed in the presence of IL2 (89.1% without treatment and 91.7% with the combination of venetoclax and 5-azacytidine; data not shown).

[0277] Finally, freshly isolated human PBMCs (HD, n = 6) from healthy donors were labeled with CellTrace Violet stain (CTV, Invitrogen) to determine cell proliferation upon treatment with ICT01 or its isotype control (hIgG1S) at 1 μg / mL in the presence or absence of increasing concentrations of venetoclax (0–0.1 μM), 5-azacytidine (0–0.4 μM), or their combination in PBMC culture medium (RPMI 1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin, and 1 mM sodium pyruvate). Half of the cells were additionally treated with 50 IU / mL IL-2. Cells were harvested on day 4, centrifuged at 1800 rpm for 5 min, and then incubated with 25 μl of FcR blocking reagent (Miltenyi Biotec) for 10 min at RT. Afterwards, 25 μL of the following antibody mix prepared in PBS + 2% FBS + 2 mM EDTA (FACS buffer) was added: anti-CD4-BV650, CD56-FITC, Vδ2-PE, CD3-PE-CF594, and CD8-A700 antibodies for immune subset identification, and a viability marker (Live / Dead™ Fixable Dead Cell Stain) to distinguish live from dead cells. Cells were incubated for 20 min at 4°C, washed twice with FACS buffer, and then analyzed by flow cytometry.

[0278] Venetoclax increased ICT01-induced Vγ9Vδ2 T cell proliferation by approximately 10%, which was not observed with 5-azacytidine alone or in combination (Figure c). ICT01- and IL2-induced Vγ9Vδ2 T cell proliferation was unaffected by venetoclax and 5-azacytidine alone or in combination (Figure d).

[0279] Taken together, the data indicate that venetoclax and 5-azacytidine treatment, either as single agents or in combination, does not interfere with ICT01-induced Vy9V52 T cell activation or proliferation. Thus, the combined treatment appears to protect Vy9V52 T cells from venetoclax-induced cell death and induce proliferation, activation, and potentially an increase in the numbers of cytotoxic Vy9V52 T cells in patients.

[0280] 2.3 Combination treatment of Vγ9Vδ2 T cells with venetoclax, HMA, and BTN3A activating antibodies increases killing of AML The effect of venetoclax and 5-azacytidine on anti-BTN3A antibody-induced Vy9V52 T cell killing of AML cell lines was assessed by measuring the relative number of viable target cells in HD-PBMC-AML cell cocultures using flow cytometry.

[0281] To first assess the sensitivity of the KG1a AML cell line to venetoclax and 5-azacytidine treatment in vitro, cells were treated for 2 days with increasing concentrations of venetoclax (0–5 μM), 5-azacytidine (0–10 μM), or their combination in cell culture medium (α-MEM + Glutamax supplemented with 20% fetal bovine serum (FBS) and 1 mM sodium pyruvate). On day 2, cells were centrifuged at 1800 rpm for 5 minutes, and the supernatant was discarded. Cells were lysed with Cell Titer Glo reagent (Promega), and ATP levels were monitored using luminescence as a measure of viable cells.

[0282] KG1a AML cells were isolated from EC50 The mean viability of venetoclax at 5 μM was 1.82 μM and that of 5-azacytidine at 4.72 μM, with a mean viability of 20.2% for venetoclax at 5 μM and 47.2% for 5-azacytidine at 10 μM. The combination of venetoclax at 5 μM and 5-azacytidine at 10 μM resulted in a mean viability of 0.9% for KG1a AML cells, demonstrating a synergistic effect of venetoclax and 5-azacytidine treatment (Figure a).

[0283] KG1a AML cells were then stained with CellTrace CFSE Proliferation dye (ThermoFisher) for specific detection by flow cytometry, counted, and cocultured with thawed HD-PBMCs. They were treated with the anti-BTN3A mAb ICT01 or m20.1 or their respective isotype controls (hIgG1S or mIgG1) in PBMC medium (RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and 1 mM sodium pyruvate). On day 1 of culture, increasing concentrations of venetoclax (0-0.4 μM), 5-azacytidine (0-0.4 μM), or their combination were added. Cells were harvested on day 3 of coculture, centrifuged at 1800 rpm for 5 min, and then incubated with 25 μl of FcR blocking reagent (Miltenyi Biotec) for 10 min at room temperature (RT). Then, 25 μL of the following antibody mix prepared in PBS + 2% FBS + 2 mM EDTA (FACS buffer) was added: anti-CD56-BV421, CD19-BV421, CD14-BV785, Tγδ-PE, and CD3-AF700 antibodies for immune subset identification, and a viability marker (Live / Dead™ Fixable Dead Cell Stain) for distinguishing live from dead cells. Cells were incubated for 20 min at room temperature and washed once with FACS buffer and once with Annexin V staining buffer. To assess apoptosis and determine relative cell numbers, Annexin V-APC probe mixed with CountBright Absolute Counting Beads (Invitrogen) was added, followed by a 15-minute incubation at RT before flow cytometry analysis.

[0284] In this assay, KG1a cells were highly resistant to killing by BTN3A-activated antibody-mediated Vγ9Vδ2 T cells (although there was considerable variability within PBMC donors, the number of viable KG1a cells was reduced by an average of approximately 20% in the ICT01 condition compared to the hIgG1S condition) (Figure b). In the hIgG1S condition, treatment with 5-azacytidine had no effect on KG1a survival (Figure d). In contrast, in the hIgG1S treatment condition, venetoclax reduced the relative number of KG1a cells by approximately 28% at the highest concentration and by 44% in combination with 5-azacytidine. When activated with ICT01, treatment with venetoclax and 5-azacytidine alone had the effect of slightly increasing the killing activity of Vγ9Vδ2 T cells compared to the hIgG1S condition. However, the combination of venetoclax and 5-azacytidine significantly reduced the relative number of viable KG1a cells by approximately 46.5% compared to the hIgG1S condition (p<0.05) and by approximately 32.9% compared to no venetoclax and 5-azacytidine treatment, demonstrating a synergistic effect of the combination treatment.

[0285] Similar results were obtained with mAb 20.1, a BTN3A-activating antibody (Figure 6). In the mIgG1 condition, treatment with 5-azacytidine had no effect on KG1a cell viability, whereas venetoclax at 0.1 μM reduced the relative number of KG1a cells by 41%, and in combination with 0.4 μM 5-azacytidine, by 57%. Furthermore, the combination of 20.1 mAb with venetoclax and 5-azacytidine significantly reduced the relative number of viable KG1a cells compared to m20.1 alone or venetoclax and 5-azacytidine with the isotype control, resulting in an approximately 67% reduction in viable KG1a cells compared to the control condition (Figure 6).

[0286] Collectively, these data demonstrate that activation of PBMCs from healthy donors with ICT01 increases Vy9V52 T cell survival against venetoclax-induced cell death, and that venetoclax and 5-azacytidine treatment does not affect Vy9V52 T cell activation or proliferation in HD-derived PBMCs in vitro. Furthermore, combined treatment of BTN3A antibody-activated Vy9V52 T cells in HD-PBMCs with venetoclax and 5-azacytidine significantly reduced survival of the Vy9V52 T cell-resistant AML cell line KG1a.

[0287] 2.4 Combination treatment with venetoclax, HMA, and ICT01 increases Vγ9Vδ2 T cell-mediated killing of Burkitt lymphoma and chronic B cell leukemia cell lines The effect of venetoclax and 5-azacytidine on BTN3A-activating antibody-induced Vy9V52 T cell killing of Burkitt lymphoma and chronic B-cell leukemia cell lines was assessed by measuring the relative number of viable target cells in HD-PBMC-target cell cocultures using flow cytometry.

[0288] In the presence of the BTN3A-activating antibody ICT01, 48-hour coculture of Raji (Burkitt's lymphoma) or JVM-2 (chronic B-cell leukemia) with HD-PBMCs induced a reduction in viable cells of approximately 22% and 16%, respectively, with high variability within PBMC donors (Figure 7a, b). In the hIgG1S-treated condition, the combination of venetoclax and 5-azacytidine at the indicated concentrations reduced the relative numbers of Raji and JVM2 targets by approximately 37% and 32%, respectively, compared to the hIgG1S-only condition (control). Importantly, the combination of BTN3A-activating antibodies such as ICT01 with venetoclax and 5-azacytidine for killing Burkitt's lymphoma and chronic B-cell leukemia cell lines was shown to be beneficial, as after 48 hours of co-culture, the combination of ICT01, venetoclax, and 5-azacytidine still reduced the relative number of viable target cells, and reduced the number of viable target cells by approximately 55% and approximately 46% compared to control conditions.

[0289] Collectively, these data suggest that the combination of BTN3A-activating antibody-activated Vy9V52 T cells in HD-PBMCs with venetoclax and 5-azacytidine treatment significantly reduces the survival of several hematological cancer cell lines and may be useful in the treatment of hematological malignancies.

[0290] 2.5 Combined treatment of ICT01-activated Vγ9Vδ2 T cells with Bcl-2 family member inhibitors increases killing of AML The effects of three Bcl-2 family member inhibitors, ABT-737, navitoclax, and MIK665, on ICT01-induced Vγ9Vδ2 T cell killing of AML cell lines were assessed by measuring the relative number of viable target cells in HD-PBMC-AML cell cocultures using flow cytometry.

[0291] Assays using ABT-737 or navitoclax used KG1a cells. However, this cell line does not express MCL-1 and is therefore completely resistant to MIK665 treatment. Therefore, MOLM14 cells, which express MCL-1, were used in killing assays to evaluate the activity of ICT01 in combination with MIK665.

[0292] Killing of KG1a co-cultured with HD-PBMCs in the presence of ICT01, ABT-737 or their combination (Fig. 8a). In this experiment, ICT01 induced approximately 21% KG1a cell death compared to control conditions (similar to previous results (Figure 5b)). Treatment with 0.25 μM and 0.5 μM ABT-737 alone induced approximately 34% and 54% KG1a cell death, respectively. When cocultures of KG1a and HD-PBMCs were treated with the combination of ICT01 and ABT-737, this combination proved beneficial for killing AML cell lines, reducing the number of viable KG1a cells by approximately 53% and 71% compared to controls (for ABT-737 used at 0.25 μM and 0.5 μM, respectively) (Figure 8a).

[0293] Killing of KG1a co-cultured with HD-PBMCs in the presence of ICT01, navitoclax or their combination (Fig. 8b). Treatment with navitoclax alone at 0.25 μM and 0.5 μM induced approximately 51% and 71% KG1a cell death, respectively. When cocultures of KG1a and HD-PBMCs were treated with the combination of ICT01 and navitoclax, the combination proved beneficial in killing AML cell lines, reducing the number of viable KG1a cells by approximately 65% ​​and 88% compared to controls (navitoclax used at 0.25 μM and 0.5 μM, respectively) (Figure 8b).

[0294] Killing of MOLM14 cells co-cultured with HD-PBMCs in the presence of ICT01, MIK665, or their combination (Fig. 8c).In this experiment, ICT01 induced approximately 56% MOLM14 cell death compared to control conditions. Treatment with 0.025 μM and 0.05 μM MIK665 alone induced approximately 52% and 72% KG1a cell death, respectively. When cocultures of MOLM14 and HD-PBMCs were treated with the combination of ICT01 and MIK665, the combination proved beneficial in killing AML cell lines, reducing the number of viable MOLM14 cells by approximately 85% and 93% compared to controls (MIK665 used at 0.025 μM and 0.05 μM, respectively) (Figure 8c).

[0295] Collectively, these data demonstrate that the combination of ICT01-activated Vγ9Vδ2 T cells in HD-PBMCs with Bcl-2 family member inhibitor treatment significantly reduced the survival of AML cell lines, confirming that ICT01 can be used in combination with Bcl-2 family member inhibitors to treat hematological malignancies.

[0296] 2.6 ICT01 in combination with venetoclax and 5-azacytidine significantly prolongs survival of MOLM-14-implanted NSG mice The in vivo efficacy of ICT01, in combination with venetoclax and 5-azacytidine, against the AraC-resistant human AML-derived cell line MOLM-14 was evaluated in a xenograft model using NSG mice implanted with the human tumor cell line and adoptively transferred with human Vy9V52 T cells. The aim of this study was to evaluate the effect of ICT01-activated human Vy9V52 T cells on tumor growth and mouse survival in combination with repeated iv injections and Ven / Aza.

[0297] NSG mice were injected with MOLM-14 on day 0 and randomized into homogenous groups based on the intensity of the bioluminescent signal. The different experimental groups are summarized in the table.

[0298] [Table 5]

[0299] As shown in the table, there was no basal control of MOLM-14 in vivo growth by human Vy9V52 T cells when co-injected with an irrelevant control isotype antibody (hIgG1S) (Group 1 vs. Group 3). In contrast, as indicated by the lower bioluminescence signal and median survival times (Table 6 and Figure), human Vy9V52 T cells significantly delayed tumor growth when administered with the BTN3A-activating mAb ICT01 (median survival times of 24 and 28 days for Groups 3 and 4, respectively) or in mice treated with venetoclax and 5-azacytidine (median survival times of 24 and 29 days for Groups 1 and 2, respectively). Combining venetoclax and 5-azacytidine treatment with transfer of non-activated human Vy9V52 T cells extended the median survival of mice to 32.5 days (groups 2 and 3 vs. group 5), indicating that venetoclax and 5-azacytidine treatment renders cells more susceptible to killing by human Vy9V52 T cells (Table).

[0300] Importantly, treatment of mice with ICT01, human Vy9V52 T cells, venetoclax and 5-azacytidine dramatically improved median survival to 42.5 days (group 6) (Figure 9).

[0301] These data confirm that the combination of a BTN3A-activating antibody, such as ICT01, with a Bcl2 family inhibitor, such as venetoclax, and 5-azacytidine, may be a promising approach for the treatment of patients with AML.

[0302] [Table 6]

[0303] [Table 7]

Claims

1. A BTN3A activating antibody for use in treating cancer in a subject in need thereof, wherein a therapeutically effective amount of said BTN3A activating antibody is administered to said subject in combination, simultaneously, sequentially or separately, with a therapeutically effective amount of a Bcl2 inhibitor compound, and optionally further in combination with a hypomethylating agent.

2. 2. The BTN3A activating antibody for use according to claim 1, wherein the Bcl2 inhibitor is venetoclax.

3. 3. The BTN3A activating antibody for use according to claim 1 or 2, wherein the BTN3A activating antibody comprises: (a) a variable heavy chain (VH) polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1; and (b) a variable light chain (VL) polypeptide comprising an amino acid sequence that is at least about 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:

2.

4. The BTN3A activating antibody for use according to any one of claims 1 to 3, wherein the anti-BTN3A antibody is an antibody comprising a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:

6.

5. 5. The BTN3A activating antibody for use according to any one of claims 1 to 4, wherein the BTN3A activating antibody is administered in combination with venetoclax, and further in combination with azacitidine or decitabine, either simultaneously, sequentially or separately.

6. 6. The BTN3A activating antibody for use according to any one of claims 1 to 5, wherein the cancer is a hematological malignancy, such as acute myeloid leukemia.

7. 7. The BTN3A activating antibody for use according to any one of claims 1 to 6, wherein the cancer is acute myeloid leukemia, the BTN3A activating antibody is an antibody comprising a heavy chain of SEQ ID NO: 4 and a light chain of SEQ ID NO: 6, and the BTN3A activating antibody is administered in combination with venetoclax, and further in combination with azacitidine or decitabine, either simultaneously, sequentially or separately.

8. The BTN3A activating antibody for use according to any one of claims 1 to 7, wherein the subject is not eligible for intensive chemotherapy.

9. 9. The BTN3A activating antibody for use according to any one of claims 1 to 8, wherein the BTN3A activating antibody is administered once every three weeks or once every four weeks.

10. The BTN3A activating antibody for use according to any one of claims 1 to 9, wherein said BTN3A activating antibody is administered intravenously.

11. 11. The BTN3A activating antibody for use according to any one of claims 1 to 10, wherein the BTN3A activating antibody is administered at a unit dose of about 7 to about 200 mg, e.g., 75 mg, for example, once every 21 days for 1 to 22 cycles.

12. The BTN3A activating antibody for use according to any one of claims 1 to 11, wherein venetoclax is administered orally once daily.

13. The BTN3A activating antibody for use according to any one of claims 1 to 12, wherein venetoclax is orally administered in a unit dose of about 50 mg to about 500 mg.

14. A BTN3A activating antibody for use according to any one of claims 1 to 13, wherein the hypomethylating agent comprises azacytidine.

15. The hypomethylating agent is at about 50 mg / m ２ ~Approx. 100mg / m ２ 15. The BTN3A activating antibody for use according to any one of claims 1 to 14, administered at a dose of

Images