Combination of BTN3A-activating antibodies, BCL2 inhibitors and demethylating agents for treatment of cancer

Abstract

The present invention relates to a therapeutic combination of a BTN3A-activating antibody, a Bcl-2 family inhibitor and a demethylating agent, which is useful, inter alia, in the treatment of cancer, in particular hematological malignancies. The present disclosure more particularly relates to the combined use of BTN3A-activating antibodies that activate the cytolytic function of V [gamma] 9V [delta] 2T cells, WiNtural that selectively inhibits the Bcl2 receptor, and a demethylating agent, such as azacitidine, to synergistically and specifically promote the anti-cancer activity of V [gamma] 9V [delta] 2T cells.

Description

Technical Field

[0001] Disclosed below is a therapeutic combination of a BTN3A activating antibody, a Bcl-2 inhibitor, and a demethylating agent, which can be particularly used for treating cancer, especially hematological malignancies. The present disclosure more particularly relates to the combined use of a BTN3A activating antibody that activates the cytolytic function of Vγ9Vδ2 T cells, Venetoclax that selectively inhibits the Bcl2 receptor, and a demethylating agent (such as Azacytidine) to synergistically and specifically promote the anti-cancer activity of Vγ9Vδ2 T cells. Background Art

[0002] To date, various therapeutic and vaccine strategies for treating cancer have been proposed, which rely on mobilizing the patient's immune response, and more particularly T lymphocytes, to combat malignant cells. Several immunomodulatory antibodies against CTLA-4, PD-1, and PD-L1 have been approved by multiple regulatory agencies worldwide for clinical use. Although these drugs represent a major advance in cancer therapy, the medical needs of a large patient population of cancer patients who are unresponsive to currently available treatments remain unmet.

[0003] Gamma delta (γδ) T cells are an unconventional subset of T cells with characteristics of both innate and adaptive immune responses and play an important role in immune surveillance against malignancies and infections (Holtmeier W et al., eds. Chem Immunol Allergy [Internet]. Basel: KARGER; 2005 [cited August 19, 2022]. pp. 151-183. Available from: https: / / www.karger.com / Article / FullText / 86659). In the blood of healthy adults, Vγ9Vδ2 T cells constitute the majority (50 to 90%) of circulating γδ T cells (Kabelitz D et al. Cell Mol Immunol. September 2020; 17(9):925-939) and account for 1 to 5% of total blood lymphocytes (Pauza CD et al. GammaDelta T Cell Therapy for Cancer: It Is Good to be Local. Front Immunol [Internet]. 2018 [cited November 19, 2018]). Different from conventional αβ T cells, which represent the major T cell subset in circulating T cells and recognize MHC-restricted antigens, γδ T cells are activated in a non-MHC-restricted manner. More specifically, activation of Vγ9Vδ2 T cells is triggered by the intracellular accumulation of phosphoantigens (pAg) that are overproduced in response to viral and bacterial infections, metabolic stress, or genetic dysregulation during carcinogenesis. Vγ9Vδ2 T cell activation in these pathophysiological situations induces a wide range of functional activities, including the production of cytokines and chemokines, the 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 ofγδT Cells by BTN3A and Other BTN / BTNL in Tumor Immunity. Front Immunol [Internet]. 2018 [cited August 6, 2018]).Due to their potent anti-tumor activity (Pauza CD et al., see above) and the association between their infiltration of malignant tissues and favorable prognosis (Gentles AJ et al. Nat Med. August 2015; 21(8):938-945; Tosolini M et al. OncoImmunology. March 4, 2017; 6(3):e1284723. PMID: 28405516), Vγ9Vδ2 T cells represent an attractive target for cancer immunotherapy.

[0004] In recent years, various Vγ9Vδ2 T cell-based immuno-oncology therapies have been explored in multiple tumors, activating Vγ9Vδ2 T cells in vivo using amino bisphosphonates (ABPs) (such as zoledronate) or synthetic phosphoantigens (i.e., BrHPP) in combination with IL-2, or adoptively transferring autologous or allogeneic Vγ9Vδ2 T cells to patients after in vitro / ex vivo expansion (Kabelitz D et al. 2020, see above). 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, see above). Especially in leukemia, only low responses have been observed in clinical trials using ABPs to stimulate Vγ9Vδ2 T cells in vivo. In contrast, the expansion and transfer of Vγ9Vδ2 T cells have a response rate of over 50% (Künkele KP et al. Cells. March 30, 2020; 9(4):829; Barros M de S et al. Front Immunol. September 22, 2021; 12:729085), enhancing the utility of Vγ9Vδ2 T cells especially in AML therapy, and their use has been further investigated in numerous clinical trials (Saura-Esteller J et al. Front Immunol. June 16, 2022; 13:915837). Despite apparent success, the in vitro / ex vivo expansion and transfer of Vγ9Vδ2 T cells are laborious, indicating the need for novel strategies to enhance the clinical benefits of anti-tumor immunity mediated by Vγ9Vδ2 T cells using in vivo activation and expansion.

[0005] Patent publications WO2012080351A1, WO2012080769A1, WO20200257031A1, and WO2020136218 mention various antibodies against BTN3A that can activate the cytokine production, proliferation, and cytolytic functions of Vγ9Vδ2 T cells.

[0006] Previous studies have shown that Venetoclax and the demethylating agent 5-azacytidine have direct anti-leukemia activity. Additionally, 5-azacytidine has been shown to improve the recognition of cancer cells by immune effector cells via induction of stress ligand expression (Gang AO et al. Blood Cancer J. March 2014;4(3):e197-e197; Lee JB et al. Blood. July 22, 2021;138(3):234-245), and Venetoclax has been described to enhance T cell- and NK cell-mediated cytotoxicity against AML blasts (Lee et al. 2021, supra; Wu et al. Int Immunopharmacol. March 2022;104:108497). However, Venetoclax treatment results in lymphopenia in AML patients, which may be clinically disadvantageous given that Vγ9Vδ2 T cells typically account for <5% of total T cells in adult peripheral blood.

[0007] The inventors have shown that activation of Vγ9Vδ2 T cells by ICT01 partially protects them from Venetoclax-induced cell death, and the combination of ICT01 with Venetoclax and 5-azacytidine significantly improves AML cell killing.

[0008] The present invention relies on the combined use of Venetoclax and a demethylating agent with ICT01, which combination shows superior AML killing compared to single agents. By triggering the anti-cancer activity of Vγ9Vδ2 T cells, thereby protecting them from cell death and apoptosis of chemotherapy-induced AML cells. Summary of the Invention

[0010] Specific embodiments

[0011] 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 and a therapeutically effective amount of a Bcl2 family inhibitor compound (such as a Bcl2 inhibitor) are administered to the subject simultaneously, sequentially, or separately, optionally further in combination with a demethylating agent.

[0012] E2. The BTN3A-activating antibody for use according to embodiment E1, wherein the Bcl2 inhibitor is Venetoclax.

[0013] E3. The BTN3A-activating antibody for use according to embodiment E1 or E2, wherein the BTN3A-activating antibody binds to human BTN3A with a K D , preferably with a K D of 10 nM or lower as measured by surface plasmon resonance.

[0014] E4. A BTN3A activating antibody for use as in any one of embodiments E1 to E3, wherein the BTN3A activating antibody induces activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with BTN3A-expressing cells, wherein, as measured in a degranulation assay, the EC 50 is less than 5 μg / ml, preferably 1 μg / ml or less.

[0015] E5. A BTN3A activating antibody for use as in any one of embodiments E1 to E4, wherein the BTN3A activating antibody:

[0016] - 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 or SEQ ID NO:3;

[0017] - comprises HCDR1-3 of SEQ ID NO:12-14 and LCDR1-3 of SEQ ID NO:15-17;

[0018] - comprises HCDR1-3 of SEQ ID NO:18-20 and LCDR1-3 of SEQ ID NO:21-23; or

[0019] - competes for binding with an antibody selected from: mAb 20.1, which is produced by a hybridoma deposited under CNCM accession number I-4401; mAb 7.2, which is produced by a hybridoma deposited under CNCM accession number I-4402; and an antibody having a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

[0020] E6. A BTN3A activating antibody for use as in any one of embodiments E1 to E5, wherein the BTN3A antibody comprises a mutated or chemically modified IgG1 constant region, wherein the mutated or chemically modified IgG1 constant region does not confer or reduces binding to Fcγ receptors as compared to a corresponding antibody having a wild-type IgG1 isotype constant region.

[0021] E7. A BTN3A activating antibody for use as in any one of embodiments E1 to E6, wherein the mutated IgG1 constant region is the IgG1 triple mutant L247F, L248E and P350S.

[0022] E8. A BTN3A activating antibody for use as in any one of embodiments E1 to E7, 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.

[0023] E9. A BTN3A activating antibody for use as in any one of embodiments E1 to E8, wherein the BTN3A activating antibody is administered concomitantly, sequentially or separately in combination with venetoclax, and further in combination with azacitidine or decitabine.

[0024] E10. A BTN3A activating antibody for use as in any one of embodiments E1 to E9, wherein the cancer is a hematological malignancy.

[0025] E11. An anti-BTN3A activating antibody for use as in any one of embodiments E1 to E10, wherein the cancer is acute myeloid leukemia.

[0026] E12. A BTN3A activating antibody for use as in any one of embodiments E1 to E11, wherein the cancer is acute myeloid leukemia, and wherein 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 concomitantly, sequentially or separately in combination with venetoclax, and further in combination with azacitidine or decitabine.

[0027] E13. A BTN3A activating antibody for use as in any one of embodiments E1 to E12, wherein the subject is not suitable for intensive chemotherapy.

[0028] E14. A BTN3A activating antibody for use as in any one of embodiments E1 to E13, wherein the subject is older than 75 years.

[0029] E15. A BTN3A activating antibody for use as in any one of embodiments E1 to E14, wherein the BTN3A activating antibody is administered once every three weeks or once every four weeks.

[0030] E16. A BTN3A activating antibody for use as in any one of embodiments E1 to E15, wherein the BTN3A activating antibody is administered intravenously.

[0031] E17. A BTN3A activating antibody for use as in any one of embodiments E1 to E16, wherein the BTN3A activating antibody is administered in a unit dose of about 7 to about 200 mg, such as about 75 mg, for example once every 21 days for 1 to 22 cycles.

[0032] A BTN3A activating antibody for use as in any one of embodiments E1 to E17, wherein Venetoclax is administered orally once daily.

[0033] E19. A BTN3A activating antibody for use as in any one of embodiments E1 to E18, wherein Venetoclax is administered orally in unit doses of from about 50 mg to about 500 mg.

[0034] E20. A BTN3A activating antibody for use as in any one of embodiments E1 to E19, wherein azacitidine or decitabine is administered as a demethylating agent.

[0035] E21. A BTN3A activating antibody for use as in any one of embodiments E1 to E20, wherein azacitidine is administered as a demethylating agent.

[0036] E22. A BTN3A activating antibody for use as in any one of embodiments E1 to E21, wherein the demethylating agent is administered at a dose of from about 50 mg / m 2 to about 100 mg / m 2 .

[0037] E23. A BTN3A activating antibody for use as in any one of embodiments E1 to E22, wherein the demethylating agent is administered at a dose of about 75 mg / m 2 .

[0038] E24. A BTN3A activating antibody for use as in any one of embodiments E1 to E23, wherein the demethylating agent is administered once daily.

[0039] E25. A BTN3A activating antibody for use as in any one of embodiments E1 to E24, wherein the demethylating agent is administered for 5 to 7 consecutive days.

[0040] E26. A BTN3A activating antibody for use as in any one of embodiments E1 to E25, wherein the demethylating agent is administered

[0041] (a) for 7 consecutive days from day 1 to day 7 of a 28-day cycle,

[0042] (b) for 5 consecutive days from day 1 to day 5 of a 28-day cycle, followed by a 2-day break, then for 2 consecutive days from day 8 to day 9 of the 28-day cycle, or

[0043] (c) for 6 consecutive days from day 1 to day 6 of a 28-day cycle, followed by a 1-day break, then optionally administered once on day 8 of the 28-day cycle.

[0044] A BTN3A activating antibody for use as in any one of embodiments E1 to E26, wherein the demethylating agent is administered subcutaneously or intravenously.

[0045] E28. A BTN3A activating antibody for use as in any one of embodiments E1 to E27, wherein venetoclax is administered for at least one cycle first, followed by a recovery period, and the BTN3A activating antibody is administered, for example, at least 10 to 14 days after the recovery period or together with the second cycle of the administration of venetoclax.

[0046] E29. A BTN3A activating antibody for use as in any one of embodiments E1 to E27, wherein the first administration of venetoclax occurs after the first cycle of treatment with a BTN2A activating antibody, preferably 21 days after the first administration of the BTN3A activating antibody.

[0047] E30. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a BTN3A activating antibody and a therapeutically effective amount of a Bcl2 family inhibitor compound (such as a Bcl2 inhibitor), optionally further in combination with a demethylating agent, simultaneously, sequentially or separately.

[0048] E31. The method of embodiment E30, wherein the Bcl2 inhibitor is venetoclax.

[0049] E32. The method of embodiment E30 or E31, wherein, as measured by surface plasmon resonance, the BTN3A activating antibody binds to human BTN3A with a K D , preferably with a K D of 10 nM or lower.

[0050] E33. The method of any one of embodiments E30 to E32, wherein the BTN3A activating antibody induces activation of γδ T cells, typically Vγ9Vδ2 T cells, in co-culture with BTN3A-expressing cells, wherein the EC 50 is below 5 μg / ml, preferably 1 μg / ml or lower, as measured in a degranulation assay.

[0051] E34. The method of any one of embodiments E30 to E33, wherein the BTN3A activating antibody:

[0052] - comprising (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 or SEQ ID NO:3;

[0053] - comprising HCDR1-3 of SEQ ID NO:12-14 and LCDR1-3 of SEQ ID NO:15-17;

[0054] - comprising HCDR1-3 of SEQ ID NO:18-20 and LCDR1-3 of SEQ ID NO:21-23; or

[0055] - competing for binding with an antibody selected from: mAb 20.1, which is produced by a hybridoma deposited under CNCM accession number I-4401; mAb 7.2, which is produced by a hybridoma deposited under CNCM accession number I-4402; and an antibody having a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

[0056] E35. The method according to any one of embodiments E30 to E34, wherein the BTN3A antibody comprises a mutated or chemically modified IgG1 constant region, and wherein the mutated or chemically modified IgG1 constant region does not confer or reduces binding to Fcγ receptors as compared to a corresponding antibody having a wild-type IgG1 isotype constant region.

[0057] E36. The method according to any one of embodiments E30 to E35, wherein the mutated IgG1 constant region is the IgG1 triple mutant L247F L248E and P350S.

[0058] E37. The method according to any one of embodiments E30 to E36, 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.

[0059] E38. The method according to any one of embodiments E30 to E37, wherein the BTN3A activating antibody is administered simultaneously, sequentially or separately in combination with venetoclax, and further in combination with azacitidine or decitabine.

[0060] E39. The method according to any one of embodiments E30 to E38, wherein the cancer is a hematological malignancy.

[0061] E40. A method according to any one of embodiments E30 to E39, wherein the cancer is acute myeloid leukemia.

[0062] E41. A method according to any one of embodiments E30 to E40, wherein the cancer is acute myeloid leukemia, and wherein the BTN3A activating antibody is an antibody comprising the heavy chain of SEQ ID NO: 4 and the light chain of SEQ ID NO: 6, and the BTN3A activating antibody is administered in combination with venetoclax simultaneously, sequentially, or separately, and further in combination with azacitidine or decitabine.

[0063] E42. A method according to any one of embodiments E30 to E41, wherein the subject is not suitable for intensive chemotherapy.

[0064] E43. A method according to any one of embodiments E30 to E42, wherein the subject is older than 75 years.

[0065] E44. A method according to any one of embodiments E30 to E43, wherein the BTN3A activating antibody is administered once every three weeks or once every four weeks.

[0066] E45. A method according to any one of embodiments E30 to E44, wherein the BTN3A activating antibody is administered intravenously.

[0067] E46. A method according to any one of embodiments E30 to E45, wherein the BTN3A activating antibody is administered in a unit dose of about 7 to about 200 mg, such as about 75 mg, for example, once every 21 days for 1 to 22 cycles.

[0068] E47. A method according to any one of embodiments E30 to E46, wherein venetoclax is administered orally once a day.

[0069] E48. A method according to any one of embodiments E30 to E47, wherein venetoclax is administered orally in a unit dose of about 50 mg to about 500 mg.

[0070] E49. A method according to any one of embodiments E30 to E48, wherein azacitidine or decitabine is administered as a demethylating agent.

[0071] E50. A method according to any one of embodiments E30 to E49, wherein azacitidine is administered as a demethylating agent.

[0072] E51. A method according to any one of embodiments E30 to E50, wherein the demethylating agent is administered at a dose of about 50 mg / m 2 to about 100 mg / m 2 of the dose.

[0073] E52. A method according to any one of embodiments E30 to E51, wherein the demethylating agent is administered at a dose of about 75 mg / m 2 .

[0074] E53. A method according to any one of embodiments E30 to E52, wherein the demethylating agent is administered once daily.

[0075] E54. A method according to any one of embodiments E30 to E53, wherein the demethylating agent is administered for 5 to 7 consecutive days.

[0076] E55. A method according to any one of embodiments E30 to E54, wherein the administration of the demethylating agent is carried out

[0077] (a) for seven consecutive days from day 1 to day 7 of a 28-day cycle,

[0078] (b) for five consecutive days from day 1 to day 5 of a 28-day cycle, followed by a two-day break, and then for two consecutive days from day 8 to day 9 of the 28-day cycle, or

[0079] (c) for six consecutive days from day 1 to day 6 of a 28-day cycle, followed by a one-day break, and then optionally administered once on day 8 of the 28-day cycle.

[0080] E56. A method according to any one of embodiments E30 to E55, wherein the demethylating agent is administered subcutaneously or intravenously.

[0081] E57. A method according to any one of embodiments E30 to E56, wherein Venetoclax is administered first for at least one cycle, followed by a recovery period, and the BTN3A activating antibody is administered, for example, at least 10 to 14 days after the recovery period or together with the second cycle of the administration of Venetoclax.

[0082] E58. A method according to any one of embodiments E30 to E56, wherein the first administration of Venetoclax occurs after the first cycle of treatment with the BTN2A activating antibody, preferably 21 days after the first administration of the BTN3A activating antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1: Activation of Vγ9Vδ2 T cells with ICT01 protects them from Venetoclax-induced cell death. A) Mean ± SEM frequencies of live (black), apoptotic (light gray), or dead (dark gray) Vγ9Vδ2 T cells in HD-PBMCs 48 hours after treatment with Venetoclax (left) or 5-azacytidine (right). N = 4 HD. B) Mean frequencies of live Vγ9Vδ2 T cells in HD-PBMCs 48 hours after treatment with Venetoclax, 5-azacytidine, or combination. N = 4 HD. C) Mean ± SEM frequencies of live (black), apoptotic (light gray), or dead (dark gray) Vγ9Vδ2 T cells in HD-PBMCs 48 hours after treatment with Venetoclax in the presence of ICT01 (right) or its isotype control (hIgG1S, left). N = 4 HD. D) Mean ± SEM frequencies of live Vγ9Vδ2 T cells in HD-PBMCs 48 hours after treatment with Venetoclax in the presence of ICT01 (black) or its isotype control (hIgG1S, light gray), normalized to the no-Venetoclax condition. N = 4 HD, each point represents a healthy donor. ***p < 0.005, two-way ANOVA. E) Mean ± SEM median fluorescence intensity (MFI) of CD69 in Vγ9Vδ2 T cells in HD-PBMCs treated with ICT01 (black) or its isotype control (hIgG1S, light gray) for 48 hours. N = 5 HD, each point represents a healthy donor.

[0084] Figure 2 : Activation of Vγ9Vδ2 T cells with ICT01 protects them from cell death induced by Bcl-2 family member inhibitors (Navitoclax, ABT-737, and MIK665). A) Heatmap showing mean frequencies of live Vγ9Vδ2 T cells in HD-PBMCs assessed by flow cytometry 48 hours after treatment with increasing concentrations of the indicated Bcl-2 family member inhibitors. For ABT-737, N = 3 HD, and for Navitoclax and MIK665, N = 5. B-D) Frequencies (mean ± SEM) of live Vγ9Vδ2 T cells in HD-PBMCs assessed by flow cytometry 48 hours after treatment with the indicated concentrations of ABT-737 (N = 3 HD) (B), Navitoclax (N = 5 HD) (C), or MIK665 (N = 5 HD) (D) in the presence of 1 μg / mL ICT01 (black) or its isotype control hIgG1S (light gray). Data were normalized to no treatment with ABT-737, Navitoclax, or MIK665, and each point represents a healthy donor. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA.

[0085] Figure 3 : Pretreatment with ICT01-mediated activation reduces the Venetoclax sensitivity of Vγ9Vδ2 T cells. A) Schematic icon of the treatment sequence. B) Mean ± SEM frequency of CD25+Vγ9Vδ2 T cells in HD-PBMC on day 3 of treatment with ICT01 (black) or its isotype control (hIgG1S, light gray) and Venetoclax. N = 6 HD. C) Mean ± SEM relative number of viable Vγ9Vδ2 T cells and D) Mean ± SEM % of viable Vγ9Vδ2 T cells relative to the condition without Venetoclax on day 3 of treatment with ICT01 (black) or its isotype control (hIgG1S, light gray) and Venetoclax. N = 5 HD. Each point represents a healthy donor. *p < 0.05, two-way ANOVA.

[0086] Figure 4 : Phenotypic activation and proliferation of Vγ9Vδ2 T cells in HD-PBMC stimulated with ICT01 are mainly not affected by treatment with Venetoclax and 5-azacytidine: A) Schematic icon of the treatment sequence (left) and mean ± SEM frequency of CD25-expressing Vγ9Vδ2 T cells in HD-PBMC on day 4 of treatment with Venetoclax, 5-azacytidine, or combination in the presence of ICT01 (black) or its isotype control (hIgG1S, light gray) (right). N = 6 HD, each point represents a healthy donor. B) Schematic icon of the treatment sequence (left) and mean ± SEM frequency of CD25-expressing Vγ9Vδ2 T cells in HD-PBMC on day 3 of treatment with Venetoclax, 5-azacytidine, or combination followed by treatment with ICT01 (black) or its isotype control (hIgG1S, light gray) (right). N = 6 HD, each point represents a healthy donor. C) and D) Mean ± SEM frequency of Cell Trace Violet (CTV)-dim Vγ9Vδ2 T cells in HD-PBMC on day 4 of treatment with Venetoclax, 5-azacytidine, or combination and ICT01 (black) or its isotype control (hIgG1S, light gray) in the absence (C) or presence of IL2 (D). N = 6 HD, each point represents a healthy donor, *p < 0.05, one-way ANOVA for RN.

[0087] Figure 5: Treatment with Vγ9Vδ2 T cells activated sequentially with ICT01, followed by treatment with venetoclax and 5-azacytidine, significantly reduced the number of cells in the resistant AML cell line. A) Sensitivity of the KG1a cell line to venetoclax, 5-azacytidine, or the combination. Shown is the mean luminescence signal relative to the untreated condition reflecting viable KG1a AML cells after 48 hours of treatment with venetoclax, 5-azacytidine, or the combination. N = 2 experiments. B) Mean ± SEM relative number of viable KG1a AML cells monitored by flow cytometry after co-culture for 3 days with HD-PBMC (E:T ratio 25:1) treated with ICT01 (black) or its isotype control (hIgG1S, light gray) at 0.1 or 1 μg / mL. N = 6, each point represents a healthy donor. C) Schematic illustration of the treatment protocol for HD-PBMC to kill KG1a AML cells. D) Relative number of viable KG1a AML cells (mean ± SEM) (left) and frequency relative to viable KG1a AML cells without venetoclax or 5-azacytidine treatment (mean ± SEM) (right) after co-culture with HD-PBMC for 3 days in the presence of ICT01 (black) or its isotype control (hIgG1S, light gray) and treatment with venetoclax, 5-azacytidine, or the combination. N = 6, each point represents a healthy donor, *p < 0.05, Wilcoxon test.

[0088] Figure 6 : Treatment with anti-BTN3A m20.1 and venetoclax and 5-azacytidine significantly improved the killing of the AML cell line KG1a mediated by Vγ9Vδ2 T cells. KG1a cells were co-cultured with HD-PBMC at a ratio of 5:1 in the presence of m20.1 or isotype control (0.1 μg / mL). After 6 hours, the indicated concentrations of venetoclax, 5-azacytidine, or the combination were added to the culture. The relative number of viable KG1a was monitored by flow cytometry after 48 hours. Shown is the relative number of viable KG1a AML cells (mean ± SEM) after co-culture with HD-PBMC for 2 days in the presence of m20.1 (black) or its isotype control mIgG1 (light gray) and treatment with venetoclax, 5-azacytidine, or the combination. N = 6, each point represents a healthy donor, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, two-way ANOVA.

[0089] Figure 7: Treatment with ICT01-activated Vγ9Vδ2 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 ratio of 5:1 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 combination were added to the cultures. The relative number of viable Raji (A) or JVM2 (B) was monitored by flow cytometry after 48 hours. The figure shows the relative number of viable Raji (A) or JVM2 (B) (mean ± SEM) after co-culture with HD-PBMCs for 2 days 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 point represents a healthy donor, *p < 0.05, **p < 0.01, two-way ANOVA.

[0090] Figure 8 : Treatment with ICT01-activated Vγ9Vδ2 T cells, followed by treatment with inhibitors of Bcl-2 family members, significantly reduced the number of viable AML cell lines. KG1a or MOLM14 AML cell lines were co-cultured with HD-PBMCs at a ratio of 5:1 in the presence of ICT01 or isotype control (0.1 μg / mL). After 6 hours, the indicated concentrations of ABT-737, navitoclax, or MIK665 were added to the cultures. A) The relative number of viable KG1a cells (mean ± SEM) after co-culture with HD-PBMCs for 2 days in the presence of ICT01 (black) or its isotype control hIgG1S (light gray) and treatment with the indicated concentration of ABT-737. B) The relative number of viable KG1a cells (mean ± SEM) after co-culture with HD-PBMCs for 2 days in the presence of ICT01 (black) or its isotype control hIgG1S (light gray) and treatment with the indicated concentration of navitoclax. C) The relative number of viable MOLM14 cells (mean ± SEM) after co-culture with HD-PBMCs for 2 days in the presence of ICT01 (black) or its isotype control hIgG1S (light gray) and treatment with the indicated concentration of MIK665. N = 6 HD, each point represents a healthy donor, *p < 0.05, two-way ANOVA.

[0091] Figure 9: ICT01 in combination with Venetoclax and 5-Azacytidine significantly prolongs the survival of NSG mice engrafted with MOLM-14: The Kaplan-Meier survival curves of each treatment group show the improved efficacy of ICT01 in combination 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

[0093] Definitions

[0094] To make the present disclosure more readily understandable, certain terms are first defined. Additional definitions are set forth throughout the detailed description.

[0095] As used herein, the terms "polypeptide", "protein", or "peptide" refer to any chain of amino acid residues, regardless of its length or post-translational modifications such as glycosylation.

[0096] As used herein, the term "BTN3A" has its general meaning in the art. In certain embodiments, it refers to a human BTN3A polypeptide, including BTN3A1 of SEQ ID NO:24, BTN3A2 of SEQ ID NO:25, or BTN3A3 of SEQ ID NO:26.

[0097] As used herein, the term "antibody" refers to an immunoglobulin molecule and the immunologically active portion of an immunoglobulin molecule, i.e., a molecule that contains an antigen-binding site that immunospecifically binds an antigen. The terms "antibody" or "immunoglobulin" have the same meaning and will be used interchangeably in the present disclosure. Thus, the term antibody encompasses not only whole antibody molecules, but also antibody fragments and variants (including derivatives) of antibodies. As used herein, the term "antibody" also includes bispecific or multispecific molecules. An antibody can be derivatized or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand of a receptor), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. In fact, an antibody can be derivatized or linked to more than one other functional molecule to produce 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 produce a bispecific molecule, an antibody of the present disclosure can be functionally linked (e.g., by chemical conjugation, gene fusion, non-covalent binding, or otherwise) to one or more other binding molecules (such as another antibody, antibody fragment, peptide, or binding mimetic), thereby producing a bispecific molecule. Additionally, for embodiments in which the bispecific molecule is multispecific, in addition to the first and second target epitopes, the molecule can further include a third binding specificity.

[0098] In the natural antibodies of rodents and primates, the 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, λ (lambda) and κ (kappa). 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 different sequence domains. In a typical IgG antibody, the light chain includes two domains: a variable domain (VL) and a constant domain (CL). The heavy chain includes 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 the binding recognition and specificity for antigens. The constant regions of the light chain (CL) and the heavy chain (CH) confer important biological properties such as antibody chain association, secretion, transplacental mobility, complement binding, and binding to Fc receptors (FcR).

[0099] The Fv fragment is the N-terminal portion of the Fab fragment of an immunoglobulin and consists of the variable portions of one light chain and one heavy chain. The specificity of an antibody resides in the structural complementarity between the antibody binding site and the antigenic determinant. The antibody binding site is composed of residues mainly from the hypervariable regions or complementarity-determining regions (CDRs). Occasionally, residues from non-hypervariable regions or framework regions (FRs) can participate in the antibody binding site or affect the overall domain structure and thus the binding site. The complementarity-determining region or CDR refers to the amino acid sequences that together define the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light chain and heavy chain of an immunoglobulin each have three CDRs, called L-CDR1, L-CDR2, L-CDR3 and H-CDR1, H-CDR2, H-CDR3, respectively. Thus, the antigen binding site typically includes six CDRs, which contain a set of CDRs from each of the heavy chain and light chain V regions. The framework region (FR) refers to the amino acid sequences inserted between the CDRs. Accordingly, the variable regions of the light chain and heavy chain usually contain four framework regions and three CDRs in the following sequence: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

[0100] Residues in the variable domains of antibodies are routinely numbered according to the system devised by Kabat et al. This system is set forth 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 in this specification. Kabat residue names do not always directly correspond to the linear numbering of amino acid residues in the SEQ ID sequences. For deletions or insertions in the structural components (whether framework or complementarity determining regions (CDRs)) corresponding to the basic variable domain structure, the actual linear amino acid sequence may contain fewer amino acids or contain additional amino acids compared to the strict Kabat numbering. For a given antibody, the correct Kabat numbering of residues can be determined by aligning the homologous residues in the antibody sequence with the "standard" Kabat numbered sequence. According to the Kabat numbering system, the CDRs of the heavy chain variable domain are located at residues 31 to 35 (H-CDR1), residues 50 to 65 (H-CDR2), and residues 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), residues 50 to 56 (L-CDR2), and residues 89 to 97 (L-CDR3).

[0101] It is currently well recognized in the art that non-CDR regions of mammalian antibodies can be replaced by similar regions of isospecific or xenospecific antibodies while retaining the epitope specificity of the original antibody. This is most clearly exemplified in the development and use of "humanized" antibodies, in which non-human CDRs are covalently joined to human FR and / or Fc / pFc' regions to produce a functional antibody.

[0102] As used herein, "humanized" describes an antibody in which some, most, or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, the methods of humanization described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762, and 5,859,205. U.S. Pat. Nos. 5,585,089 and 5,693,761, as well as WO90 / 07861, also present four criteria of likelihood that can be used to design humanized antibodies. The first recommendation is to use, for the acceptor, a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or a common framework from multiple human antibodies. The second recommendation is that if the amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for the human sequence, the donor amino acid can be selected over the acceptor. The third recommendation is that, among positions immediately adjacent to the three CDRs in the humanized immunoglobulin chain, the donor amino acid can be selected over the acceptor amino acid. The fourth recommendation is to use the donor amino acid present at a framework position where the amino acid is predicted to have a side-chain atom within 3 Å of a CDR in the three-dimensional model of the antibody and is predicted to be able to interact with the CDR. The above methods are only illustrative of some of the methods that can be used by those skilled in the art to prepare humanized antibodies. Those skilled in the art will be familiar with other methods for antibody humanization. In some humanized forms of the antibody, some, most, or all of the amino acids outside the CDR regions can be replaced with amino acids from human immunoglobulin molecules, but some, most, or all of the amino acids within one or more of the CDR regions remain unchanged. Minor additions, deletions, insertions, substitutions, or modifications of amino acids are permitted, provided that they do not eliminate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules include IgGl, IgG2, IgG3, IgG4, IgA, and IgM molecules. A "humanized" antibody retains antigen specificity similar to that of the original antibody. However, using certain methods of humanization, the affinity and / or specificity of antibody binding can be increased using methods of "directed evolution", as described by Wu et al., Mol. Biol. 294:151, 1999.

[0103] Fully human monoclonal antibodies can also be prepared by immunizing transgenic mice that have most of the human immunoglobulin heavy and light chain loci. See, for example, U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584 and the references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that the production of endogenous (e.g., murine) antibodies is functionally disrupted. The animals are further modified to contain all or part of the human germline immunoglobulin loci such that immunization of these animals will result in the production of fully human antibodies to 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 techniques. These monoclonal antibodies will have human immunoglobulin amino acid sequences and thus will not elicit a human anti-mouse antibody (HAMA) response when administered to humans.

[0104] There are also in vitro methods for generating human antibodies. These methods include phage display techniques (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.

[0105] As used herein, the term "antigen-binding fragment" of an antibody (or simply "antibody fragment") refers to the full length of an antibody or to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., the BTN3A protein as defined above). In certain embodiments, the antibodies provided herein are antibody fragments, and more specifically, include any protein that comprises the antigen-binding domain of the antibodies disclosed herein. Well-known antibody fragments include: Fab fragments, monovalent fragments consisting of the VL, VH, CL, and CH1 domains; F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by disulfide bridges in the hinge region; Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al., 1989 Nature 341:544-546), which consist of a VH domain or any fusion protein comprising such antigen-binding fragments; diabodies, which are smaller antibody fragments having two antigen-binding sites and which comprise a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain and create two antigen-binding sites. In addition, although the two domains VL and VH of an Fv fragment are encoded by separate genes, they can be joined by recombinant methods using a synthetic linker so that they can be made into a single-chain protein in which the VL and VH regions pair to form a monovalent molecule (called 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 within the term "antigen-binding fragment" of an antibody (also simply referred to herein as an antibody fragment). More generally, antibody fragments as used herein are also intended to encompass single-domain antibodies, which are antibody fragments that comprise all or a portion of the heavy-chain variable domain or all or a portion of the light-chain variable domain of an antibody. In certain embodiments, the single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1). These antibody fragments are obtained using conventional techniques known to those of skill in the art and are screened for use in the same manner as intact antibodies. Particularly 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 prepared by a variety of techniques, including (but not limited to) proteolytic cleavage of intact antibodies and production by recombinant host cells as described herein.

[0106] As used herein, the term "monoclonal antibody" refers to a preparation of antibody molecules having a single specificity. Monoclonal antibodies exhibit a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody that exhibits a single binding specificity and has variable and constant regions that are derived from or based on human germline immunoglobulin sequences or are derived from fully synthetic sequences. The method of preparing a monoclonal antibody is not relevant to the binding specificity.

[0107] A "recombinant antibody" is an antibody that has been produced, expressed, generated, or isolated by recombinant means, such as 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 that is transgenic for human immunoglobulin genes (e.g., a mouse); or an antibody produced, expressed, generated, or isolated by any other means in which a particular immunoglobulin gene sequence (such as a human immunoglobulin gene sequence) is assembled with other DNA sequences. Recombinant antibodies include, for example, chimeric antibodies and humanized antibodies. In some embodiments, the recombinant human antibodies of the present disclosure have the same amino acid sequence as the corresponding naturally occurring human antibody but are structurally different from the naturally occurring human antibody. For example, in some embodiments, the glycosylation pattern is different due to the recombinant production of the recombinant human antibody. In some embodiments, the recombinant human antibody is chemically modified by adding or subtracting at least one covalent chemical bond relative to the structure of the human antibody that naturally occurs in humans.

[0108] As used herein, an "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen 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 cross-react with other antigens, such as related BTN3A molecules from other species. Additionally, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0109] The phrases "antibody that recognizes an antigen" and "antibody that is specific for an antigen" are used interchangeably herein with the term "antibody that specifically binds to an antigen". The terms "anti-BTN3A antibody" or "BTN3A antibody" are also used herein for simplicity and have the meaning of "antibody that recognizes BTN3A".

[0110] As used herein, the term "activating antibody" refers to an antibody that can directly or indirectly induce the immune function of effector cells. Specifically, as used herein, an activating anti-BTN3A antibody has at least the ability to induce the activation of γδ T cells (usually Vγ9Vδ2 T cells) in co-culture with cells expressing BTN3, wherein, as measured in a degranulation assay, the EC50 is less than 5 μg / ml, preferably 1 μg / ml or less (for a detailed assay, see WO / 2020 / 025703).

[0111] As used herein, in the context of an antibody binding to a predetermined antigen or epitope, particularly BTN3A, the term "binding" generally refers to a binding affinity corresponding to about 10 -7 M or lower, such as about 10 -8 M or lower, such as about 10 -9 M or lower, about 10 -10 M or lower or about 10 -11 M or even lower K D d when measured by surface plasmon resonance (SPR) technology, for example, in a BIAcore 3000 instrument, typically using the soluble form of the antigen as the ligand and the antibody as the analyte. (GE Healthcare, Piscaataway, NJ) is one of the various forms of surface plasmon resonance assays routinely used for epitope binning of monoclonal antibodies. Generally, an antibody binds to a predetermined antigen with an affinity corresponding to a K D d that is at least ten-fold lower, such as at least 100-fold lower, for example, at least 1,000-fold lower; such as at least 10,000-fold lower, for example, at least 100,000-fold lower, than the K D d of its binding to a non-specific antigen (e.g., BSA, casein), where the non-specific antigen is not the same as or closely related to the predetermined antigen. If the K D d of the antibody is extremely low (i.e., the antibody has a high affinity), then the K D d of the antibody binding to the antigen is usually at least 10,000-fold lower than the K D d of its binding to a non-specific antigen. D

[0112] As used herein, in the context of an antibody, the term "affinity" refers to the strength of binding of the antibody to an epitope.

[0113] As used herein, the term "K on " or "Kass (K a )" is intended to refer to the association rate of a specific antibody-antigen interaction, while the term "K dis (Kd)" or "K off " is intended to refer to the dissociation rate of a specific antibody-antigen interaction.

[0114] As used herein, the term "K" D is intended to refer to the equilibrium dissociation constant, which is obtained from the ratio of k off to k on (i.e., k off / k on ) and is expressed as molar concentration (M). The K D value is related to the concentration of the antibody (the amount of antibody required for a particular experiment), and thus, the lower the K D value (lower concentration) and the higher the affinity of the antibody. The K D value of an antibody can be determined using methods long established in the art. Preferred methods for determining the K D value of an mAb can be found in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988), Coligan et al., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y., (1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), the disclosures of which are incorporated herein by reference in their entireties. One method for determining the K D of an antibody is by using surface plasmon resonance or by using a biosensor system such as (see also Rich RL, Day YS, Morton TA, Myszka DG. High-resolution and high-throughput protocols for measuring drug / human serum albumin interactions using Anal Biochem. September 15, 2001; 296(2):197-207) or systems. The platform is based on the technology of Biolayer Interferometry (BLI). The principle of BLI technology is based on the optical interference pattern of white light reflected from two surfaces (the immobilized protein layer and the internal reference layer). The binding between the ligand immobilized on the surface of the biosensor tip and the analyte in the solution results in an increase in the optical thickness at the biosensor tip, which causes a shift in the interference pattern measured in nanometers. The wavelength shift (Δλ) is a direct measure of the change in the optical thickness of the biolayer. When this shift is measured over a period of time and its magnitude is plotted as a function of time, a classical binding / dissociation curve is obtained. This interaction is measured in real time, allowing the monitoring of binding specificity, binding rate, dissociation rate, and concentration (see Abdiche et al. 2008 and details in the results). Affinity measurements are typically performed at 25 °C.

[0115] As used herein, the term "specificity" refers to the ability of an antibody to detectably bind to an epitope presented on an antigen (such as BTN3A). In some embodiments, the term is intended to refer to an antibody that binds to human BTN3A expressed on peripheral blood mononuclear cells (PBMCs), which, as determined in the examples, preferably has an EC 50 (The assays and protocols are generally disclosed in WO / 2020 / 025703, especially with reference to Table 4). In other embodiments, it has a K of 100 nM or lower, 10 nM or lower, 1 nM or lower, 100 pM or lower, or 10 pM or lower D Binds to the antigen recombinant polypeptide, measured by SPR measurement as mentioned above and for details also see Table 4 of WO / 2020 / 025703.

[0116] An antibody that "cross-reacts with an antigen other than BTN3A" is intended to refer to an antibody that binds to the antigen other than BTN3A with a K of 10 nM or lower, 1 nM or lower, or 100 pM or lower D An antibody that binds to an antigen other than BTN3A. An antibody that "does not cross-react with a specific antigen" is intended to refer to an antibody that binds to the antigen with a K of 1 μM or greater D or a K of 10 μM or greater D An antibody that binds to the antigen. In certain embodiments, such antibodies that do not cross-react with the antigen exhibit substantially undetectable binding to these proteins in a standard binding assay. In a particular embodiment, for example, as measured in a Biacore assay (especially see the relevant assay exemplified in Table 26 of WO / 2020 / 025703), the humanized antibodies of the present disclosure (such as mAb1) cross-react with cynomolgus BTN3A1, BTN3A2, and BTN3A3 of SEQ ID NO:27, SEQ ID NO:28, and SEQ ID NO:29, respectively.

[0117] Specificity can be further demonstrated by, for example, a ratio of the affinity / avidity for binding to a specific antigen (in this case the BTN3A polypeptide) compared to non-specific binding to other unrelated molecules of about 10:1, about 20:1, about 50:1, about 100:1, 10,000:1 or greater.

[0118] As used herein, the term "avidity" refers to an informative measure of the overall stability or strength of an antibody-antigen complex. It is controlled by three main factors: antibody epitope affinity; the valency of both the antigen and the antibody; and the structural arrangement of the interacting parts. Ultimately, these factors define the specificity of an antibody, i.e., the likelihood that a particular antibody will bind to an exact antigen epitope.

[0119] As used herein, the term "subject" includes any human or non-human animal. The term "non-human animal" includes all vertebrates, such as mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, etc.

[0120] As used herein, the term "optimized" means that a nucleotide sequence has been used to encode an amino acid sequence with codon changes that are preferred in a cell or organism (generally a eukaryotic cell, such as a Chinese Hamster Ovary cell (CHO) or a human cell). The optimized nucleotide sequence is engineered to fully or as much as possible retain the amino acid sequence originally encoded by the starting nucleotide sequence. The amino acid sequence encoded by the optimized nucleotide sequence is also referred to as optimized.

[0121] As used herein, the term "identity" with respect to a polypeptide sequence refers to amino acid sequence identity between two molecules. Molecules are identical at an amino acid position if that position in both molecules 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 to obtain maximal matching (including gaps as necessary). The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., identity % = number of identical positions / total number of positions × 100), taking into account the number of gaps that need to be introduced to achieve the best alignment of the two sequences and the length of each gap. Comparison of sequences and determination of the percent identity between two sequences can be accomplished using mathematical algorithms, as described below.

[0122] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4:11-17, 1988), which has been 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 publicly available techniques and widely available computer programs such as BLASTP, FASTA (Atschul et al., J. Mol. Biol. 215:403, 1990) or the Needleman and Wunsch (J. Mol, Biol. 48:444-453, 1970) algorithm, which has been 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 a Blossom 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.

[0123] The percent identity between two nucleotide amino acid sequences can also be determined using, for example, an algorithm such as the BLASTN program for nucleic acid sequences, using a word length (W) of 11, an expectation value (E) of 10, M = 5, N = 4 and a comparison of both strands as a preset.

[0124] Additional antibodies can be identified in standard antigen-binding assays, such as ELISA binding assays, based on their ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with other antibodies of the present disclosure. The ability of a test antibody to inhibit the binding of an antibody of the present disclosure to a target indicates that the test antibody can compete with that antibody for binding to the target; according to non-limiting theory, such antibodies can bind to the same or a related (e.g., structurally similar or spatially proximate) epitope on the target as the antibody with which they are competing. Accordingly, another aspect of the present disclosure provides antibodies that bind to the same antigen as the antibodies disclosed herein and compete with the antibodies disclosed herein. As used herein, an antibody "competes" for binding when, in the presence of a competing antibody at equimolar concentration, the competing antibody inhibits the target binding of an antibody or antigen-binding fragment of the present 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%.

[0125] "Combination therapy", "co-administer", "combination administration" or "concomitant administration" refers to the administration of a combination of at least two therapeutic agents, wherein a first agent (usually a BTN3A activating compound) is administered simultaneously or separately within a time interval with a second agent (e.g., a Bcl2 family inhibitor) and optionally a third agent (e.g., a demethylating agent) in the same subject in need thereof, wherein these time intervals allow the combination of agents to exhibit a cooperative or synergistic effect for treating a disorder (e.g., cancer and more particularly hematological malignancies). The term is not intended to imply that the therapeutic agents must be administered simultaneously and / or formulated to be delivered together, although these delivery methods are within the scope of the present disclosure. A BTN3A activating antibody (e.g., ICT01 disclosed herein) can be administered in parallel with or before or after one or more other additional therapies or therapeutic agents. The term is also intended to cover treatment regimens in which the agents are not necessarily administered by the same route of administration.

[0126] As used herein, the term "synergistic" or "synergy", when used in connection with a description of the efficacy of a combination of agents, refers to any measured effect of the combination that is greater than the effect predicted from the sum of the effects of the individual agents (i.e., greater than an additive effect). In some embodiments, the tumor growth rate or tumor size (e.g., the rate of change of tumor size (e.g., volume, mass)) is used to determine whether a combination of drugs is synergistic (e.g., if a combination of drugs produces an additive effect, the combination of drugs is synergistic when the tumor growth rate is slower than the expected rate). In some embodiments, the median overall survival time (e.g., <12 months) is used to determine whether a combination of drugs is synergistic (e.g., if a combination of drugs produces an additive effect, the combination of drugs is synergistic when the median overall survival time of a subject or group of subjects is longer than the expected median overall survival time). In some embodiments, complete remission (CR) and complete remission with incomplete count recovery (CRi) rates are used to determine whether a combination of drugs is synergistic (compared to single therapy). In some embodiments, T cell expansion can also be used to determine whether a combination of drugs is synergistic (e.g., if a combination of drugs produces an additive effect, the combination of drugs is synergistic when the expansion rate of a specific T cell subset (measured as the percentage increase in the population or the absolute number of cells increased compared to the baseline value) is higher than the expected expansion rate).

[0127] Activated BTN3A antibody

[0128] The activated anti-BTN3A antibodies of the present disclosure generally exhibit one or more of the following characteristics:

[0129] (i) As measured by SPR (e.g., as described in the examples of patent application WO2020025703), it binds to BTN3A with a K of 10 nM or lower, preferably with a K of 1 nM or lower; D preferably with a K of 1 nM or lower; D and binds to BTN3A;

[0130] (ii) As measured by SPR (e.g., as described in the examples of patent application WO2020025703), it cross-reacts with cynomolgus monkey BTN3A with a K of 100 nM or lower, preferably with a K of 10 nM or lower; D preferably with a K of 10 nM or lower; D and cross-reacts with cynomolgus monkey BTN3A;

[0131] (iii) As measured in a flow cytometry assay (e.g., as described in the examples of patent application WO2020025703), it binds to human PBMCs with an EC of 50 μg / ml or lower, preferably 10 μg / ml or lower; 50 and binds to human PBMCs;

[0132] (iv) It induces the activation of γδ-T cells (usually Vγ9Vδ2 T cells) in co-culture with cells expressing BTN3, wherein the EC 50 is less than 5 μg / ml, preferably 1 μg / ml or lower, as described in the examples of patent application WO2020025703;

[0133] (v) It induces the in vitro activation of Vγ9Vδ2 T cells in human PBMCs, as measured by the surface expression of the activation marker CD69, and the EC 50 is less than 0.1 μg / mL, preferably 0.01 μg / mL or lower, for example, it is 100 pg / mL - 0.1 μg / mL.

[0134] Examples of BTN3A activating antibodies are described in the paragraphs below. In some embodiments, the BTN3A activating antibody is selected from the group consisting of, such as the BTN3A antibodies described in international patent applications WO2012080769; WO2012080351 and WO2020025703. In some specific embodiments, the BTN3A activating antibody is selected from the humanized antibodies described in WO2020025703 or is a humanized form of the BTN3A activating antibodies described in WO2012080769 and WO2012080351. In some embodiments, the BTN3A activating antibody can be selected from mAb 20.1 and mAb 7.2, which can be obtained from one of the hybridomas deposited under CNCM accession numbers I-4401 and I-4402 as described in WO2012080769 and WO2012080351 or their humanized forms; and from the humanized mAb 1-6 described in WO2020025703.

[0135] In some embodiments, the BTN3A activating antibody comprises the six CDRs (VH CDR1 (also known as HCDR1), VH CDR2 (also known as HCDR2), VH CDR3 (also known as HCDR3), VL CDR1 (also known as LCDR1), VL CDR2 (also known as LCDR2), VL CDR3 (also known as LCDR3)) of the antibody 20.1 or 7.2 described in WO2012080769 and WO2012080351 or mAb 1-6 as described in WO2020025703. In a particular embodiment, the anti-BTN3A activating antibody comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 as shown in Table 1 below:

[0136] Table 1: CDR regions of mAb1, mAb2, mAb4, and mAb5, parental murine mAb 7.2, and murine mAb 20.1 antibodies according to the Kabat numbering defined in WO2020025703.

[0137]

[0138] In some embodiments of the antibodies used as disclosed herein, the 6 CDR regions are 100% identical to the 6 CDR regions of antibody 20.1 or 7.2 described in WO2012080769 and WO2012080351 or mAb 1 - 6 described in WO2020025703. In particular, in some embodiments, the 6 CDR regions of the antibodies as disclosed herein are 100% identical to the 6 CDR regions of Table 1 (in particular mAb 7.2; 1; 2; 4; and 5).

[0139] Other antibodies used as disclosed herein include those having amino acids that have been mutated by amino acid deletion, insertion, or substitution, but that still have at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity in the CDR regions compared to the 6 CDR regions of antibody 20.1 or 7.2 described in WO2012080769 and WO2012080351 or mAb 1 - 6 described in WO2020025703, and in particular compared to the 6 CDR regions defined in Table 1. Generally, in accordance with the present disclosure, compared to the CDR sequences of antibody 20.1 or 7.2 described in WO2012080769 and WO2012080351 or mAb 1 - 6 described in WO2020025703, and in particular compared to the CDR sequences of Table 1, and more specifically compared to the CDR sequences of mAb 7.2; 1; 2; 4; and 5, the antibody may have amino acid changes (including deletions, insertions, or substitutions) between 1, 2, 3, or 4 in one or more CDRs.

[0140] In a particular embodiment, the antibody used according to the present disclosure comprises a variant CDRH2 of mAb 20.1 having substitutions N5S and K10N (also known as N53S, K58N according to Kabat numbering).

[0141] In other particular embodiments, the antibody used according to the present disclosure comprises a variant CDRL1 of mAb 20.1 having substitution L8V (also known as L31V according to Kabat numbering).

[0142] In more specific embodiments, the antibodies used according to the present disclosure comprise variant CDRH2 of mAb 20.1 having substitutions N5S and K10N (also known as N53S, K58N according to Kabat numbering) and variant CDRL1 of mAb 20.1 having substitution L8V (also known as L31V according to Kabat numbering).

[0143] In one specific embodiment, the BTN3A-activating antibody for use according to the present disclosure comprises heavy chain CDR1-3 sequences of SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively, and light chain CDR1-3 sequences of SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39.

[0144] In one specific embodiment, the BTN3A-activating antibody for use according to the present disclosure comprises variable heavy chain VH of SEQ ID NO:40 and variable light chain of SEQ ID NO:41.

[0145] In specific embodiments, the BTN3A-activating antibody is a bispecific antibody, characterized by comprising a first binding portion that specifically binds to human BTN3A and a second binding portion that specifically binds to a tumor antigen, which is typically a tumor antigen targeting malignant cells of the targeted hematological malignancy. For example, the first binding portion is a full-length bivalent antibody having heavy chain CDR sequence CDRH1 of SEQ ID NO:34, CDRH2 of SEQ ID NO:35, CDRH3 of SEQ ID NO:36, and light chain CDR sequences CDRL1 of SEQ ID NO:37, CDRL2 of SEQ ID NO:38, CDRL3 of SEQ ID NO:39.

[0146] Other anti-BTN3A-activating antibodies, especially variants of the mAb20.1-activating antibody, are disclosed in WO2023 / 161457 (Evobright GmbH), the content of which is incorporated herein by reference in its entirety. The antibodies used according to the present disclosure also include antibodies having at least 90%, especially at least 95%, 96%, 97%, 98%, 99, or 100% identity to the VH and VL regions defined in Table 2. More specifically, the antibodies of the present disclosure include the selected humanized recombinant antibodies mAb1, mAb2, mAb4, and mAb5, which are structurally characterized by their variable heavy and light chain amino acid sequences and human constant regions (isotypes) as described in Table 2 below:

[0147] Table 2: Variable heavy and light chain amino acid sequences of mAb1-mAb6

[0148]

[0149]

[0150] mAb3 and mAb6 are humanized antibodies of the parental murine BTN3A activating antibody, designated mAb 20.1 as described in WO2012 / 080351.

[0151] The corresponding amino acid and nucleotide coding sequences of the constant isotype regions of IgG1, IgG4 and their mutant forms IgG1L247F / L248E / P350S and IgG4S241P / 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; thus, this residue can additionally be deleted in the constructs of mAb1 to mAb6.

[0152] The full-length light and heavy chains of mAb1, mAb2, mAb4 and mAb5 and the corresponding coding sequences are shown in Table 3 below.

[0153] Table 3: Full-length heavy and light chain DNA coding sequences

[0154]

[0155] In certain embodiments, which can be combined with the previous embodiments, the antibodies provided herein are antibody fragments of the antibodies as defined above. Antibody fragments include, for example (but are not limited to), Fab, Fab', Fab'-SH, F(ab')2, Fv, monovalent antibodies and scFv fragments, bispecific antibodies, single-domain or nanobodies and other fragments. Preferably, they are monovalent antibodies, such as Fab of scFv fragments.

[0156] In some embodiments, the antibodies of the present disclosure competitively bind to the BTN3A antibodies described above. In particular, the antibodies of the present disclosure compete with antibodies selected from mAb 20.1 and mAb 7.2 (which can be obtained from one of the hybridomas available under CNCM deposit numbers I-4401 and I-4402 as described in WO2012080769 and WO2012080351) and antibodies selected from mAb 1-6 described in WO2020025703. In more specific embodiments, the antibodies of the present disclosure compete with antibodies selected from: mAb 7.2 produced by the hybridoma deposited under deposit number I-4402 at the CNCM and the antibody having a heavy chain of SEQ ID NO:4 and a light chain of SEQ ID NO:6.

[0157] In some embodiments, the antibodies of the present disclosure are chimeric antibodies, humanized antibodies or human antibodies. In a preferred embodiment of the present disclosure, the BTN3A antibody is a humanized antibody. Generally, non-human antibodies are humanized to reduce their immunogenicity in humans while having the same (or superior) affinity as the parental 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 antibody of the parental antibody mAb 7.2 as disclosed in WO2012080351. Generally, a humanized antibody comprises one or more variable domains in which the CDRs (or portions thereof) are derived from a non-human antibody (e.g., murine mAb 7.2), and the FRs (or portions thereof) are derived from a murine antibody sequence with mutations to reduce immunogenicity. The humanized antibody optionally further comprises at least a portion of a human constant region. Preferably, the recombinant antibody according to the present disclosure is a humanized silent antibody, typically a humanized silent IgG1 or IgG4 antibody. Highly suitable humanized anti-BTN3A antibodies according to the present disclosure are generally described in WO2020025703 and include mAbs having the VH / VL polypeptide sequences of Table 2 and mAbs having the light / heavy chains of Table 3.

[0158] As used herein, the term "silent" antibody refers to an antibody that does not exhibit or exhibits reduced 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 and / or C1q binding" means that the silent antibody exhibits FcγR and / or C1q binding that is at least 50% lower, e.g., 80% lower, than the FcγR and / or C1q binding observed with the corresponding antibody having a wild-type human IgG1 or IgG4 isotype.

[0159] Framework or Fc engineering

[0160] The antibodies of the present disclosure may include modifications to framework residues within VH and VL to reduce their immunogenicity.

[0161] In some specific embodiments, the antibody of the present disclosure is a humanized monoclonal antibody of the parental murine antibody mAb 7.2, which includes the following amino acid mutations in at least the VH framework region: V5Q; V11L; K12V; R66K; S74F; I75S; E81Q; S82AR; R82BS; R83T; D85E; T87S; L108S; and the following amino acid mutations in at least the Vκ framework region: T5N; V15L; R18T; V19I; K42N; A43I; D70G; F73L; Q100G.

[0162] In other specific embodiments, the antibodies of the present disclosure are humanized monoclonal antibodies of the parental murine antibody mAb 7.2, which compared to mAb 7.2 include the following amino acid mutations in at least the VH framework region: V5Q; V11L; K12V; R66K; S74F; I75S; E81Q; S82AR; R82BS; R83T; D85E; T87S; L108S; and the following amino acid mutations in at least the Vκ framework region: T5N; V15L; R18T; V19I; K42N; A43I; S63T; D70G; F73L; Q100G.

[0163] In addition to the modifications made in the framework region, the antibodies of the present disclosure can be engineered to include modifications in the Fc region, typically altering one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cell cytotoxicity.

[0164] 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 their glycosylation, thereby altering one or more functional properties of the antibody. Each of these embodiments is further described in detail below.

[0165] As used herein, the terms "isotype constant region" or "Fc region" are used interchangeably to define the C-terminal region of the immunoglobulin heavy chain, including the native sequence Fc region and variant Fc regions. The human IgG heavy chain Fc region is generally defined as comprising the amino acid residues from position C226 or from P230 to the carboxyl terminus of the IgG antibody, where the numbering is according to the EU numbering system. The C-terminal lysine (residue K447) of the Fc region can be removed, for example, during the production or purification of the antibody or when its corresponding codon is deleted in the recombinant construct. Thus, the composition of the antibodies of the present disclosure can comprise a population of antibodies in which all K447 residues are removed, a population of antibodies in which the K447 residues are not removed, and a population of antibodies having a mixture of antibodies with or without the K447 residue.

[0166] In some specific embodiments, the hinge region of CH1 is modified such that the number of cysteine residues in the hinge region is altered, for example increased or decreased. This method is further described in U.S. Patent No. 5,677,425 to Bodmer et al. The number of cysteine residues in the CH1 hinge region is altered, for example, to facilitate light chain and heavy chain assembly or to increase or decrease the stability of the antibody.

[0167] In other embodiments, the Fc hinge region of the antibody is mutated to reduce the in vivo half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc hinge fragment such that the binding of the antibody to Staphylococcyl protein A (SpA) is reduced relative to the native Fc hinge domain SpA binding. This method is described in further detail in U.S. Patent No. 6,165,745 to Ward et al.

[0168] In other embodiments, the Fc region is altered by substituting at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids may be substituted with different amino acid residues such that the antibody has an altered affinity for an effector ligand, while retaining the antigen-binding ability of the parental antibody. The effector ligand with altered affinity may be, for example, an Fc receptor or the C1 component of complement. This method is described in further detail in U.S. Patents Nos. 5,624,821 and 5,648,260 to Winter et al.

[0169] In another embodiment, one or more amino acids selected from the amino acid residues may be substituted with different amino acid residues such that the antibody has altered C1q binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This method is described in further detail in U.S. Patent No. 6,194,551 to Idusogie et al.

[0170] In another embodiment, one or more amino acid residues are altered to alter the ability of the antibody to fix complement. This method is described in further detail in PCT Publication WO 94 / 29351 to Bodmer et al.

[0171] 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 cytotoxicity (ADCC) and / or to reduce the affinity of the antibody for Fcγ receptors. Such antibodies with reduced effector function and particularly reduced ADCC include silent antibodies.

[0172] In certain embodiments, the Fc domain of the IgG1 isotype is used. In some specific embodiments, a mutant variant of the IgG1 Fc fragment is used, such as a silent IgG1 Fc that reduces or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cytotoxicity (ADCC) and / or to bind to Fcγ receptors.

[0173] In certain embodiments, the Fc domain of the IgG4 isotype is used. In some specific embodiments, a mutant variant of the IgG4 Fc fragment is used, such as a silent IgG4 Fc that reduces or eliminates the ability of the fusion polypeptide to mediate antibody-dependent cytotoxicity (ADCC) and / or to bind to Fcγ receptors.

[0174] Silencing effector functions can be obtained by mutations in the Fc constant portion of antibodies and have been described in the art (Baudino et al., J. Immunol. 2008; Strohl, Curr. Opin. Biotechnol. 2009). Examples of silenced IgG1 antibodies include the triple mutant variant IgG1 L247F L248E P350S. Examples of silenced IgG4 antibodies include the double mutant variant IgG4 S241P L248E.

[0175] In certain embodiments, the Fc domain is a silenced Fc mutant that prevents glycosylation at position 314 of the Fc domain. For example, the Fc domain contains an amino acid substitution of asparagine at position 314. Examples of such amino acid substitutions include replacement of N314 with glycine or alanine.

[0176] In other embodiments, the glycosylation of the antibody is modified. For example, non-glycosylated antibodies (i.e., antibodies lacking glycosylation) can be produced. Glycosylation can be altered, for example, to increase the affinity of the antibody for an antigen. Such carbohydrate modifications can be accomplished, for example, by altering one or more glycosylation sites within the antibody sequence. For example, one or more amino acid substitutions can be made to eliminate one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such non-glycosylation can increase the affinity of the antibody for an antigen. Such methods are described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861 to Co et al.

[0177] Another modification of the antibodies herein covered by the present disclosure is pegylation or hydroxyethyl starchylation 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 similar reactive water-soluble polymer). As used herein, the term "polyethylene glycol" is intended to encompass any PEG form 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 pegylated antibody is a non-glycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies of the present disclosure. See, for example, EP 0 154 316 to Nishimura et al. and EP 0 401 384 to Ishikawa et al.

[0178] Another possibility is that at least the antigen-binding region of an antibody of the present disclosure is fused 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 methods are described, for example, in Nygren et al., EP 0486 525.

[0179] In certain embodiments, the C-terminal lysine that is normally present on the human IgG heavy chain constant domain is engineered out to reduce heterogeneity, which is due to the reduced cleavage that is typically observed during manufacture or storage. Such modifications do not appreciably alter the desired function of these antibodies while conferring a stability benefit to these molecules.

[0180] A nucleic acid molecule encoding an antibody of the present disclosure

[0181] Also disclosed herein are nucleic acid molecules encoding the anti-BTN3A antibodies of the present disclosure. Examples of the variable light and heavy chain nucleotide sequences are those nucleotide sequences encoding the variable light and heavy chain amino acid sequences of any of mAb1, mAb2, mAb4, and mAb5, which can be readily derived from Tables 1 and 2 and optionally taking into account codon bias using the genetic code and depending on the host cell species.

[0182] The present disclosure also relates to nucleic acid molecules derived from the variable light and heavy chain amino acid sequences that have been optimized for protein expression in mammalian cells (such as the CHO cell line).

[0183] The nucleic acid can be present in whole cells, cell lysates, or can be nucleic acid in a partially purified or substantially pure form. A nucleic acid is "isolated" or "made substantially pure" when it is purified away from other cellular components or other contaminants, such as other cellular nucleic acids or proteins, 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)). The nucleic acids of the present disclosure can be, for example, DNA or RNA and can contain or not contain intron sequences. In one embodiment, the nucleic acid can be present in a vector, such as a phage display vector or present in a recombinant plasmid vector.

[0184] The nucleic acids of the present disclosure can be obtained using standard molecular biology techniques. Once DNA fragments encoding, for example, VH and VL segments are obtained, these DNA fragments can be further manipulated by standard recombinant DNA techniques, such as converting variable region genes into full-length antibody chain genes, Fab fragment genes, or scFv genes. In these manipulations, a DNA fragment encoding VL or VH (such as VL and VH as defined in Table 1) is operably linked to another DNA molecule or a fragment encoding another protein, such as an antibody constant region or a flexible linker. As used in this context, the term "operably linked" is intended to mean that two DNA fragments are joined in a functional manner, such that the amino acid sequences encoded by the two DNA fragments remain in-frame, or such that a protein is expressed under the control of a desired promoter.

[0185] An isolated DNA encoding a VH region can be converted into a full-length heavy chain gene by operably linking the DNA encoding VH to another DNA molecule encoding the heavy chain constant regions (CH1, CH2, and CH3). The sequences of human heavy chain constant region genes are known in the art (Kabat et al., K.S. (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 the IgG1 isotype, such as the human IgG1 isotype. In other embodiments, the heavy chain constant region is selected from the IgG4 isotype, such as the human IgG4 isotype. For a Fab fragment heavy chain gene, the DNA encoding VH is operably linked to another DNA molecule encoding only the heavy chain CH1 constant region.

[0186] An isolated DNA encoding a VL region can be converted into a full-length light chain gene (and into a Fab light chain gene) by operably linking the DNA encoding VL 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, see above) and DNA fragments encompassing these regions can be obtained by standard PCR amplification. The light chain constant region can be a κ or λ constant region.

[0187] To generate the scFv gene, DNA fragments encoding VH and VL are operably linked to another fragment encoding a flexible linker (e.g., encoding the amino acid sequence (Gly4-Ser)3) such that the VH and VL sequences can be expressed as a continuous single-chain protein, where the VL and VH regions are joined by the flexible linker (Bird et al., 1988, supra; Huston et al., 1988, supra; McCafferty, J. et al., 1990. Nature 348, 552-554).

[0188] Generation of transfected tumors producing monoclonal antibodies

[0189] The antibodies of the present disclosure can be produced in host cell transfected tumors using, for example, a combination of recombinant DNA techniques and gene transfection methods well known in the art (Morrison, 1985; Science 229, 1202-1207).

[0190] For example, to express an antibody or an antibody fragment thereof, DNA encoding a partial or full-length light chain and heavy chain can be obtained by standard molecular biology or biochemical techniques (e.g., DNA chemical synthesis, PCR amplification, or cDNA cloning using a hybridoma expressing the antibody of interest) and the 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 the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the antibody gene. An expression vector and expression control sequences compatible with the expression host cell used are selected. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors, or more commonly, the two genes are inserted into the same expression vector. The antibody gene is inserted into the expression vector by standard methods (e.g., ligating the antibody gene fragment and complementary restriction sites on the vector, or blunt-end ligation if no restriction sites are present). By inserting an expression vector encoding the heavy chain constant and light chain constant regions of the desired isotype, the light chain and heavy chain variable regions of the antibodies described herein can be used to generate full-length antibody genes of any antibody isotype such that the VH segment is operably linked to one or more CH segments within the vector and the VL segment is operably linked to the CL segment within the vector. Alternatively or additionally, the recombinant expression vector can encode a signal peptide that facilitates secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into the vector such that the signal peptide is in-frame linked 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).

[0191] In addition to the antibody chain gene, the recombinant expression vectors disclosed herein also carry regulatory sequences that control the expression of the antibody chain gene in a host cell. The term "regulatory sequence" is intended to include promoters, enhancers, and other expression control elements (such as polyadenylation signals) that control the transcription or translation of the antibody chain gene. Such regulatory sequences are described, for example, in the disclosure of Goeddel (Goeddel, D. V. (1990). [1] Systems for heterologous gene expression. Methods in Enzymology, (Academic Press), pp. 3-7). Those skilled in the art will appreciate that the design of the expression vector, including the selection of regulatory sequences, may depend on factors such as the choice of the host cell to be transformed, the level of expression of the desired protein, etc. Regulatory sequences for mammalian host cell expression include 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., adenovirus major late promoter (AdMLP)), and polyomavirus. Alternatively, non-viral regulatory sequences, such as the ubiquitin promoter or the P-globin promoter, may be used. Furthermore, regulatory 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).

[0192] In addition to the antibody chain gene and regulatory sequences, the recombinant expression vectors of the present disclosure may also carry additional sequences, such as sequences that regulate the replication of the vector in a host cell (e.g., an origin of replication) and selectable marker genes. Selectable marker genes facilitate the selection of host cells into which the vector has been introduced (see, for example, U.S. Pat. Nos. 4,399,216, 4,634,665, and 5,179,017, all to Axel et al.). For example, on host cells into which the vector has been introduced, selectable marker genes typically confer resistance to drugs such as G418, hygromycin, or methotrexate. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for dhfr- host cells with methotrexate selection / amplification) and the neo gene (for G418 selection).

[0193] For the expression of light and heavy chains, one or more expression vectors encoding the heavy and light chains are transfected into host cells by standard techniques. The various forms of the term "transfection" are intended to cover the various techniques commonly used to introduce exogenous DNA into prokaryotic or eukaryotic host cells, such as electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. It is theoretically possible to express the antibodies of the present disclosure in prokaryotic or eukaryotic host cells. The expression of antibodies in eukaryotic cells, such as mammalian host cells, yeast, or filamentous fungi, is discussed because such eukaryotic cells, and especially mammalian cells, are more likely to assemble and secrete properly folded and immunologically active antibodies than prokaryotic cells.

[0194] In one particular embodiment, the cloning or expression vector of the present disclosure comprises one of the coding sequences of the heavy and light chains of any one of mAb1, mAb2, mAb4, and mAb5 operably linked to a suitable promoter sequence.

[0195] Mammalian host cells for expressing the recombinant antibodies of the present disclosure include Chinese hamster ovary (CHO) cells, including dhfr-CHO cells (described in Urlaub and Chasin, 1980) used in conjunction with a DHFR selectable marker (described in Kaufman and Sharp, 1982), the CHOK1 dhfr+ cell line, NSO myeloma cells, COS cells, and SP2 cells, such as the GS CHO cell line together with the GS Xceed TM Gene Expression System (Lonza). When the recombinant expression vector encoding the antibody gene is introduced into mammalian host cells, the antibody is produced by culturing the host cells for a period sufficient for the antibody to be expressed in the host cells and optionally for the antibody to be secreted into the culture medium in which the host cells are growing. After antibody secretion, standard protein purification methods can be used, such as recovering and purifying the antibody from the culture medium (Shukla et al., 2007, J. Chromatogr. B 848, 28-39).

[0196] In one particular embodiment, the host cell of the present disclosure is a host cell transfected with an expression vector having coding sequences suitable for the expression of mAb1, mAb2, mAb4, and mAb5 operably linked to a suitable promoter sequence, respectively.

[0197] For example, the present disclosure relates to a host cell comprising at least the nucleic acids of SEQ ID NO: 8 and 10 encoding the heavy and light chains of mAb1, respectively.

[0198] The latter host cells can then be further cultured under suitable conditions for the expression and production of the antibodies of the present disclosure selected from mAb1, mAb2, mAb3, mAb4, and mAb5, respectively.

[0199] Alternatively, a cell-free expression system can be used to produce any of mAb1, mAb2, mAb3, mAb4, and mAb5. Generally, methods for cell-free expression of proteins or antibodies have been described (Stech et al., 2017, Sci. Rep. 7, 12030).

[0200] Anti-BTN3A immunoconjugate

[0201] In another aspect, the present disclosure provides an anti-BTN3A antibody or fragment thereof as disclosed herein that is conjugated to a therapeutic moiety. Such conjugates are referred to herein as "immunoconjugates". An immunoconjugate that includes one or more cytotoxins is referred to as an "immunotoxin". A cytotoxin or cytotoxic agent includes any reagent that is harmful (e.g., kills) to cells.

[0202] Linker technologies available in the art can be used to conjugate a cytotoxin to an antibody of the present disclosure. Examples of types of linkers 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. Linkers can be selected, for example, that are cleavable easily by low pH within the lysosomal compartment or that are cleavable easily by proteases, such as proteases that are preferentially expressed in tumor tissue, such as cathepsins (e.g., cathepsin B, C, D).

[0203] For further discussion of the types of cytotoxins, linkers, and methods for conjugating therapeutic agents to antibodies, see also the review by Panowski et al., 2013 on antibody-drug conjugates.

[0204] The antibodies of the present disclosure can also be conjugated to radioisotopes to generate cytotoxic radiopharmaceuticals, also referred to as radioimmunoconjugates. Examples of radioisotopes that can be conjugated to an antibody for diagnostic or therapeutic use include (but are not limited to) iodine 131 indium 111 yttrium 90 and lutetium 177 . Methods for preparing radioimmunoconjugates are well established in the art.

[0205] Bispecific or multispecific anti-BTN3A antibodies

[0206] In another aspect, the present disclosure further discloses bispecific or multispecific molecules that comprise the anti-BTN3A antibodies of the present disclosure. The antibody can be derived or linked to another functional molecule, such as another peptide or protein (e.g., another antibody or ligand of a receptor), to produce a bispecific molecule that binds to at least two different binding sites or target molecules. In fact, the antibody can be derived or linked to more than one other functional molecule to produce 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 produce a bispecific molecule, the antibodies of the present disclosure can be functionally linked (e.g., by chemical coupling, gene fusion, non-covalent binding, or otherwise) to one or more other binding molecules (such as another antibody, antibody fragment, peptide, or binding mimetic), thereby producing a bispecific molecule.

[0207] Accordingly, the present disclosure includes bispecific molecules that comprise at least one first binding specificity against BTN3A, such as an antigen-binding portion of any one of mAb1, mAb2, mAb3, mAb4, mAb5, and mAb6 (or functional variants thereof), and a second binding specificity against a second target epitope.

[0208] In addition, for embodiments in which the bispecific molecule is multispecific, in addition to the first and second target epitopes, the molecule can further comprise a third binding specificity.

[0209] In one embodiment, the bispecific molecules disclosed herein comprise at least one antibody or an antibody fragment thereof as a binding specificity, including, for example, Fab, Fab', F(ab')2, Fv, monovalent antibody, or single-chain Fv. The antibody can also be a light or heavy chain dimer or any minimal fragment thereof, such as an Fv or single-chain construct, as described in U.S. Patent No. 4,946,778 to Ladner et al.

[0210] Other antibodies that can be used in the bispecific molecules disclosed herein are murine, chimeric, and humanized monoclonal antibodies.

[0211] The bispecific molecules of the present disclosure can be prepared by combining component binding specificities using methods known in the art. For example, the respective binding specificities of the bispecific molecule can be generated separately and then bound to each other. When the binding specificities are proteins or peptides, various coupling agents or crosslinking agents can be used for covalent binding. Examples of crosslinking 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)cyclohexane-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.

[0212] Alternatively, the two binding specificities can be encoded in the same vector and expressed and assembled in the same host cell. This method is particularly applicable when the bispecific molecule is an mAb×mAb, mAb×scFv, mAb×Fab, mAb×F(ab')2, or ligand×Fab fusion protein. In certain embodiments, the bispecific molecule can include a fusion of an anti-BTN3A antibody, the anti-BTN3A antibody including a full-length heavy chain and a light chain and an scFv that binds to a target epitope. In a related more specific embodiment, the scFv is fused to the C-terminal portions of the heavy and light chains of the antibody (bivalent binding specificity for the target epitope). The bispecific molecules of the present disclosure can be single-chain molecules comprising a single-chain antibody and a binding determinant, or single-chain bispecific molecules comprising two binding determinants.

[0213] Binding of the bispecific molecule to its specific target can be confirmed by, for example, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, biological assays (such as growth inhibition and apoptosis), or Western Blot assay. Each of these assays typically detects the presence of a protein-antibody complex of particular interest by employing a labeled reagent (such as an antibody) that is specific for the complex of interest.

[0214] Bcl2 family inhibitor

[0215] The present disclosure relates to the use of an activated BTN3A antibody in combination with at least one Bcl2 family inhibitor.

[0216] In cells, apoptosis is induced by a caspase activation cascade, which can be triggered via extrinsic and intrinsic pathways. The intrinsic pathway is caused by the disruption of mitochondrial integrity regulated by BCL-2 family proteins. B-cell lymphoma 2 (BCL-2) was the first protein identified in this family, hence its name. Members are characterized by the expression of four BCL homology (BH) domains. Six anti-apoptotic (BCL-2, BCL-XL, BCL-w, BCL-2-associated protein A1 (Bfl-1 / A1), myeloid cell leukemia 1 (MCL-1), and BCL-B / Boo) and ten pro-apoptotic family members (including BAX and BAK) have been identified in humans. Under normal conditions, anti-apoptotic proteins bind to the key apoptosis-inducing proteins BAX and BAK, which ensures cell survival. Under stress (such as loss of survival signals, DNA damage, chemicals, or therapeutic agents), this balance is disrupted, mitochondrial membrane integrity is lost, and apoptosis begins (Perini et al., 2018 J Hematol Oncol. 11(1):65; Roberts, Hematology Am Soc Hematol Educ Program. December 4, 2020; 2020(1):1-9).

[0217] 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.

[0218] In one embodiment, the Bcl2 family inhibitor is a Bcl2 inhibitor selected from: (i) BCL-2 and BCL-XL inhibitors; and (ii) selective BCL-2 inhibitors.

[0219] In certain embodiments, inhibitors of BCL-2 family proteins include inhibitors of MCL-1, such as AZD5991 (e.g., CAS number 2143010-83-5) and S64315 (MIK665) (e.g., CAS number 1799631-75-6).

[0220] In certain embodiments, the Bcl2 inhibitor is selected from BCL-2 and BCL-XL inhibitors, and the BCL-2 and BCL-XL inhibitors include: ABT-737 (e.g., CAS number 852808-04-9), navitoclax (e.g., CAS number 923564-51-6), and AZD4320 (e.g., CAS number 1357576-48-7).

[0221] Preferably, the Bcl-2 inhibitor is selected from the group consisting of venetoclax, navitoclax, obatoclax, and even more preferably venetoclax.

[0222] Venetoclax (DCI) (also known as ABT-199) selectively binds to and inhibits the B-cell lymphoma-2 (BCL-2) protein. In some blood cancers, BCL-2 prevents cancer cells from undergoing apoptosis. The IUPAC name of venetoclax is 4-[4-[[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-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.

[0223] Venetoclax has the following formula:

[0224]

[0225] Venetoclax is sold as VENCLEXTA TM in tablet form. Venetoclax is indicated in the United States for: (i) the treatment of adult patients with chronic lymphocytic leukemia (CLL) or small lymphocytic lymphoma (SLL); (ii) in combination with injectable 5-azacytidine or decitabine or low-dose cytarabine for the treatment of newly diagnosed acute myeloid leukemia (AML) in patients 75 years of age or older, or for the treatment of adults with comorbidities that preclude the use of intensive induction chemotherapy. In some embodiments, patients receive 20 mg / m 2 subcutaneously once daily for 10 consecutive days every 4 weeks as low-dose cytarabine.

[0226] Navitoclax (also known as ABT-263) selectively binds to the apoptosis inhibitor proteins Bcl-2, Bcl-XL, and Bcl-w and prevents their binding to the apoptosis effector proteins Bax and Bak. The IUPAC name of navitoclax is 4-[4-[[2-(4-chlorophenyl)-5,5-dimethylcyclohex-1-en-1-yl]methyl]piperazin-1-yl]-N-[4-[[(2R)-4-morpholin-4-yl-1-phenylsulfanylbutan-2-yl]amino]-3-(trifluoromethylsulfonyl)phenyl]sulfonylbenzamide.

[0227] Obatoclax (also known as GX15-070) is a pan-inhibitor of Bcl-2 family proteins and has pro-apoptotic activity. Obatoclax is a selective antagonist of the BH3-binding groove of Bcl-2 family proteins, which are overexpressed in some cancers. The IUPAC name of obatoclax is (2Z)-2-[(5Z)-5-[(3,5-dimethyl-1H-pyrrol-2-yl)methylene]-4-methoxypyrrol-2-ylidene]indole.

[0228] S64315 (also known as MIK665) is a potent and selective inhibitor of MCL-1 and has pro-apoptotic and anti-tumor activities due to the high expression of MCL-1 in a variety of human cancers, including cancers of hematopoietic and lymphoid origin. The IUPAC name of 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.

[0229] In a preferred embodiment, the Bcl-2 inhibitor used in the combination of the present disclosure is Venetoclax.

[0230] Demethylating agent

[0231] In certain embodiments, the combination therapies described herein include a demethylating agent. Demethylating agents, also known as HMAs or demethylating agents, inhibit DNA methylation. In certain embodiments, the demethylating agent blocks the activity of DNA methyltransferases. In certain embodiments, the demethylating agent includes (but is not limited to) azacitidine and decitabine.

[0232] Azacitidine is also known as 5-AC, 5-azacitidine, 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

[0233] Azacitidine is a pyrimidine nucleoside analogue of cytidine with antitumor activity. Azacitidine incorporates into DNA, where it reversibly inhibits DNA methyltransferase, thereby blocking DNA methylation. Demethylation of DNA by azacitidine can activate tumor suppressor genes silenced by hypermethylation, thus producing an antitumor effect. Azacitidine can also incorporate into RNA, thereby disrupting normal RNA function and weakening the activity of tRNA cytosine-5-methyltransferase.

[0234] In some embodiments, azacitidine is administered at a dose of: about 25 mg / m 2 to about 150 mg / m 2 , such as about 50 mg / m 2 to about 100 mg / m 2 , about 70 mg / m 2 to about 80 mg / m 2 , about 50 mg / m 2 to about 75 mg / m 2 , about 75 mg / m 2 to about 125 mg / m 2 , about 50 mg / m 2 , about 75 mg / m 2 , about 100 mg / m 2 , about 125 mg / m 2 or about 150 mg / m 2 . 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, for example, at a dose of about 50 mg / m 2 to about 100 mg / m 2 (such as about 75 mg / m 2 ) for about 5 to 7 consecutive days, for example, in a 28-day cycle. For example, azacitidine can be administered at a dose of about 75 mg / m 2 for seven consecutive days from day 1 to day 7 of a 28-day cycle. As another example, azacitidine can be administered at a dose of about 75 mg / m 2 for five consecutive days from day 1 to day 5 of a 28-day cycle, followed by a two-day break, and then administered for two consecutive days on days 8 to 9. As another example, azacitidine can be administered at a dose of about 75 mg / m 2 for six consecutive days from day 1 to day 6 of a 28-day cycle, followed by a one-day break, and then one administration is allowed on day 8.

[0235] Combination kits and compositions

[0236] Combination kit

[0237] The combinations of the present disclosure that include an anti-BTN3A antibody and a Bcl2 family inhibitor (such as a Bcl2 inhibitor) as previously defined may be presented in the form of a combination kit. As used herein, the term "combination kit" or "kit of parts" according to the present disclosure refers to a pharmaceutical composition or composition for administration, a BTN3A activating antibody (such as mAb1) and a Bcl2 family inhibitor (such as a Bcl2 inhibitor (such as venetoclax)). When the two compounds are administered simultaneously, the combination kit may contain, for example, the components in separate pharmaceutical compositions (suitably an anti-BTN3A antibody and a Bcl2 family inhibitor). When the components (suitably a BTN3A activating antibody and a Bcl2 inhibitor) are not administered simultaneously, the combination kit will contain the active ingredients in separate pharmaceutical compositions (in a single package or in separate packages of separate pharmaceutical compositions).

[0238] In one aspect, there is provided a kit of parts comprising:

[0239] (i) a BTN3A activating antibody, such as mAb1, bound to a pharmaceutically acceptable excipient, diluent or carrier, typically a composition comprising a BTN3A activating antibody as previously defined; and

[0240] (ii) a Bcl2 inhibitor, such as venetoclax, bound to a pharmaceutically acceptable excipient, diluent and / or carrier, typically a composition comprising a Bcl2 inhibitor as previously defined; and

[0241] (iii) an optional demethylating agent, such as azacitidine or decitabine.

[0242] In one embodiment of the present disclosure, the kit of parts comprises:

[0243] (i) a BTN3A activating antibody, such as mAb1, bound to a pharmaceutically acceptable excipient, diluent or carrier, typically a composition comprising a BTN3A activating antibody as previously defined; and

[0244] (ii) a Bcl2 inhibitor, such as venetoclax, bound to a pharmaceutically acceptable excipient, diluent and / or carrier, typically a composition comprising a Bcl2 inhibitor as previously defined; and

[0245] (iii) an optional demethylating agent, such as azacitidine or decitabine, bound to a pharmaceutically acceptable excipient, diluent and / or carrier,

[0246] wherein the components are provided in a form suitable for sequential, separate and / or simultaneous administration.

[0247] In one embodiment, the kit of parts comprises:

[0248] (i) A first container containing a BTN3A activating antibody, said BTN3A activating antibody being combined with a pharmaceutically acceptable excipient, diluent, and / or carrier, typically a composition comprising a BTN3A activating antibody as previously defined; and

[0249] (ii) A second container containing a Bcl2 family inhibitor, such as a Bcl2 inhibitor, said Bcl2 family inhibitor being combined with a pharmaceutically acceptable excipient, diluent, and / or carrier, typically a composition comprising a Bcl2 family inhibitor, such as a Bcl2 inhibitor, as previously defined; and

[0250] (iii) An optional third container containing a demethylating agent as previously defined, such as azacitidine or decitabine.

[0251] The combination kit may also be provided with instructions, such as dosage and administration instructions. Such dosage and administration instructions may be of the type provided to a doctor, or they may be of the type provided by a doctor, such as instructions provided to a patient.

[0252] Composition

[0253] Thus, according to the present disclosure, a BTN3A activating antibody as defined herein and a Bcl2 family inhibitor as defined herein may be formulated separately into independent compositions, for example, with a pharmaceutically acceptable carrier (such as a pharmaceutical composition).

[0254] As used herein, "pharmaceutically acceptable carrier" refers to a diluent, adjuvant, or excipient and includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents and the like that are physiologically compatible. In addition to the active compound (i.e., the BTN3A activating antibody and / or the Bcl2 family inhibitor), the composition may further comprise one or more of the following compounds.

[0255] Sterile phosphate buffered saline is an example of a pharmaceutically acceptable carrier. Other suitable carriers are well known 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.

[0256] The form, route of administration, dosage, and regimen of the pharmaceutical composition naturally depend on the condition being treated, the severity of the disease, the age, weight, and gender of the patient, etc.

[0257] The pharmaceutical compositions of the present disclosure may be formulated for topical, oral, parenteral, intranasal, intravenous, intramuscular, subcutaneous, or intraocular administration, etc.

[0258] Preferably, the pharmaceutical composition contains a pharmaceutically acceptable vehicle for a formulation capable of being injected. These vehicles may in particular be isotonic sterile physiological saline solutions (monosodium or disodium phosphate, sodium chloride, potassium chloride, calcium chloride or magnesium chloride, etc. or mixtures of such salts), or dry, in particular lyophilized, compositions which, when bacteriostatic water or physiological saline is added as appropriate, allow an injectable solution to be constituted.

[0259] The dosage for administration can be adjusted according to various parameters, and in particular according to the mode of administration used, the associated pathology or the duration of treatment desired.

[0260] Compositions and dosing regimens comprising BTN3A activating antibodies

[0261] In some embodiments, a BTN3A activating antibody as defined herein can thus be formulated in a composition (such as the pharmaceutical composition defined above), the composition containing one or a combination of the antibodies disclosed herein, such as an antibody selected from mAb1, mAb2, mAb3, mAb4 and mAb5 or an antigen-binding portion thereof, formulated together with a pharmaceutically acceptable carrier.

[0262] The antibodies of the present disclosure can be formulated into compositions in neutral or salt form as defined above. Pharmaceutically acceptable salts include acid addition salts (formed from the free amino groups of the protein) and are formed from inorganic acids (such as hydrochloric acid or phosphoric acid) or organic acids (such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc.). Salts formed from free carboxyl groups can also be derived from inorganic bases, such as sodium hydroxide, potassium hydroxide, ammonium hydroxide, calcium hydroxide or ferric hydroxide; and organic bases, such as isopropylamine, trimethylamine, histidine, procaine, etc. Suitable formulations for solutions of antibodies for infusion or subcutaneous injection have been 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 as defined above is formulated for intravenous infusion.

[0263] The pharmaceutical composition comprising a BTN3A activating antibody can be formulated at various concentrations. For example, the formulation may contain an activated BTN3A activating antibody at a concentration of 0.1 μΜ - 1 mM, more preferably 1 μΜ - 500 μΜ, 500 μΜ - 1 mM, 300 μΜ - 700 μΜ, 1 μΜ - 200 μΜ, 100 μΜ - 200 μΜ, 200 μΜ - 300 μΜ, 300 μΜ - 400 μΜ, 400 μΜ - 500 μΜ, 500 μΜ - 600 μΜ, 600 μΜ - 700 μΜ, 800 μΜ - 900 μΜ or 900 μΜ - 1 mM. Generally, the formulation contains a BTN3A activating antibody at a concentration of 300 μΜ - 700 μΜ.

[0264] Typically, the therapeutic dose of an activated BTN3A activating antibody in a human patient will be in the range of 100 pg to 700 mg per administration (based on a body weight of 70 kg). For example, the maximum therapeutic dose can be in the range of 0.1 to 10 mg / kg per administration, such as 0.1 - 5 mg / kg or 1 - 5 mg / kg or 0.1 - 2 mg / kg. It should be understood that, as determined by the oncologist / physician, such doses can be administered at different intervals; for example, the dose can be administered daily, twice a week, weekly, every two weeks, every three weeks, or monthly.

[0265] Typically, in certain embodiments, the activated BTN3A activating antibody is administered intravenously at a dose of 20 μg - 200 mg per dose, particularly 1 mg - 200 mg or 7 mg - 200 mg per dose, usually every 21 days.

[0266] In certain embodiments, suitable doses for intravenous administration of an activated anti - BTN3A antibody can be selected from 1, 7, 10, 20, 50, 75, 100, 125, 150, 175, and 200 mg.

[0267] In certain embodiments, the activated BTN3A activating antibody (preferably mAb1 as described below) used according to the methods of the present disclosure is administered intravenously at a dose of 20 μg - 200 mg per dose, and preferably, the second dose is administered at least 15 days (usually about 21 days) after the first dose.

[0268] Compositions and dosing regimens comprising Bcl2 family inhibitors

[0269] Therapy with a Bcl2 family inhibitor can be initiated according to a weekly incremental schedule, reaching the recommended daily dose within a specific time of several days or weeks.

[0270] For the treatment of chronic lymphocytic leukemia (CLL) and small lymphocytic lymphoma (SLL), Venetoclax is currently administered at a daily dose of 20 mg in the first week, 50 mg in the second week, 100 mg in the third week, 200 mg in the fourth week, and 400 mg in the fifth week and beyond the fifth week. For combination therapy of AML with another agent (such as 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 day 3 and beyond day 3. (5 - Azacytidine for injection) is administered intravenously or subcutaneously at a dose of 75 mg / m 2 on days 1 to 7 of each cycle, starting from day 1 of Venetoclax treatment, in a 28 - day cycle.

[0271] In some embodiments, Venetoclax is administered orally. In some embodiments, Venetoclax is administered in the form of tablets. In some embodiments, Venetoclax is administered daily. In some embodiments, Venetoclax is administered at a dose of about 20 mg to about 400 mg, such as 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.

[0272] In some embodiments, 5-azacytidine and Venetoclax are co-administered. 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 in separate dosage forms, such as injectables suitable for intravenous or subcutaneous use and / or tablets or capsules for oral use. In some embodiments, 5-azacytidine and Venetoclax are co-formulated into a single unit dosage form, such as an injectable suitable for intravenous or subcutaneous use or a tablet or capsule for oral use.

[0273] One of ordinary skill in the art can determine other specific dosing regimens with other Bcl2 family inhibitors, particularly with respect to the regimens specified for the therapeutic indications being treated by the combination therapies of the present disclosure.

[0274] Uses and methods of the combinations of the present disclosure

[0275] The present disclosure provides a therapeutic combination comprising a BTN3A activating antibody (e.g., mAb1) and a Bcl2 family inhibitor (e.g., a Bcl2 inhibitor (e.g., Venetoclax)) and optionally a demethylating agent (e.g., azacytidine) as defined previously for treating cancer, particularly hematological malignancies, and more preferably acute myeloid leukemia.

[0276] 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) and a therapeutically effective amount of a Bcl2 family inhibitor (e.g., a Bcl2 inhibitor (e.g., Venetoclax)) and optionally a therapeutically effective amount of a demethylating agent (such as azacytidine or decitabine) in combination, simultaneously, sequentially, or separately.

[0277] As used herein, the terms "treat", "treating", or "treatment" refer to one or more of the following: (1) inhibiting a disease; e.g., inhibiting a disease, disorder, or condition in a subject who is experiencing or displaying a lesion or symptom of the disease, disorder, or condition (i.e., arresting further progression of the lesion and / or symptom); and (2) alleviating a disease; e.g., alleviating a disease, disorder, or condition in a subject who is experiencing or displaying a lesion or symptom of the disease, disorder, or condition (i.e., reversing the lesion and / or symptom), such as reducing the severity of the disease or reducing or alleviating one or more symptoms of the disease. Specifically, with respect to the treatment of acute myeloid leukemia, the term "treatment" may refer to inhibiting the proliferation of AML blasts or reducing the number of AML blasts.

[0278] The antibodies of the present disclosure are BTN3A activating antibodies and can activate the cytolytic function, cytokine production, and / or proliferation of Vγ9Vδ2 T cells, and thus can be used to overcome the immunosuppressive mechanisms observed in cancer patients and during chronic infections (see, in particular, WO2012 / 080769, WO2012 / 080351, and WO2020 / 025703). The results of the present disclosure now show that the combinations of the present disclosure promote further synergistic and specific Vγ9Vδ2 T cell killing of AML blasts in human PBMCs, highlighting their therapeutic importance, particularly for the treatment of hematological malignancies.

[0279] As used herein, the terms "cancer", "hyperproliferation", and "neoplastic" refer to a state or condition in which cells have the ability to grow autonomously, i.e., an abnormal state or condition characterized by rapid proliferative cell growth. Hyperproliferative and neoplastic disease states can be classified as pathological, i.e., characterizing or constituting a disease state, or can be classified as non-pathological, i.e., a deviation from normal but not associated with a disease state. The terms refer to include all types of neoplastic growth or carcinogenic processes, metastatic tissue, or malignantly transformed cells, tissues, or organs (regardless of histopathological type or stage of invasion).

[0280] Examples of cancers 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 blood cancer is myelodysplastic syndrome (MDS) (e.g., lower-risk MDS, such as very low-risk MDS, low-risk MDS, or intermediate-risk MDS, or higher-risk myelodysplastic syndromes, such as high-risk MDS or very high-risk MDS).

[0281] In some embodiments, the subject in need of such treatment is a subject with a hematologic malignancy (such as acute myeloid leukemia) who is not suitable for intensive induction chemotherapy.

[0282] In some embodiments, the subject in need of such treatment is 75 years of age or older and / or has comorbidities.

[0283] In some embodiments, the subject in need of such treatment is a subject indicated for treatment with venetoclax in combination with a demethylating agent according to standard of care.

[0284] Each therapeutic agent in the combination therapy of the present disclosure (i.e., the BTN3A activating antibody, the Bcl2 inhibitor, and optionally the demethylating agent as previously defined) can be administered in separate pharmaceutical agents that are administered sequentially in any order. The anti-BTN3A antibody (such as mAb1), the Bcl2 family inhibitor (such as the Bcl2 inhibitor (such as venetoclax)), and the demethylating agent (such as azacitidine) are typically formulated as separate compositions as previously described.

[0285] Sequential administration (such as in separate pharmaceutical compositions) is particularly applicable when the therapeutic agents in the combination therapy are in different dosage forms (one agent is a tablet or capsule and the other agent is a sterile liquid) and / or are administered according to different dosing schedules (such as the chemotherapeutic agent is administered at least daily and the biotherapeutic agent is administered at a lower frequency, such as once a week, once every two weeks, or once every three weeks).

[0286] In some embodiments, the BTN3A activating antibody (such as mAb1) is administered before the first administration of the Bcl2 family inhibitor (such as the Bcl2 inhibitor (such as venetoclax)), while in other embodiments, the BTN3A activating antibody (such as mAb1) is administered after the administration of the Bcl2 family inhibitor (such as the Bcl2 inhibitor (such as venetoclax)).

[0287] The dosing regimen (also referred to herein as the administration regimen) selected for the combination therapies of the present disclosure depends on a number of factors, including the serum or tissue turnover rate of the entity, the degree of the symptoms, the immunogenicity of the entity, and the accessibility of the target cells, tissues, or organs in the subject being treated. Preferably, the dosing regimen maximizes the amount of each therapeutic agent delivered to the patient while conforming to an acceptable level of side effects. Thus, the dose and dosing frequency of each biotherapeutic and chemotherapeutic agent in the combination depends in part on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Guidance for selecting appropriate doses of antibodies, cytokines, and small molecules is available. See, e.g., Wawrzynczak (1996) Antibody Therapy, BiosScientific 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 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 appropriate dosing regimen can be determined by a clinician, e.g., using parameters or factors known or suspected to affect treatment or predicted to affect treatment in the art, and will depend, e.g., on the patient's clinical history (e.g., prior therapies), the type and stage of the cancer being treated, and biomarkers of response to one or more of the therapeutic agents in the combination therapy.

[0288] Any suitable dosage range may be determined by the attending medical staff. The dosing regimen may be adjusted to provide the desired optimal response (e.g., therapeutic or prophylactic response). Dosages have been disclosed in the previous sections related to Bcl2 family inhibitors. In some embodiments of the combination therapy, a suitable dosage range for a Bcl2 family inhibitor as defined herein and in particular venetoclax may be, for example, from 10 mg to 600 mg. In some embodiments, an exemplary dosage is from about 100 mg to 400 mg. In some embodiments, the dosing regimen includes providing the Bcl2 family inhibitor (e.g., a Bcl2 inhibitor (e.g., venetoclax)) daily.

[0289] In some embodiments, at least one therapeutic agent in the combination therapy is administered using the same dosing regimen (dosage, frequency, and duration of treatment) that is typically employed when the agent is used as a single therapy for treating the same cancer.

[0290] For example, venetoclax is administered in combination with a demethylating agent (such as azacitidine) using the same dosing regimen recommended for treating patients recently diagnosed with acute myeloid leukemia (see “Practical Dosing Considerations for Venetoclax”, Cancernetwork.com, Oncology, Volume 33, Issue 9, Volume 33, Issue 9).

[0291] When a Bcl2 family inhibitor (e.g., a Bcl2 inhibitor (e.g., venetoclax)) is administered in combination with a demethylating agent (e.g., azacitidine), the demethylating agent may be administered subcutaneously once daily at 75 mg / m 2 on days 1 to 5 and days 8 to 9 of each cycle. In the event of lymphopenia, a recovery period of up to 14 days between cycles is recommended (Jonas and Pollyea, 2019; Leukemia 33:2795 - 2804), and growth factors are administered to treat at least neutropenia. Subsequent cycles are determined by the patient's response, but are very similar to Cycle 1.

[0292] In other embodiments, the patient receives a lower total amount (e.g., a smaller dose, less frequent dosing, and / or a shorter treatment duration) of at least one therapeutic agent in the combination therapy than when the agent is used as a single therapy.

[0293] Regarding the dosing regimen for the BTN3A activating antibody used in the treatment combinations of the present disclosure, any suitable dosage range determined by the attending medical staff may be used.

[0294] The dosing regimen can be adjusted to provide the optimal desired response (e.g., therapeutic or prophylactic response). The antibodies of the present disclosure can be formulated in a therapeutic mixture to contain from about 1 to 200.0 milligrams. It should be understood that such doses can be administered at different intervals as determined by the oncologist / physician; for example, the dose can be administered daily, twice a week, weekly, bi-weekly, tri-weekly, or monthly.

[0295] Generally, in certain embodiments, the BTN3A activating antibody (e.g., mAb1) is typically administered intravenously every 21 days at a dose of 1 mg - 200 mg per dose, such as 20 - 100 mg. In certain embodiments, suitable doses for intravenous administration of the activated BTN3A antibody (e.g., mAb1) can be selected from 1, 7, 10, 20, 50, 75, 100, 125, 150, 175, and 200 mg.

[0296] In certain embodiments, the activated BTN3A antibody (preferably mAb1 as described below) used in the combination methods of the present disclosure is administered intravenously at a dose of 7 mg - 200 mg per dose, preferably, the second dose is administered at least 15 days after the first dose, typically once every 21 days, for 1 to 22 cycles.

[0297] In some embodiments, a Bcl-2 family inhibitor (e.g., a Bcl-2 inhibitor (e.g., venetoclax)) is administered in combination, separately, sequentially, or simultaneously at a dose of from about 10 mg to about 500 mg, such as from about 20 mg to about 400 mg, from about 50 mg to about 350 mg, from about 100 mg to about 300 mg, from about 150 mg to about 250 mg, 50 mg to about 500 mg, from about 100 mg to about 500 mg, from about 150 mg to about 500 mg, from about 200 mg to about 500 mg, from about 250 mg to about 500 mg, from about 300 mg to about 500 mg, from about 350 mg to about 500 mg, from about 400 mg to about 500 mg, from about 450 mg to about 500 mg, from about 10 mg to about 400 mg, from about 10 mg to about 350 mg, from about 10 mg to 300 mg, from about 10 mg to about 250 mg, from about 10 mg to about 200 mg, from about 10 mg to about 150 mg, from about 10 mg to about 100 mg, from about 10 mg to about 50 mg, from about 50 mg to about 150 mg, from about 150 mg to about 250 mg, from about 250 mg to about 350 mg, or from about 350 mg to about 400 mg. In some embodiments, a 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, a Bcl-2 inhibitor (e.g., venetoclax) is administered once daily. In some embodiments, a Bcl-2 family inhibitor (e.g., a Bcl-2 inhibitor) is administered orally.

[0298] In some embodiments, a Bcl-2 family inhibitor (e.g., a Bcl-2 inhibitor (e.g., venetoclax)) is administered orally once daily, such as daily during a 21-day cycle, at a dose of from about 350 mg to about 450 mg (e.g., about 400 mg). In some embodiments, the dose of the Bcl-2 inhibitor is escalated over a 4-day period in the first cycle to reach a dose of about 400 mg / day. For example, the doses on days 1, 2, 3, and 4 and thereafter in cycle 1 are about 100 mg, about 200 mg, about 300 mg, and about 400 mg, respectively.

[0299] In some embodiments, a Bcl-2 family inhibitor (e.g., a Bcl-2 inhibitor such as venetoclax) is administered for, e.g., about 5 weeks during a ramp-up period, followed by administration at a fixed dose for, e.g., at least about 24 months. In some embodiments, the Bcl-2 inhibitor is administered at the following doses: about 10 mg to about 30 mg once daily (e.g., about 20 mg) for, e.g., about 1 week; then about 40 mg to about 60 mg once daily (e.g., about 50 mg) for, e.g., about 1 week; then about 80 mg to about 120 mg once daily (e.g., about 100 mg) for, e.g., about 1 week; then about 150 mg to about 250 mg once daily (e.g., about 200 mg) for, e.g., about 1 week; then about 350 mg to about 450 mg once daily (e.g., about 400 mg) for, e.g., about 1 week; and then at a fixed dose, about 350 mg to about 450 mg once daily (e.g., about 400 mg) for, e.g., at least about 24 months.

[0300] In certain embodiments, a Bcl2 family inhibitor (e.g., a Bcl-2 inhibitor such as venetoclax) is administered once daily at a unit dose of 10 mg - 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.

[0301] In certain embodiments, a Bcl2 family inhibitor (e.g., a Bcl-2 inhibitor such as venetoclax) and a demethylating agent (e.g., azacitidine) are first administered for at least one cycle, followed by a recovery period of at least 10 to 14 days, and the BTN3A activating antibody is administered, e.g., after at least 10 to 14 days of the recovery period, or together with the second cycle of administration of the Bcl2 inhibitor and the demethylating agent.

[0302] In other certain embodiments, the first administration of a Bcl2 family inhibitor (e.g., a Bcl-2 inhibitor such as venetoclax) and a demethylating agent (e.g., azacitidine) occurs after the first cycle of treatment with a BTN3A activating antibody (e.g., mAb1), preferably after 21 days after the first administration of the BTN3A activating antibody.

[0303] In certain embodiments, the activating BTN3A antibody (preferably mAb1 as described below) used in accordance with the methods of the present disclosure is administered intravenously at a dose of 7 mg - 200 mg per 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; and the Bcl2 family inhibitor (such as a Bcl-2 inhibitor (such as venetoclax)) is administered, for example, at a unit dose of 10 mg - 600 mg, optionally in combination with azacitidine or decitabine once daily for at least 5 to 9 days, followed by a recovery period of at least 14 days, and wherein venetoclax and the demethylating agent (such as azacitidine) are administered first 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, after said recovery period of at least 10 to 14 days.

[0304] In certain embodiments, the combination therapies as disclosed herein (generally mAb1 and a Bcl2 family inhibitor (such as a Bcl-2 inhibitor, particularly venetoclax) as previously described) can be administered in combination with other anti-tumor agents.

[0305] In other certain embodiments, the combination therapies as disclosed herein (generally mAb1 and the Bcl2 family inhibitor (such as a Bcl-2 inhibitor, particularly venetoclax) as previously described) can be administered in combination with cell therapies (particularly γδ T cell therapies).

[0306] The present disclosure thus relates to a combination as defined herein for enhancing in vivo tumor cells in γδ T cell therapy in a subject in need (generally suffering from cancer), wherein the BTN3A activating antibody and the Bcl2 family inhibitor (such as a Bcl-2 inhibitor) can be administered to the subject concomitantly, simultaneously, in parallel, or sequentially.

[0307] As used herein, the term γδ T cell therapy refers to a therapy comprising administering to a subject in need at least an effective amount of γδ T cells. Such γδ T cells can be allogeneic or autologous. In certain embodiments, the γδ T cells can be genetically engineered by deletion or knockout or insertion or knock-in of specific genes. In certain embodiments, the γδ T cells include γδ T cells expressing a chimeric antigen receptor. The γδ T cells can have been expanded and / or purified in vitro. Alternatively, the γδ T cells can also be included in a cell composition that includes other blood cells and other cells of the immune system, for example. For references regarding γδ T cell therapy, see Pauza CD. et al., Front Immunol. June 8, 2018; 9:1305. doi:10.3389; Saudemont A. et al., Front Immunol. February 5, 2018; 9:153. doi:10.3389.

[0308] Accordingly, the present disclosure relates to a method of treating a subject having cancer and having tumor cells (such as hematological tumor cells), said cancer including solid tumors or hematological malignancies, in particular leukemia (such as acute myeloid leukemia), said method comprising:

[0309] (i) administering to the subject a combination of an effective amount of an anti-BTN3A activating antibody as disclosed herein (usually mAb1, mAb2, mAb3, mAb4 or mAb5), an effective amount of a Bcl2 inhibitor (such as venetoclax), and optionally a demethylating agent (such as azacitidine), and,

[0310] (ii) administering to the subject an effective amount of a γδT cell composition,

[0311] wherein the combination of the effective amount of the anti-BTN3A antibody and the effective amount of the Bcl2 inhibitor has the ability to enhance the anti-tumor cytolysis against the tumor cells mediated by the γδT cell composition.

[0312] The present disclosure, which has been fully described, is further illustrated by the following examples, which are illustrative only and are not intended to further limit.

[0313] Table 4: Sequences used in the present disclosure

[0314]

[0315]

[0316]

[0317]

[0318]

[0319]

[0320]

[0321]

[0322] Detailed Description

[0323] Examples

[0324] 1. Methods and assays of the present disclosure

[0325] Method for characterizing a BTN3A activating antibody for use according to the present disclosure

[0326] 1.1 Binding affinity determination: Multi-cycle kinetics assay (SPR)

[0327] The anti-BTN3A antibody can be subjected to multi-cycle kinetic analysis using a Biacore T200 instrument (serial number 1909913) running Biacore T200 evaluation software V2.0.1 (Uppsala, Sweden).

[0328] The purified antibody was diluted to a concentration of 2 μg / ml in 2% BSA / PBS. At the start of each cycle, each antibody was captured on Protein A at a density (RL) of approximately 146.5 RU (theoretical value to obtain an RMax of approximately 50 RU). After capture, the surface was stabilized before injecting the BTN3A1 antigen (Sino Biological catalog number 15973-H08H). BTN3A1 was titrated in a two-fold dilution range from 25 to 0.78 nM in 0.1% BSA / HBS-P+ (running buffer). The binding phase was monitored for 400 seconds and the dissociation phase was monitored for 35 minutes (2100 seconds). Kinetic data was obtained using a flow rate of 50 μl / min to minimize any potential mass transfer effects. At the end of each cycle, two injections of 10 mM glycine-HCL pH 1.5 were used to regenerate the Protein A surface. Two blank tests (without BTN3A1) and one replicate test of a single concentration of analyte were performed for each test antibody to check the stability of the surface and analyte within the kinetic cycles. The signal from Fc2, Fc3, and Fc4 was subtracted from the signal from the reference channel Fc1 to correct for differences in non-specific binding to the reference surface. Additionally, a blank run was subtracted for each Fc to correct for any antigen-independent signal variations, such as offsets. The sensorgrams were fitted using a one-to-one binding mathematical model under the global RMax parameter and no bulk signal (constant RI = 0 RU).

[0329] 1.2 Binding assay of human PBMCs by flow cytometry

[0330] The BTN3A activating antibodies for use according to the present disclosure can also be characterized for their binding to human PBMCs (isolated from the blood of healthy donors). PBMCs were isolated from the buffy coat by density centrifugation using Lymphoprep (Axis-shield, Dundee, UK). Subsequently, the PBMCs were frozen and stored at -80 °C or in liquid nitrogen until needed.

[0331] 100 μl of cells (1×10 6 cells / ml) were transferred to each well of a freshly made U-bottom 96-well plate, and then the plate was centrifuged and the supernatant was discarded.

[0332] Prepare serial dilutions of the antibody from 0.001 μg / ml to 150 μg / ml in PBS containing 2 mM EDTA. Resuspend human PBMCs in 50 μl of the prepared diluted test antibody titration series.

[0333] After incubating for 30 minutes at 4°C in the dark, centrifuge the plate and wash twice with 150 μl / well of PBS + 2 mM EDTA, then resuspend the cells in 50 μl of a mixture consisting of goat anti-human antibody (PE-labeled) diluted 1 / 100 in PBS + 2 mM EDTA and Live / Dead Fixable Violet diluted 1 / 500.

[0334] After incubating for 15 minutes at 4°C in the dark, centrifuge the plate and wash once with 150 μl / well PBS + 2 mM EDTA, then resuspend the cells in 200 μl of PBS + 2 mM EDTA. Analyze the cells on a BD LSR Fortessa cell counter. Analyze the data using FlowJo software (version 10, FlowJo, LLC, Ashland, USA).

[0335] The same protocol can be performed on cynomolgus macaque PBMCs and on the Daudi Burkitt's lymphoma cell line.

[0336] 1.3 In vitro functional efficacy: γδ-T cell degranulation assay

[0337] The assay consists of measuring the activation or inhibition of anti-BTN3A antibodies on γδ-T cell degranulation against the Daudi Burkitt lymphoma cell line ((Harly et al., 2012, Blood Vol. 120, No. 11, pp. 2269-2279). γδ-T cells were expanded from PBMC of healthy donors by culturing with zoledronic acid (1 μM) and IL2 (200 UI / mL) for 11 to 13 days. IL2 was added on day 5, day 8, and every 2 days thereafter. The percentage of γδ-T cells was determined at the start of the culture and the culture time was evaluated by flow cytometry until it reached at least 80%. Subsequently, cryopreserved or fresh γδ-T cells were used for the degranulation assay against the Daudi cell line (effector:target (E:T) ratio of 1:1), and the cells were co-cultured at 37 °C for 4 hours in the presence of 10 μg / mL 7.2 and / or 20.1 humanized variants and / or their chimeric forms. Activation with PMA (20 ng / mL) plus ionomycin (1 μg / mL) served as a positive control for γδ-T cell degranulation, and medium alone served as a negative control. At the end of the 4-hour co-incubation, the cells were analyzed by flow cytometry to assess the percentage of γδ-T cells positive for CD107a (LAMP-1, lysosome-associated membrane protein-1) + CD107b (LAMP-2). CD107 is mobilized to the cell surface after activation-induced granule exocytosis, and thus measurement of surface CD107 is a sensitive marker for identifying recently degranulated cytolytic T cells.

[0338] The same protocol can be performed using AML blasts isolated from the patient instead of Daudi cells as target cells.

[0339] 1.4 In vitro functional efficacy: Vγ9Vδ2 T cell activation in PBMC

[0340] The assay consists of measuring the activation of BTN3A activating antibodies on Vγ9Vδ2 T cells in PBMC. Human PBMC were isolated by Ficoll density gradient centrifugation of peripheral blood (EDTA-buffy coat or heparinized whole blood). When using whole blood, RBC were depleted using 1× RBC lysis buffer (eBioscience) for 10 minutes at room temperature and then washed with PBS + 1% FBS.

[0341] At 37 °C, 5% CO2, in a volume of 200 μL, in a 96-well round-bottom plate, PBMC or RBC-depleted cells were incubated with increasing concentrations of BTN3A activating antibodies (dose range from 0.00001 to 100 μg / mL) in RPMI 1640 containing 10% FBS, 1% penicillin / streptomycin at 1.5 to 3×10 6Cultured at [X] cells / mL. After culturing for two days, the activation status was monitored by analyzing the surface expression of activation markers by flow cytometry. Cells were washed in PBS + 2% FBS and 2 mM EDTA (FACS buffer). Cells were centrifuged at 1800 rpm for 5 minutes and then incubated with 10 μL of FcR blocking reagent (Miltenyi Biotec) for 10 minutes at room temperature (RT), followed by the addition of 30 to 50 μL of an appropriate antibody mixture prepared in FACS buffer, which contained at least fluorescent-conjugated anti-CD3, anti-Vγ9 or Vδ2 TCR, and anti-CD69 antibodies. A viability marker (LIVE / DEAD Fixable Dead Cell Stain) was added in all experiments to exclude dead cells from the analysis. Cells were incubated at 4 °C for 30 minutes and washed twice in FACS buffer, then fixed in Cytofix Fixation Buffer (BD Bioscience) and subjected to flow cytometry analysis. Data were analyzed using flowjo V-10.6 software. Activated Vγ9Vδ2 T cells were defined as CD3+Vδ2+ (or Vγ9+ or Vδ2+Vγ9+)CD69+.

[0342] 1.5 In vitro Vγ9Vδ2 T cell expansion

[0343] Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation of peripheral blood obtained from the French Blood Establishment (Etablissement du Sang, EFS) in Provence-Alpes-Côte d'Azur (France). To expand Vγ9Vδ2 T cells, 300×10 6 PBMCs were resuspended at 1.5×10 6 cells / mL in RPMI 1640 supplemented with 10% FBS and 1% sodium pyruvate in a 75 cm2 flask for 10 to 14 days in the presence of rHuIL-2 (200 UI / mL) and an amino bisphosphonate (zoledronate, 1 μM). Starting from day 5, rHuIL-2 was renewed every 2 or 3 days and the cells were maintained at 1×10 6 cells / mL. At the end of the expansion phase, the purity of Vγ9Vδ2 T cells was evaluated by flow cytometry, and if the number of Vγ9Vδ2 T cells reached 80% of the live cells, these cells were then frozen in CryoStor CS10 for future use.

[0344] 1.6 Mouse model

[0345] The selected mouse strain was the highly immunodeficient NSG mouse lacking mature T cells, B cells, and natural killer (NK) cells and also lacking multiple cytokine signaling pathways, thus making it an ideal choice for human cell transplantation. Female NSG mice at six to eight weeks of age were co-housed in disposable standard cages on a ventilated rack in the TrGET platform facility (Cancer Research Center of Marseille, France). The mice were housed under sterile conditions with autoclaved food and water provided ad libitum and maintained on a 12-hour light and 12-hour dark cycle with temperature and humidity controlled. The cages had an enriched environment and bedding material.

[0346] NSG mice were intravenously (iv) injected via the tail vein with 0.2 × 10 6 MOLM-14 (CVCL_7916) cells expressing luciferase (luc2) per mouse, with a volume of 100 μl, on day 0. Bioluminescence analysis was performed on day 0 using a PhotonIMAGER (Biospace Lab) after adding endotoxin-free luciferin (30 mg / kg), and the mice were randomly divided into homogeneous groups of 6 to 8 mice based on the bioluminescence signal intensity (Table 5). On days 1, 8, 15, and 23, 3 × 10 6 human in vitro-expanded Vγ9Vδ2 T cells and rHuIL-15 / IL-15Rα-Fc complex were iv injected. On days 1, 5, 8, 11, 15, 18, 23, and 25, ICT01 or hIgG1S was iv injected.

[0347] The rHuIL-15 / IL-15Rα-Fc complex was pre-complexed for 30 minutes 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.

[0348] Venetoclax was administered to the mice via oral gavage (og) from day 1 to day 4 and then to the mice 5 days a week for a total of 3 weeks. 5-Azacytidine was administered via intraperitoneal (ip) injection concomitantly with venetoclax treatment from day 1 to day 4. Venetoclax and 5-azacytidine were administered 4 to 6 hours after Vγ9Vδ2 T cell transfer.

[0349] At days 0, 7, 14, 21, and 28 after cell injection, the bioluminescence signals emitted by MOLM-14 cells were measured to track tumor growth. The disease symptoms of the mice (significant weight loss, wrinkled fur, hunchback, weakness, and reduced activity) were monitored daily to determine the killing time of the injected animals with signs of distress. Survival curves were estimated by the Kaplan–Meier method and compared using the log-rank test.

[0350] 2. Results

[0351] 2.1 Activation of Vγ9Vδ2 T cells mediated by ICT01 increases resistance to inhibitors of Bcl-2 family members.

[0352] In peripheral blood mononuclear cells (HD-PBMC) from healthy donors, the effect of ICT01 on the survival of Vγ9Vδ2 T cells treated with inhibitors of Bcl-2 family members was evaluated using the apoptosis marker caspase 3 / 7 and the dead cell marker in flow cytometry analysis.

[0353] Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll density gradient centrifugation (EDTA-buffy coat) of peripheral blood from healthy donors (HD, n = 4–6), 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) used 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 the combination or ABT-737 (0–1.875 μM), navitoclax (0–1.875 μM), or MIK665 (0–1.875 μM). The cells were harvested on day 2, centrifuged at 1800 rpm for 5 minutes and then incubated with 25 μl of FcR blocking reagent (Miltenyi Biotec) for 10 minutes at room temperature (RT), followed by the addition of 25 μL of the following antibody mixture prepared in PBS + 2% FBS + 2 mM EDTA (FACS buffer): anti-CD4-BUV395, CD56-BV421, Vd2-PE, CD3-PE-CF594, and CD8-A700 antibodies for identifying immune subsets, anti-CD25-BV650 and CD69-APC antibodies to monitor the activation status of Vγ9Vδ2 T cells, and the viability marker (LIVE / DEAD TMA fixable dead cell stain was used to distinguish live and dead cells. Cells were incubated at room temperature (RT) for 20 minutes and washed twice with FACS buffer. An apoptosis protease 3 / 7 green detection reagent (ThermoFisher) was added to measure apoptosis and cells were incubated at room temperature for 30 minutes, followed by flow cytometry analysis.

[0354] Although 5-azacytidine had no effect on the survival of Vγ9Vδ2 T cells, treatment with venetoclax led to apoptosis and death of Vγ9Vδ2 T cells in a concentration-dependent manner (treatment with 0.01 μM venetoclax reduced viable cells by approximately 29% and treatment with 0.1 μM venetoclax reduced cells by up to approximately 49% compared to the no-venetoclax condition) ( Figure 1 a, Figure 1 b). In contrast, co-treatment with ICT01 significantly reduced venetoclax-induced cell death (treatment with 0.01 μM venetoclax reduced the survival rate by approximately 11% and treatment with 0.1 μM venetoclax reduced the survival rate by approximately 30% compared to the no-venetoclax condition) ( Figure 1 c, Figure 1 d). As expected, specific activation of Vγ9Vδ2 T cells induced by ICT01 showed increased expression of CD69 and CD25 2 days after treatment compared to hIgG1S ( Figure 1 e and data not shown). This indicates that activation of Vγ9Vδ2 T cells by ICT01 partially protects them from venetoclax-induced cell death.

[0355] As described for venetoclax, treatment with other Bcl-2 family member inhibitors induced apoptosis and death of Vγ9Vδ2 T cells in a concentration-dependent manner, with EC 50 values for ABT-737, navitoclax, and MIK665 of 0.24 μM, 0.16 μM, and 0.24 μM, respectively ( Figure 2 a). When PBMC were co-treated with ICT01, a significant decrease in Vγ9Vδ2 T cell death induced by Bcl-2 family member inhibitors was observed. For ABT-737, the EC 50 for Vγ9Vδ2 T cell death increased from 0.23 μM in the presence of the isotype control to 0.46 μM in the presence of ICT01 ( Figure 2 b), for navitoclax, the EC 50 for Vγ9Vδ2 T cell death increased from 0.16 μM in the presence of the isotype control to 0.20 μM in the presence of ICT01 ( Figure 2 c), and for MIK665, the EC 50Increased from 0.24 μM in the presence of the isotype control to 0.78 μM in the presence of ICT01( Figure 2 d).

[0356] In a similar experiment, freshly isolated PBMCs were stimulated with ICT01 or its isotype control hIgG1S for 1 day prior to 2-day treatment with venetoclax( Figure 3 a). At the end of the incubation period, the number of viable Vγ9Vδ2 T cells was evaluated by flow cytometry. The results showed that activation with ICT01 (confirmed by increased surface expression of CD25( Figure 3 b)) significantly reduced venetoclax-induced cell death of Vγ9Vδ2 T cells( Figure 3 c, Figure 3 d).

[0357] Collectively, these results indicate that in vitro activation of Vγ9Vδ2 T cells by ICT01 protects Vγ9Vδ2 T cells from cell death induced by venetoclax and inhibitors of other Bcl-2 family members. This would provide an advantage for monotherapy in terms of the number of Vγ9Vδ2 T cells, which are important in anti-cancer immunity and can cooperate with venetoclax (or other Bcl-2 family member inhibitors) to kill AML blasts.

[0358] 2.2 Venetoclax and 5-azacytidine do not affect ICT01-induced activation and proliferation of Vγ9Vδ2 T cells

[0359] ICT01 has been shown to induce activation and proliferation of resting Vγ9Vδ2 T cells. To further evaluate the effects of venetoclax and 5-azacytidine on Vγ9Vδ2 T cells, ICT01-induced phenotypic activation, IFNγ production, and proliferation were measured in healthy donor-derived PBMCs cultured with or without venetoclax, 5-azacytidine, or combinations thereof.

[0360] Phenotypic activation of ICT01-induced Vγ9Vδ2 T cells is indicated by upregulation of CD25. Thus, in the presence or absence of increasing concentrations of venetoclax (0 - 0.1 μM), 5-azacytidine (0 - 0.4 μM), or combinations thereof, human PBMCs from healthy donors (HD, n = 6) 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) used at 1 μg / mL. Cells were harvested on day 4, centrifuged at 1800 rpm for 5 minutes and then incubated with 25 μl of FcR blocking reagent (Miltenyi Biotec) for 10 minutes at room temperature (RT), followed by addition of 25 μL of the following antibody mixture prepared in PBS + 2% FBS + 2 mM EDTA (FACS buffer): anti-CD4-BV650, CD56-FITC, Vδ2-PE, CD3-PE-CF594, and CD8-A700 antibodies for identification of immune subsets, anti-CD25-BV650 antibody to monitor activation status, and viability marker (LIVE / DEAD TM Fixable Dead Cell Stain) to distinguish live and dead cells. Cells were incubated at 4 °C for 20 minutes and washed twice in FACS buffer, followed by flow cytometry analysis.

[0361] 5-azacytidine and venetoclax had no effect on the upregulation of CD25 of ICT01-induced Vγ9Vδ2 T cells and thus had no effect on their phenotypic activation ( Figure 4 a).

[0362] In a similar experiment, freshly isolated healthy donor-derived PBMCs were treated with increasing concentrations of venetoclax (0 - 0.1 μM), 5-azacytidine (0 - 0.4 μM), or combinations thereof for one day prior to stimulation with ICT01 or its isotype control hIgG1S for two days. Additionally, activation of Vγ9Vδ2 T cells by ICT01 was not affected by venetoclax and 5-azacytidine treatment as indicated by similar induction of CD25 expression under all tested conditions ( Figure 4 b).

[0363] The function of Vγ9Vδ2 T cells is tightly regulated by activating (NKG2D and DNAM-1) and inhibitory receptors (PD-1, NKG2A, BTLA, TIM3). To evaluate the effect of venetoclax and 5-azacytidine treatment on the expression of these receptors on Vγ9Vδ2 T cells, frozen human PBMCs from healthy donors (HD, n = 6) were thawed and treated with ICT01 or its isotype control (hIgG1S) used 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 IL2) in the presence or absence of increasing concentrations of venetoclax (0 - 0.1 μM), 5-azacytidine (0 - 0.4 μM), or combinations. Cells were harvested on day 5, centrifuged at 1800 rpm for 5 minutes and then incubated with 25 μl of FcR blocking reagent (Miltenyi Biotec) for 10 minutes at room temperature, followed by the addition of 25 μL of the following antibody mixture prepared in PBS + 2% FBS + 2 mM EDTA (FACS buffer): anti-CD4-BUV395, Vδ2-PE, CD3-PE-CF594, and CD8-A700 antibodies for identifying immune subsets, anti-BTLA-BV421, PD1-BV650, DNAM-1-BV785, TIM3-BB515, NKG2A-PE-Vio770, and NKG2D-APC antibodies for detecting inhibitory and activating receptors, and addition of a viability marker (LIVE / DEAD TM Fixable Dead Cell Stain) to distinguish live and dead cells. Cells were incubated at 4 °C for 20 minutes and washed twice in FACS buffer, followed by flow cytometry analysis.

[0364] ICT01 induced an increase in the expression of all analyzed activating and inhibitory receptors on Vγ9Vδ2 T cells on day 5 of culture, which was not affected by treatment with venetoclax and 5-azacytidine (data not shown).

[0365] To evaluate the effects of venetoclax and 5-azacytidine on cytokine production, freshly isolated human PBMCs from healthy donors (HD, n = 6) 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) used 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 the combination. Half of the cells were additionally treated with 50 IU / mL IL2. Golgi Stop was added after 6 to 8 hours of co-culture and the cells were harvested after 16 hours, centrifuged at 1800 rpm for 5 minutes and then incubated for 10 minutes at room temperature (RT) with 25 μl of FcR blocking reagent (Miltenyi Biotec), followed by addition of 25 μL of the following antibody mixture prepared in PBS + 2% FBS + 2 mM EDTA (FACS buffer): anti-CD4-BV650, Vγ9-FITC, CD3-PE-CF594, CD56-PE-Vio770, and CD8-AF700 antibodies for identification of immune subsets, and addition of a viability marker (LIVE / DEAD TM Fixable Dead Cell Stain) to distinguish live and dead cells. After 20 minutes, 100 μL of CytoFix solution (BD Bioscience) was added and the cells were incubated at 4°C for 20 minutes. 100 μL of Perm / Wash (BD Bioscience) was added, the cells were centrifuged and washed once in 200 μL of Perm / Wash. After washing, the cells were stained with anti-IFNγ-APC antibody at 4°C for 30 minutes, washed twice with Perm / Wash and analyzed by flow cytometry.

[0366] Treatment with venetoclax or 5-azacytidine alone had no effect on IFNγ production by ICT01-induced IL2-independent Vγ9Vδ2 T cells (data not shown). When activated with ICT01 alone, treatment with the combination of venetoclax and 5-azacytidine reduced IFNγ production by Vγ9Vδ2 T cells from 30.4% to 25.2%, which was not observed in the presence of IL2 (untreated was 89.1% and treated with the combination of venetoclax and 5-azacytidine was 91.7%, data not shown).

[0367] Finally, freshly isolated human PBMCs from healthy donors (HD, n = 6) were labeled with CellTrace Violet stain (CTV, Invitrogen) to determine cell proliferation in PBMC medium (RPMI 1640 medium supplemented with 10% FBS, 1% penicillin / streptomycin, and 1 mM sodium pyruvate) in the presence or absence of increasing concentrations of venetoclax (0 - 0.1 μM), 5-azacytidine (0 - 0.4 μM), or the combination, after treatment with ICT01 or its isotype control (hIgG1S) used at 1 μg / mL. Half of the cells were additionally treated with 50 IU / mL IL2. Cells were harvested on day 4, centrifuged at 1800 rpm for 5 minutes and then incubated with 25 μl of FcR blocking reagent (Miltenyi Biotec) for 10 minutes at room temperature, followed by addition of 25 μL of the following antibody mixture prepared in PBS + 2% FBS + 2 mM EDTA (FACS buffer): anti-CD4-BV650, CD56-FITC, Vδ2-PE, CD3-PE-CF594, and CD8-A700 antibodies for identification of immune subsets, and addition of a viability marker (LIVE / DEAD TM Fixable Dead Cell Stain) to distinguish live and dead cells. Cells were incubated at 4 °C for 20 minutes and washed twice in FACS buffer, followed by flow cytometry analysis.

[0368] Venetoclax increased the proliferation of ICT01-induced Vγ9Vδ2 T cells by approximately 10%, which was not observed when using 5-azacytidine alone or in combination with both reagents ( Figure 4 c). The proliferation of Vγ9Vδ2 T cells induced by ICT01 and IL2 was not affected by venetoclax and 5-azacytidine used alone or in combination ( Figure 4 d).

[0369] In summary, the data indicate that venetoclax and 5-azacytidine as single agents or in combination do not interfere with the activation or proliferation of ICT01-induced Vγ9Vδ2 T cells. Thus, the combination treatment will protect Vγ9Vδ2 T cells from venetoclax-induced cell death, induce their proliferation and activation, and potentially increase the number of cytotoxic Vγ9Vδ2 T cells in patients.

[0370] 2.3 Treatment of Vγ9Vδ2 T cells with a combination of venetoclax, HMA, and BTN3A activating antibodies increases AML killing

[0371] The effects of venetoclax and 5-azacytidine against anti-BTN3A antibody-induced Vγ9Vδ2 T cell killing of AML cell lines were evaluated by measuring the relative number of viable target cells in HD-PBMC-AML cell co-cultures using flow cytometry.

[0372] First, to evaluate the sensitivity of the KG1a AML cell line to venetoclax and 5-azacytidine treatment in vitro, cells were treated with increasing concentrations of venetoclax (0 - 5 μM), 5-azacytidine (0 - 10 μM), or the combination in cell culture medium (α-MEM+Glutamax supplemented with 20% fetal bovine serum (FBS) and 1 mM sodium pyruvate) for 2 days. On day 2, the cells were centrifuged at 1800 rpm for 5 minutes and the supernatant was discarded. The cells were lysed with Cell Titer Glo reagent (Promega), and the amount of ATP was monitored using fluorescence as a measure of viable cells.

[0373] KG1a AML cells showed an EC 50 value of 1.82 μM for venetoclax and an EC 50 value of 4.72 μM for 5-azacytidine, where the average viability of venetoclax at 5 μM was 20.2% and the average viability of 5-azacytidine at 10 μM was 47.2%. The combination of 5 μM venetoclax and 10 μM 5-azacytidine gave an average viability of 0.9% for KG1a AML cells, indicating a synergistic effect of venetoclax and 5-azacytidine treatment ( Figure 5 a).

[0374] Next, KG1a AML cells were stained with CellTrace CFSE proliferation dye (ThermoFisher) for specific detection by flow cytometry, counted, co-cultured with thawed HD-PBMC, and treated with 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 the first day of culture, venetoclax (0 - 0.4 μM), 5-azacytidine (0 - 0.4 μM), or a combination was added at increasing concentrations. Cells were harvested on day 3 of co-culture, centrifuged at 1800 rpm for 5 minutes and then incubated with 25 μl of FcR blocking reagent (Miltenyi Biotec) for 10 minutes at room temperature (RT), followed by the addition of 25 μL of the following antibody mixture prepared in PBS + 2% FBS + 2 mM EDTA (FACS buffer): anti-CD56-BV421, CD19-BV421, CD14-BV785, Tγδ-PE, and CD3-AF700 antibodies for discriminating immune subsets, and a viability marker (LIVE / DEAD TM Fixable Dead Cell Stain) to distinguish live and dead cells. Cells were incubated for 20 minutes at room temperature, washed once in FACS buffer and once in Annexin V staining buffer. Annexin V-APC probe mixed with CountBright absolute counting beads (Invitrogen) was added to measure apoptosis and relative cell number, followed by incubation for 15 minutes at room temperature, and then flow cytometry analysis was performed.

[0375] In this assay, KG1a was rather resistant to BTN3A-activated antibody-mediated Vγ9Vδ2 T cell killing (ICT01 reduced the number of live KG1a cells by an average of about 20% compared to the hIgG1S condition, with greater variability among PBMC donors)( Figure 5 b). Under the hIgG1S condition, treatment with 5-azacytidine had no effect on KG1a survival( Figure 5d). In contrast, under the hIgG1S treatment condition, venetoclax reduced the relative number of KG1a by approximately 28% at the highest concentration and reduced the relative number of KG1a by 44% in combination with 5-azacytidine. Compared with the hIgG1S condition, after activation with ICT01, treatment with venetoclax alone and 5-azacytidine alone had a slight increase in the killing activity of Vγ9Vδ2 T cells. However, compared with the hIgG1S condition, the combination of venetoclax and 5-azacytidine significantly reduced the relative number of viable KG1a cells to approximately 46.5% (p < 0.05), and reduced it to approximately 32.9% compared with the treatment without venetoclax and 5-azacytidine, indicating a synergistic effect of the combination treatment.

[0376] The mAb 20.1BTN3A activating antibody obtained similar results ( Figure 6 ). Under the mIgG1 condition, although treatment with 5-azacytidine had no effect on KG1a survival, when combined with 0.4 μM of 5-azacytidine, 0.1 μM of the venetoclax used reduced the relative number of KG1a by 41% and 57%. In addition, compared with the combination of m20.1 used alone or venetoclax and 5-azacytidine used with an isotype control, the combination of 20.1 mAb with venetoclax and 5-azacytidine significantly reduced the relative number of viable KG1a cells, and compared with the control condition, the viable KG1a cells were reduced by approximately 67% ( Figure 6 ).

[0377] In summary, these data indicate that activation of PBMC from healthy donors with ICT01 increases the survival rate of Vγ9Vδ2 T cells against venetoclax-induced cell death, and treatment with venetoclax and 5-azacytidine does not affect the activation or proliferation of Vγ9Vδ2 T cells in HD-derived PBMC in vitro. In addition, the combination of BTN3A antibody-activated Vγ9Vδ2 T cells with venetoclax and 5-azacytidine treatment in HD-PBMC significantly reduces the survival rate of the Vγ9Vδ2 T cell-resistant AML cell line KG1a.

[0378] 2.4 Treatment with the combination of venetoclax, HMA, and ICT01 increases the killing of Burkitt's lymphoma and chronic B-cell leukemia cell lines mediated by Vγ9Vδ2 T cells

[0379] The effects of venetoclax and 5-azacytidine on the killing of Burkitt's lymphoma and chronic B-cell leukemia cell lines by BTN3A activating antibody-induced Vγ9Vδ2 T cells were evaluated by measuring the relative number of surviving target cells in HD-PBMC-target cell co-cultures using flow cytometry.

[0380] In the presence of the BTN3A-activating antibody ICT01, co-culture of Raji (Burkitt lymphoma) or JVM-2 (chronic B-cell leukemia) with HD-PBMC for 48 hours induced a reduction in viable cells of approximately 22% and approximately 16%, respectively, with a large variability among PBMC donors ( Figure 7 a, Figure 7 b). Under hIgG1S treatment conditions, compared to the hIgG1S-alone condition (control), at the indicated concentrations, the combination of venetoclax and 5-azacytidine reduced the relative number of targets in Raji and JVM2 by approximately 37% and approximately 32%, respectively. Importantly, after 48 hours of co-culture, the combination of ICT01, venetoclax, and 5-azacytidine still reduced the relative number of viable target cells, and compared to the control condition, the number of viable target cells was reduced by approximately 55% and approximately 46%, indicating that the combination of a BTN3A-activating antibody (such as ICT01) with venetoclax and 5-azacytidine is beneficial for killing Burkitt lymphoma and chronic B-cell leukemia cell lines.

[0381] In summary, these data indicate that the combination of BTN3A-activating antibody-activated Vγ9Vδ2 T cells in HD PBMC with venetoclax and 5-azacytidine treatment significantly reduces the viability of several hematological cancer cell lines and provides an advantage for the treatment of hematological malignancies.

[0382] 2.5 Treatment of ICT01-activated Vγ9Vδ2 T cells with combinations of Bcl-2 family member inhibitors increases AML killing

[0383] 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 evaluated by measuring the relative number of viable target cells in HD-PBMC-AML cell co-cultures using flow cytometry.

[0384] For assays using ABT-737 or navitoclax, KG1a cells were used. However, this cell line does not express MCL-1 and is thus completely resistant to MIK665 treatment. Therefore, MOLM14 cells, which express MCL-1, were used in the killing assay to assess the activity of the combination of ICT01 and MIK665.

[0385] In the presence of ICT01, ABT-737, or the combination, killing of KG1a co-cultured with HD-PBMC ( Figure 8 a). In this experiment, compared to the control condition, ICT01 induced death of approximately 21% of KG1a cells (similar to the results obtained previously ( Figure 5b)). Treatment with 0.25 μM and 0.5 μM ABT-737 alone induced approximately 34% and 54% cell death of KG1a cells, respectively. When the co-culture of KG1a and HD-PBMC was treated with the combination of ICT01 and ABT-737, the number of viable KG1a cells decreased by approximately 53% and 71% compared to the control (for ABT-737 used at 0.25 μM and 0.5 μM, respectively), indicating the benefit of this combination for killing AML cell lines ( Figure 8 a).

[0386] Killing of KG1a co-cultured with HD-PBMC in the presence of ICT01, navitoclax or combination Figure 8 b)). Treatment with 0.25 μM and 0.5 μM navitoclax alone induced approximately 51% and 71% cell death of KG1a cells, respectively. When the co-culture of KG1a and HD-PBMC was treated with the combination of ICT01 and navitoclax, the number of viable KG1a cells decreased by approximately 65% and 88% compared to the control (for navitoclax used at 0.25 μM and 0.5 μM, respectively), indicating the benefit of this combination for killing AML cell lines ( Figure 8 b).

[0387] Killing of MOLM14 co-cultured with HD-PBMC in the presence of ICT01, MIK665 or combination Figure 8 c). In this experiment, ICT01 induced approximately 56% cell death of MOLM14 cells compared to the control condition. Treatment with 0.025 μM and 0.05 μM MIK665 alone induced approximately 52% and 72% cell death of MOLM14 cells, respectively. When the co-culture of MOLM14 and HD-PBMC was treated with the combination of ICT01 and MIK665, the number of viable MOLM14 cells decreased by approximately 85% and 93% compared to the control (for MI-665 used at 0.025 μM and 0.05 μM, respectively), indicating the benefit of this combination for killing AML cell lines ( Figure 8 c).

[0388] In summary, these data indicate that the combination of ICT01-activated Vγ9Vδ2 T cells in HD-PBMC and treatment with Bcl-2 family member inhibitors significantly reduces the survival rate of AML cell lines, confirming the potential of using the combination of ICT01 and Bcl-2 family member inhibitors for treating hematological malignancies.

[0389] 2.6 The combination of ICT01 with venetoclax and 5-azacytidine significantly prolongs the survival of NSG mice transplanted with MOLM-14

[0390] The in vivo efficacy of the combination of ICT01 with venetoclax and 5-azacytidine was evaluated in a xenograft model of MOLM-14, an AraC-resistant human AML-derived cell line, using NSG mice engrafted with a human tumor cell line and adoptively transferred with human Vγ9Vδ2 T cells. The aim of this study was to evaluate the effect of the combination of repeatedly iv-injected ICT01-activated human Vγ9Vδ2 T cells with Ven / Aza on tumor growth and on mouse survival.

[0391] NSG mice were injected with MOLM-14 on day 0 and randomized into homogeneous groups based on the intensity of the bioluminescence signal. The different experimental groups are outlined in Table 5.

[0392] Table 5: MOLM-14 mouse model: Group descriptions

[0393]

[0394] As shown in Table 6, human Vγ9Vδ2 T cells injected with an irrelevant control isotype antibody (hIgG1S) had no basal control of MOLM-14 in vivo growth (Group 1 vs. Group 3). In contrast, as shown by the lower bioluminescence signal and median survival (Table 6 and Figure 9 ), tumor growth was significantly delayed when human Vγ9Vδ2 T cells were administered with the BTN3A-activating mAb ICT01 (median survival of 24 days and 28 days for Groups 3 and 4, respectively) or in mice treated with venetoclax and 5-azacytidine (median survival of 24 days and 29 days for Groups 1 and 2, respectively). The combination of venetoclax and 5-azacytidine treatment with the transfer of non-activated human Vγ9Vδ2 T cells extended the median survival of the mice to 32.5 days (Groups 2 and 3 vs. Group 5), indicating that venetoclax and 5-azacytidine treatment made the cells more susceptible to killing by human Vγ9Vδ2 T cells (Table 7).

[0395] Importantly, treating mice with ICT01, human Vγ9Vδ2 T cells, venetoclax, and 5-azacytidine surprisingly increased the median survival to 42.5 days (Group 6) ( Figure 9 ).

[0396] 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 could be a promising approach for treating AML patients.

[0397] Table 6: MOLM-14 mouse model: Mean bioluminescence measurements for each group at days 0, 7, 14, 21, and 28 after tumor cell transplantation.

[0398]

[0399]

[0400] Table 7: MOLM-14 mouse model: Animal survival period. Median survival period of each group.

[0401]

Claims

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

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

3. The BTN3A activating antibody for use according to any one of claims 1 to 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 the heavy chain of SEQ ID NO:4 and the light chain of SEQ ID NO:

6.

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 simultaneously, sequentially, or separately, further in combination with azacitidine or decitabine.

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

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

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 the BTN3A activating antibody is administered intravenously.

11. The BTN3A activating antibody for use according to any one of claims 1 to 10, wherein the BTN3A activating antibody is administered in a unit dose of about 7 to about 200 mg, such as 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 as claimed in any one of claims 1 to 12, wherein Venetoclax is orally administered in a unit dose of from about 50 mg to about 500 mg.

14. The BTN3A activating antibody for use as claimed in any one of claims 1 to 13, wherein the demethylating agent comprises azacitidine.

15. The BTN3A activating antibody for use as described in any one of claims 1 to 14, wherein the demethylating agent is administered at a dose of about 50 mg / m 2 to about 100 mg / m 2 .

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