Therapeutic combinations comprising multispecific t-cell engagers

Abstract

The present invention relates to combinations and combination therapies, particularly for the treatment of myeloid malignancies, such as acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS). The combination therapy comprises (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, wherein each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binds one tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor, e.g., venetoclax or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, e.g., azacitidine or decitabine.

Description

Technical Field

[0001] This invention relates to combination therapies comprising, separately or together: (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of which specifically binds to a tumor-associated antigen suitably selected from the group consisting of CD123, CD33, and CD70; and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent. These combination therapies are particularly useful for treating malignancies, such as myeloid malignancies, such as acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS). Methods of using such combination therapies and kits comprising such combination therapies are also disclosed. Background Technology

[0002] Acute myeloid leukemia (AML) is a complex and heterogeneous disease with a poor prognosis, characterized by rapid cell proliferation, an aggressive clinical course, and high mortality. Despite the introduction of new drugs, overall survival for AML remains low, including in patients under 60 years of age, but particularly in older patients who are ineligible for stem cell transplantation. A major challenge in treating AML is the limited ability of current therapies to effectively eliminate leukemia stem cells (LSCs) while preserving healthy hematopoietic stem cells (HSCs), as LSCs exhibit resistance to chemotherapy and possess self-renewal capabilities, making them difficult to target.

[0003] Several recently introduced new drugs have provided novel treatment options for AML. For example, some of these new drugs are monoclonal antibodies that target specific proteins (e.g., CD33 or CD123) on the surface of leukemia cells, either alone or conjugated with cytotoxic agents, thereby selectively killing cancer cells. However, these therapies have shown limitations in terms of side effects and efficacy. Furthermore, immunotherapy targeting AML faces obstacles due to the lack of AML-specific target antigens and the clonal heterogeneity of tumors within and between patients. T-cell adaptor (TCE) therapies focused on a single AML antigen have been limited by toxicity from binding to healthy HSCs and myeloid cells, and by relapses caused by leukemia clones lacking the target antigen and thus evading targeted therapy. For example, while low or absent on HSCs, CD33 and CD123 are present on healthy hematopoietic cells: CD33 is present on most myeloid cells, and CD123 is primarily present on plasmacytoid dendritic cells, basophils, and endothelial cells, potentially leading to non-tumor-targeting toxicity of agents targeting a single tumor-associated antigen (TAA). To overcome these challenges, it is necessary to simultaneously target multiple tumor-associated antigens (TAAs) on AML blast cells and LSCs, aiming to enhance therapeutic efficacy without increasing toxicity.

[0004] A multispecific T-cell binding engineered ankyrin repeat protein (DARPin) has been developed to simultaneously bind CD3 and three AML tumor-associated antigens (TAAs): CD70, CD33, and CD123 on T cells. Described in WO2022190016, this DARPin has been engineered to target AML blasts and LSCs expressing these TAAs. By achieving affinity-like selectivity and efficacy, this multispecific binding protein is designed to selectively kill AML cells co-expressing at least two of these TAAs while preserving healthy cells expressing a single TAA. Experimental results demonstrate that this DARPin effectively induces selective killing of AML blasts and LSCs while minimizing the impact on healthy cells and minimizing cytokine release.

[0005] Other agents used to treat myeloid malignancies include the nucleoside analogue azacitidine and the BH3 mimic venetoclax. These agents have demonstrated favorable efficacy and safety in clinical trials and have been approved by the U.S. Food and Drug Administration (FDA) as combination therapy for newly diagnosed AML in patients aged 75 years or older or with comorbidities excluded from intensive induction chemotherapy. Azacitidine is a hypomethylating agent that works by inhibiting DNA methylation and inducing DNA damage in rapidly dividing cells, while venetoclax is a selective B-cell lymphoma-2 (Bcl-2) inhibitor that induces apoptosis by selectively targeting the Bcl-2 protein, which plays a crucial role in promoting cell survival. However, while this combination therapy has shown improved overall survival, higher complete remission rates, and reduced treatment-related mortality compared to conventional chemotherapy regimens, further advances in AML therapy are needed to address treatment resistance, relapse, and the lack of curative options, particularly for certain patient populations, ultimately aiming for higher response rates, prolonged remission, and better overall survival. Summary of the Invention

[0006] This invention relates to a combination therapy comprising, separately or together: (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of which specifically binds to a tumor-associated antigen suitably selected from the group consisting of CD123, CD33, and CD70; and (ii) a Bcl-2 inhibitor, such as veneclax or a pharmaceutically acceptable salt thereof; and / or (iii) a hypomethylating agent, such as azacitidine or decitabine. As explained elsewhere herein, veneclax is an example of a potent, selective small-molecule inhibitor of the Bcl-2 protein, and the hypomethylating agents azacitidine and decitabine induce DNA damage in dividing cells. This combination could provide a particularly effective treatment for cancers, especially myeloid malignancies such as acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS).

[0007] Based on the disclosure provided herein, those skilled in the art will recognize, or can only determine, using conventional experiments the numerous equivalents of the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following embodiments (E). Specifically, this disclosure provides, individually or in combination, the following aspects, advantageous features, and embodiments:

[0008] Implementation Plan

[0009] E1. A combination comprising, separately or together: (i) a recombinant binding protein comprising (a) a binding domain specifically binding to CD3, (b) a binding domain specifically binding to CD33 and / or a binding domain specifically binding to CD70, and (c) optionally a binding domain specifically binding to CD123, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent.

[0010] E2. The combination according to E1, wherein the recombinant binding domain is derived from an antibody or its antigen-binding fragment, an antibody mimic, a scaffold protein, and / or a repeating protein, such as an ankylosing spicule repeating protein.

[0011] E3. A combination comprising, separately or together: (i) a recombinant binding protein comprising (a) a first ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of which specifically binds to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent.

[0012] E4. The combination according to E3, wherein the recombinant binding protein comprises the first ankyrin repeat domain that specifically binds to CD3, and

[0013] i. Specifically binds to the second ankyrin repeat domain of CD123; or

[0014] ii. Specifically binds to the second ankyrin repeat domain of CD33; or

[0015] iii. Specifically binds to the second ankyrin repeat domain of CD70; or

[0016] iv. Specifically binds to the second ankyrin repeat domain of CD123 and specifically binds to the third ankyrin repeat domain of CD70; or

[0017] v. Specifically binds to the second ankyrin repeat domain of CD33 and specifically binds to the third ankyrin repeat domain of CD70; or

[0018] vi. Specifically binds to the second ankyrin repeat domain of CD123 and specifically binds to the third ankyrin repeat domain of CD33; or

[0019] vii. Specifically binds the second ankyrin repeat domain of CD123, the third ankyrin repeat domain of CD33, and the fourth ankyrin repeat domain of CD70.

[0020] E5. The combination according to any one of E1 to E4, wherein the recombinant binding protein comprises (a) the first ankyrin repeat domain that specifically binds CD3, (b) the second ankyrin repeat domain that specifically binds CD123, (c) the third ankyrin repeat domain that specifically binds CD33, and (d) the fourth ankyrin repeat domain that specifically binds CD70.

[0021] E6. The combination according to any one of E1 to E5, wherein the Bcl-2 inhibitor is Veneclare or a pharmaceutically acceptable salt thereof.

[0022] E7. The combination according to any one of E1 to E6, wherein the hypomethylating agent is azacitidine or decitabine.

[0023] E8. The combination according to any one of E1 to E7, wherein the hypomethylating agent is azacitidine.

[0024] E9. The combination according to any one of E1 to E7, wherein the hypomethylating agent is decitabine.

[0025] E10. The combination according to any one of E1 to E9, wherein the recombinant binding protein further comprises at least one or at least two binding domains that are specific for binding to human serum albumin.

[0026] E11. The combination according to E10, wherein the recombinant binding protein comprises at least one or at least two ankyrin repeat domains that are specific for binding to human serum albumin.

[0027] E12. The combination according to E11, wherein the recombinant binding protein comprises at least two ankyrin repeat domains that are specific for binding to human serum albumin.

[0028] E13. The combination according to any one of E3 to E12, wherein the CD3-specifically binding ankyrin repeating domain comprises at least about 80% of the same amino acid sequence as SEQ ID NO: 1.

[0029] E14. The combination according to any one of E3 to E13, wherein the CD33-specifically binding ankyrin repeat domain comprises at least about 80% of the same amino acid sequence as either SEQ ID NO: 2 or 3.

[0030] E15. The combination according to any one of E3 to E14, wherein the CD123-specifically binding ankyrin repeating domain comprises at least about 80% of the same amino acid sequence as any one of SEQ ID NO: 4 to 7.

[0031] E16. The combination according to any one of E3 to E15, wherein the CD70-specifically binding ankyrin repeating domain comprises at least about 80% of the same amino acid sequence as any one of SEQ ID NO: 8 to 9.

[0032] E17. The combination according to any one of E11 to E16, wherein the ankyrin repeating domain having binding specificity to human serum albumin comprises at least about 80% of the same amino acid sequence as SEQ ID NO: 10.

[0033] E18. The combination according to any one of E1 to E17, wherein the recombinant binding protein comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 12 to 18.

[0034] E19. The combination according to any one of E1 to E18, the combination comprising (i) a recombinant binding protein comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 12 to 18, and (ii) a Bcl-2 inhibitor.

[0035] E20. The combination according to E19, wherein the Bcl-2 inhibitor is Veneclare or a pharmaceutically acceptable salt thereof.

[0036] E21. The combination according to any one of E1 to E20, the combination comprising (i) a recombinant binding protein comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% of the same amino acid sequence as any one of SEQ ID NO: 12 to 18, and (ii) a hypomethylating agent.

[0037] E22. The combination according to E21, wherein the hypomethylating agent is azacitidine or decitabine.

[0038] E23. The combination according to E22, wherein the hypomethylating agent is azacitidine.

[0039] E24. The combination according to E22, wherein the hypomethylating agent is decitabine.

[0040] E25. The combination according to any one of E1 to E24, wherein the recombinant binding protein comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or 100% of the same amino acid sequence as SEQ ID NO: 12.

[0041] E26. The combination according to any one of E1 to E23 and E25, the combination comprising (i) a recombinant binding protein comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or 100% of the same amino acid sequence as SEQ ID NO: 12, (ii) Veneclare or a pharmaceutically acceptable salt thereof, and (iii) azacitidine.

[0042] E27. A combination according to any one of E1 to E22, E24 and E25, the combination comprising (i) a recombinant binding protein comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or 100% of the same amino acid sequence as SEQ ID NO: 12, (ii) Venecella or a pharmaceutically acceptable salt thereof, and (iii) decitabine.

[0043] E28. The combination according to any one of E1 to E27, wherein (i) the recombinant binding protein, (ii) the Bcl-2 inhibitor and (iii) the hypomethylating agent are separate.

[0044] E29. The combination according to any one of E1 to E28, wherein the combination comprises one or more additional antiproliferative agents, suitably anticancer agents.

[0045] E30. Combinations as defined in any of E1 through E29, used for therapeutic purposes.

[0046] E31. A combination of any of the terms defined in E1 through E30, used for the treatment of malignant tumors.

[0047] E32. A combination of any of the terms defined in E1 to E31, used for the treatment of myeloid malignancies.

[0048] E33. A combination as defined in any one of E1 to E32 for the treatment of malignant tumors, suitably myeloid malignancies, comprising administering to a subject in need a therapeutically effective amount of the therapeutic agent contained in the combination, wherein (i) the recombinant binding protein, (ii) the Bcl-2 inhibitor and / or (iii) the hypomethylating agent are administered separately, simultaneously or sequentially.

[0049] E34. A recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, wherein each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binds to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, the recombinant binding protein being used to treat malignant tumors, suitably myeloid malignant tumors, comprising administering the recombinant binding protein separately, simultaneously, or sequentially to a subject in need of it, along with (i) a Bcl-2 inhibitor and / or (ii) a hypomethylating agent.

[0050] E35. A Bcl-2 inhibitor, suitably veneclade or a pharmaceutically acceptable salt thereof, for the treatment of malignant tumors, suitably myeloid malignancies, comprising administering the Bcl-2 inhibitor separately, simultaneously or sequentially to a subject in need of it: (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two or at least three additional ankyrin repeat domains, each of said at least one, at least two or at least three additional ankyrin repeat domains specifically binding to a tumor-associated antigen, suitably selected from the group consisting of CD123, CD33 and CD70, and (ii) optionally a hypomethylating agent.

[0051] E36. A hypomethylating agent for treating malignant tumors, suitably myeloid malignant tumors, comprising administering the hypomethylating agent separately, simultaneously, or sequentially to a subject in need of it: (a) specifically binding an ankyrin repeat domain of CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of said at least one, at least two, or at least three additional ankyrin repeat domains specifically binding a tumor-associated antigen, suitably selected from the group consisting of CD123, CD33, and CD70, and (ii) optionally a Bcl-2 inhibitor.

[0052] E37. A method for treating malignant tumors, suitably myeloid malignant tumors, the method comprising administering to a subject in need of the following therapeutically effective amount: (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of said at least one, at least two, or at least three additional ankyrin repeat domains specifically binding to a tumor-associated antigen, said tumor-associated antigen suitably being a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent.

[0053] E38. The combination used according to E33, the recombinant binding protein used according to E34, the Bcl-2 inhibitor used according to E35, the hypomethylating agent used according to E36, or the method according to E37, wherein the recombinant binding protein, the Bcl-2 inhibitor, and / or the hypomethylating agent are administered in any order.

[0054] E39. The combination used according to E33 or E38, the recombinant binding protein used according to E34 or E38, the Bcl-2 inhibitor used according to E35 or E38, the hypomethylating agent used according to E36 or E38, or the method according to E37 or E38, wherein (i) the recombinant binding protein, (ii) the Bcl-2 inhibitor and / or the hypomethylating agent are administered separately.

[0055] E40. A combination of any one of E33 and E38 to E39, a recombinant binding protein of any one of E34 and E38 to E39, a Bcl-2 inhibitor of any one of E35 and E38 to E39, a hypomethylating agent of any one of E36 and E38 to E39, or the method of any one of E37 to E39, wherein (i) the recombinant binding protein and (ii) the Bcl-2 inhibitor and / or the hypomethylating agent are administered simultaneously.

[0056] E41. A combination of any one of E33 and E38 to E39, a recombinant binding protein of any one of E34 and E38 to E39, a Bcl-2 inhibitor of any one of E35 and E38 to E39, a hypomethylating agent of any one of E36 and E38 to E39, or the method of any one of E37 to E39, wherein (i) the recombinant binding protein and (ii) the Bcl-2 inhibitor and / or the hypomethylating agent are administered sequentially.

[0057] E42. A combination of any one of E33 and E38 to E41, a recombinant binding protein of any one of E34 and E38 to E41, a Bcl-2 inhibitor of any one of E35 and E38 to E41, a hypomethylating agent of any one of E36 and E38 to E41, or the method of any one of E35 to E41, wherein (i) the recombinant binding protein and (ii) the Bcl-2 inhibitor and / or the hypomethylating agent are administered in a therapeutically effective amount.

[0058] E43. A combination of any one of E33 and E38 to E42, a recombinant binding protein of any one of E34 and E38 to E42, a Bcl-2 inhibitor of any one of E35 and E38 to E43, a hypomethylating agent of any one of E36 and E38 to E42, or the method of any one of E35 to E42, wherein (i) the recombinant binding protein and (ii) the Bcl-2 inhibitor and / or the hypomethylating agent are administered in synergistically effective amounts.

[0059] E44. A combination of any one of E33 and E38 to E43, a recombinant binding protein of any one of E34 and E38 to E43, a Bcl-2 inhibitor of any one of E35 and E38 to E43, a hypomethylating agent of any one of E36 and E38 to E43, or the method of any one of E37 to E43, wherein the malignant tumor is a myeloid malignant tumor, wherein the myeloid malignant tumor is acute myeloid leukemia (AML); myelodysplastic syndrome (MDS); myeloproliferative neoplasm (MPN); chronic myeloid leukemia (CML); or chronic myelomonocytic leukemia (CMML), suitably wherein the myeloid malignant tumor is AML, AML / MDS, or MDS.

[0060] E45. The combination used according to E44, the recombinant binding protein used according to E44, the Bcl-2 inhibitor used according to E44, the hypomethylating agent used according to E44, or the method described according to E44, wherein the myeloid malignancy is AML, AML / MDS, or MDS.

[0061] E46. The combination used according to E44 or E45, the recombinant binding protein used according to E44 or E45, the Bcl-2 inhibitor used according to E44 or E45, the hypomethylating agent used according to E44 or E45, or the method described according to E44 or E45, wherein the myeloid malignancy is AML.

[0062] E47. The combination used according to E44 or E45, the recombinant binding protein used according to E44 or E45, the Bcl-2 inhibitor used according to E44 or E45, the hypomethylating agent used according to E44 or E45, or the method described according to E44 or E45, wherein the myeloid malignancy is AML / MDS.

[0063] E48. A combination of any one of E33 and E38 to E47, a recombinant binding protein of any one of E34 and E38 to E47, a Bcl-2 inhibitor of any one of E35 and E38 to E47, a hypomethylating agent of any one of E36 and E38 to E47, or the method of any one of E37 to E47, wherein the treatment is first-line or second-line treatment.

[0064] E49. A combination of any one of E33 and E38 to E48, a recombinant binding protein of any one of E34 and E38 to E48, a Bcl-2 inhibitor of any one of E35 and E38 to E48, a hypomethylating agent of any one of E36 and E38 to E48, or the method of any one of E37 to E48, wherein the subject is relapsed and / or refractory.

[0065] E50. A combination of any one of E33 and E38 to E49, a recombinant binding protein of any one of E34 and E38 to E49, a Bcl-2 inhibitor of any one of E35 and E38 to E49, a hypomethylating agent of any one of E36 and E38 to E49, or the method of any one of E37 to E49, wherein the subject is newly diagnosed and / or unsuitable for standard intensive chemotherapy.

[0066] E51. A combination of any one of E33 and E38 to E50, a recombinant binding protein of any one of E34 and E38 to E50, a Bcl-2 inhibitor of any one of E35 and E38 to E50, a hypomethylating agent of any one of E36 and E38 to E50, or the method of any one of E37 to E50, wherein the subject is 75 years of age or older.

[0067] E52. A combination of any one of E33 and E38 to E51, a recombinant binding protein of any one of E34 and E38 to E51, a Bcl-2 inhibitor of any one of E35 and E38 to E51, a hypomethylating agent of any one of E36 and E38 to E51, or the method of any one of E37 to E51, wherein the subject is not suitable for standard intensive chemotherapy, and suitably the subject has comorbidities that preclude the use of standard intensive chemotherapy.

[0068] E53. A combination of any one of E33 and E38 to E52, a recombinant binding protein of any one of E34 and E38 to E52, a Bcl-2 inhibitor of any one of E35 and E38 to E52, a hypomethylating agent of any one of E36 and E38 to E52, or a method of any one of E37 to E52, further comprising monitoring the peripheral blood and / or myeloid blast cell count of the subject.

[0069] E54. The combination used in E53, the recombinant binding protein used in E53, the Bcl-2 inhibitor used in E53, the hypomethylating agent used in E53, or the method described in E53, wherein the myeloblast count is reduced to less than about 25% and / or the percentage of myeloblasts is reduced by more than about 50% compared to pretreatment.

[0070] E55. (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of which specifically binds to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent for the manufacture of a medicament for the treatment of malignant tumors.

[0071] E56. (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of which specifically binds to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and (ii) the use of a Bcl-2 inhibitor and / or (iii) a hypomethylating agent for the treatment of malignant tumors.

[0072] E57. A medicament comprising, separately or together: (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of said at least one, at least two, or at least three additional ankyrin repeat domains specifically binding to a tumor-associated antigen, said tumor-associated antigen suitably being a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent for simultaneous, sequential, or separate administration, and wherein (i) the recombinant binding protein and (ii) the Bcl-2 inhibitor and / or hypomethylating agent are provided in a combined therapeutically effective amount, preferably in a synergistically effective amount, for treating malignant tumors.

[0073] E58. A pharmaceutical combination comprising, separately or together: (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of which specifically binds to a tumor-associated antigen, suitably selected from the group consisting of CD123, CD33, and CD70; (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent; and at least one pharmaceutically acceptable carrier or excipient.

[0074] E59. A kit comprising a combination according to any one of E1 to E33 and instructions for simultaneous or sequential administration thereto, said kit being used to treat malignant tumors, suitably myeloid malignancies, suitably acute myeloid leukemia or myelodysplastic syndrome.

[0075] E60. (i) A recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of which specifically binds to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent for use in the manufacture of a kit for treating a disease.

[0076] Other features, objects, and advantages of the invention will be apparent from the description and drawings, as well as from the claims. Attached Figure Description

[0077] Figure 1 Synergistic effect of combined treatment with recombinant binding protein (“Protein #1”) of SEQ ID NO: 12, Bcl-2 inhibitor Veneclav, and hypomethylating agent azacitidine. Cells from the AML cell line MOLM-13 were co-cultured 1:1 with PBMC-derived CTV-labeled T cells (“Nr. 53”) from healthy donors for three days and treated with the indicated EC50 concentration of each compound in 2-fold increment / decrement titrations. The number of viable MOLM-13 cells was assessed by FACS (cell number normalized to count beads). A) Protein #1 only; B) Azacitidine with Protein #1; C) Veneclav with Protein #1; D) Azacitidine and Veneclav; and E) Azacitidine, Veneclav, and Protein #1. DMSO: dimethyl sulfoxide (mediator control), Azacitidine, Ven: Veneclav.

[0078] Figure 2 Synergistic effect of combined treatment with protein #1, veneclade, and azacitidine. Cells from the AML cell line MOLM-13 were compared with PBMC-derived CTV-labeled T cells from three healthy donors. Figure 2 A, Figure 2 B. Figure 2 C) Cells were co-cultured 1:1 for three days and treated with combinations of EC50 concentrations of each compound using 2-fold incremental / decrease titrations. The number of viable MOLM-13 cells was assessed by FACS (cell number normalized to count beads), and a synergistic effect score was calculated based on the number of surviving cells versus cells treated with the medium. The synergistic effect score was calculated using CompuSyn software according to the Chou-Talalay method. CI: Combination Index, Fa: Effect Score.

[0079] Figure 3 Primary AML cells CD34 from three different donors (●, ■, ▲) were treated with a combination of protein #1, veneclade, and azacitidine. + Concordant effects were observed at LSC. Primary AML cells were purified and cultured at a 1:1 ratio with allogeneic T cells from healthy donors for 4 days in the presence of 10 pM protein #1 or control unbound (NB)-CD3 DARPin ± 1 µM azacitidine and 5 nM veneclade, as shown, then cultured for 14 days and colony formation was assessed. CFU: Colony-forming unit.

[0080] Figure 4Protein #1 induces effective killing of AML cell lines. MOLM-13 cells are engineered by CRISPR / Cas9 to express different combinations of tumor-associated antigens (TAAs). In co-cultures of T cells with MOLM-13 cell lines expressing different TAA combinations, protein #1 induces T cell activation ( Figure 4 A) and tumor cell killing ( Figure 4 B), as shown in the legend. CD8 was detected by flow cytometry. + T cell activation was assessed by upregulation of CD25 on T cells, and tumor cell killing was assessed based on the cell count of remaining viable cells. T cell activation and tumor cell killing were assessed after T cells and tumor cells were co-incubated at an effector-target ratio of 5:1 (E:T) for 48 hours.

[0081] Figure 5 Protein #1 binds to MOLM-13 clones and T cells. MOLM-13 cells are engineered with CRISPR / Cas9 to express different combinations of tumor-associated antigens (TAAs). Flow cytometry confirmed the specific binding of protein #1 to tumor-associated antigens (TAAs) on MOLM-13 cells, including (…). Figure 5 A) shows the knockout of one, two, or three TAAs, as well as CD3-specific binding to pan-T cells (…). Figure 5 B). In Figure 5 In section B, the mean ± standard deviation of protein #1 bound to T cells from three different donors is shown. Control molecules shown: NB-CD3 DARPin (non-TAA bound) and NB-TAADARPin (non-CD3 bound). MFI: mean fluorescence intensity. Detailed Implementation

[0082] definition

[0083] The selected terms are defined below and throughout the specification. It should be understood that, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art of this invention.

[0084] Unless otherwise claimed, any and all examples or exemplary language (such as "such as") provided herein are intended merely to better illustrate this disclosure and not to limit its scope.

[0085] As used in this article, the articles “a” and “a kind” can mean “one”, but they also have the same meaning as “one or more”, “at least one” and “one or more”.

[0086] Unless the context clearly indicates otherwise, the term “or” is used herein to mean the term “and / or” and is used interchangeably with that term, and should be interpreted as meaning an inclusive “or” of any one or any combination thereof.

[0087] Throughout the specification and the following claims, and unless the context otherwise requires, the terms “comprising” and “including” are used interchangeably. The terms “comprising” and “including” should be interpreted as specifying the presence of the stated features or components mentioned, but do not exclude the presence or addition of one or more features or components or groups thereof. Additionally, the terms “comprising” and “including” are intended to include examples covered by the term “consisting of”. Therefore, the term “consisting of” may be used in place of the terms “comprising” and “including” to provide more specific embodiments of the invention.

[0088] The terms “about” and “approximately” should generally be intended to indicate the acceptable degree of error for a quantity measured given the nature or precision of the measurement. An exemplary degree of error is within 10% of a given value or a range of values. For example, when a dose is mentioned as “about” a specific value, it is intended to include a range of approximately 10% plus or minus the specified value. It should be understood that, although not always explicitly stated, all numerical names are preceded by the term “about”.

[0089] It should also be understood that, although not always explicitly stated, the reagents described herein are merely examples, and equivalents of these reagents are known in the art.

[0090] The terms “combination,” “a combination,” or “combined with” refer to treatment in which two or more therapeutic agents are administered to a subject (e.g., a human subject). The “combination” described herein applies to combination therapies. The combinations disclosed herein, separately or together, comprise: (1)(i) a recombinant binding protein comprising (a) a binding domain specifically binding to CD3, (b) a binding domain specifically binding to CD33 and / or a binding domain specifically binding to CD70, and optionally (c) a binding domain specifically binding to CD123, and (ii) a Bcl-2 inhibitor, suitably veneclade or a pharmaceutically acceptable salt thereof; (2)(i) a recombinant binding protein comprising (a) a binding domain specifically binding to CD3, (b) a binding domain specifically binding to CD33 and / or a binding domain specifically binding to CD70, and optionally (c) a binding domain specifically binding to CD123, and (ii) a Bcl-2 inhibitor, suitably veneclade or a pharmaceutically acceptable salt thereof; and (iii) a recombinant binding protein comprising (a) a binding domain specifically binding to CD3, (b) a binding domain specifically binding to CD33 and / or a binding domain specifically binding to CD123. The binding domain of CD70, and optionally (c) a binding domain specifically binding to CD123, and (ii) a hypomethylating agent, suitably azacitidine or decitabine, or (3)(i) a recombinant binding protein comprising (a) a binding domain specifically binding to CD3, (b) a binding domain specifically binding to CD33 and / or a binding domain specifically binding to CD70, and optionally (c) a binding domain specifically binding to CD123, (ii) a Bcl-2 inhibitor, suitably veneclade or a pharmaceutically acceptable salt thereof, and (iii) a hypomethylating agent, suitably azacitidine or decitabine.More specifically, the combinations disclosed herein, individually or together, comprise: (1)(i) a recombinant binding protein comprising (a) a first ankyrin repeat domain specifically binding to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, wherein each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binds to a tumor-associated antigen suitably selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor, such as Veneclare or a pharmaceutically acceptable salt thereof; (2)(i) a recombinant binding protein comprising (a) a first ankyrin repeat domain specifically binding to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, wherein each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binds to a tumor-associated antigen suitably selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor, such as Veneclare or a pharmaceutically acceptable salt thereof; Each of the protein repeating domains specifically binds a tumor-associated antigen, suitably selected from the group consisting of CD123, CD33, and CD70, and (ii) a hypomethylating agent, such as azacitidine or decitabine, or (3)(i) a recombinant binding protein comprising (a) a first ankyrin repeating domain specifically binding to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeating domains, wherein each of the at least one, at least two, or at least three additional ankyrin repeating domains specifically binds a tumor-associated antigen, suitably selected from the group consisting of CD123, CD33, and CD70, (ii) a Bcl-2 inhibitor, such as veneclade or a pharmaceutically acceptable salt thereof, and (iii) a hypomethylating agent, such as azacitidine or decitabine. Therefore, this is not intended to imply that therapies or therapeutic agents must be physically mixed or administered simultaneously and / or formulated for delivery together, but such delivery methods are within the scope described herein. Therefore, the term "combination" refers to a fixed combination in the form of a single dose unit, wherein the combined therapeutic agents are administered to the patient simultaneously as a single entity or dose, or to a non-fixed combination, wherein the therapeutic agents as described herein are administered independently, simultaneously, concurrently, or sequentially, without a specific time limit, wherein such administration provides a therapeutically effective level of the individual therapeutic agents in the patient, particularly resulting in synergistic effects, such as a combined drug effect. Each component (e.g., powder or liquid) may be reconstituted or diluted to the desired dose prior to administration.

[0091] As used herein, the term "drug combination" means a product resulting from a mixture or combination of more than one therapeutic agent, and includes fixed and non-fixed therapeutic agent combinations, and also includes pharmaceutically acceptable excipients, diluents and / or carriers.

[0092] As used herein, “administer” or “administration” refers to the act of physically delivering a substance that is present outside the body into the body of a subject. Administerment includes all forms applicable to the delivery of combinations of therapeutic agents.

[0093] The term "combination therapy" refers to a treatment method in which two or more different agents, modalities of action, or interventions are administered simultaneously, sequentially, or in a coordinated manner to treat a medical condition or disease, such as cancer or malignancy as described herein. The combination of these elements is designed to produce a synergistic or complementary effect that enhances treatment outcomes, improves efficacy, reduces side effects, and / or simultaneously targets multiple aspects of the disease. In the context of this patent application, "combination therapy" specifically refers to the disclosed combination of drugs for treatment, involving the simultaneous, concurrent, or sequential administration of multiple active therapeutic agents to achieve a therapeutic effect on a specified medical condition.

[0094] As used herein, the terms “co-administration” or “combination administration” are intended to cover the administration of selected combination drugs (therapeutic agents) to a single subject (e.g., a patient) in whom they require treatment, and are intended to include treatment regimens in which the agents are not necessarily administered via the same route of administration or simultaneously. Therapeutic agents in combination therapy may be administered simultaneously with, before, or after one or more other therapies or therapeutic agents. Therapeutic agents may be administered in any order. Typically, each therapeutic agent will be administered at a dose and / or schedule determined for that agent. Such administration also includes administering each type of therapeutic agent sequentially at approximately the same time or at different times. It should also be understood that additional therapeutic agents utilized in the combination may be administered together in a single composition or separately in different compositions. Generally, additional therapeutic agents utilized in the combination are expected to be administered at levels not exceeding those used individually. In some embodiments, the levels utilized in the combination will be lower than those used as single-agent therapeutic agents. In some embodiments, Bcl-2 inhibitors and / or hypomethylators are administered at therapeutic doses or below the therapeutic dose levels relative to the single-agent dose levels. In some embodiments, when Bcl-2 inhibitors and / or hypomethylators are used or administered in combination with the recombinant binding protein described herein, the concentration of the Bcl-2 inhibitor and / or hypomethylator required to achieve the desired therapeutic effect is lower than when the Bcl-2 inhibitor and / or hypomethylator is administered alone. In some embodiments, the concentration or dose of the Bcl-2 inhibitor and / or hypomethylator required to achieve the therapeutic effect is lower than the therapeutic dose of the Bcl-2 inhibitor and / or hypomethylator as a monotherapy, for example, at least about 10% or more, at least about 20% or more, at least about 30% or more, at least about 40% or more, at least about 50% or more, at least about 60% or more, at least about 70% or more, at least about 80% or more, at least about 90% or more. In some embodiments, the concentration or dose of the recombinant binding protein described herein required to achieve the therapeutic effect is lower than the therapeutic dose of the recombinant binding protein as a monotherapy, for example, at least about 10% or more, at least about 20% or more, at least about 30% or more, at least about 40% or more, at least about 50% or more, at least about 60% or more, at least about 70% or more, at least about 80% or more, at least about 90% or more.

[0095] As used herein, the term "peptide" refers to a molecule comprising a chain of multiple amino acids linked by peptide bonds. Preferably, a polypeptide consists of eight or more amino acids linked by peptide bonds. The term "peptide" also includes multiple chains of amino acids linked together by SS bridges between cysteine ​​residues.

[0096] As used herein, the term "protein" refers to a molecule comprising a polypeptide, wherein at least a portion of the polypeptide has or is capable of acquiring a defined three-dimensional arrangement by forming secondary, tertiary, and / or quaternary structures within and / or between multiple polypeptide chains. If a protein comprises two or more polypeptide chains, the individual polypeptide chains may be non-covalently or covalently linked, for example, by disulfide bonds between the two polypeptides. A portion of a protein that individually has or is capable of acquiring a defined three-dimensional arrangement by forming secondary and / or tertiary structures is referred to as a "protein domain".

[0097] As used herein, the term "binding protein" refers to a protein containing at least one binding domain. Binding proteins may also contain two, three, four, five, or more binding domains. Preferably, the binding protein is a recombinant binding protein. Furthermore, any such binding protein may contain additional polypeptides (such as polypeptide tags, peptide linkers, fusion with other binding-specific protein domains, cytokines, hormones, or antagonists), or chemical modifications (such as coupling to polyethylene glycol, toxins, small molecules, antibiotics, etc.).

[0098] The term "binding domain" refers to a protein domain that exhibits target binding specificity. Preferably, the binding domain is a recombinant binding domain. In the context of this invention, the recombinant binding protein binds to a specific target antigen, such as CD3 and one or more tumor-associated antigens (TAAs), such as CD33, CD123, and CD70. Preferably, the recombinant binding protein "specifically binds" to its target antigen, wherein the term "specifically binds" refers to the ability of the recombinant binding protein to preferentially induce an immune response with a given target (e.g., CD3, and one or more of the TAAs CD33, CD123, and CD70). Currently provided recombinant binding proteins are "multispecific," i.e., they contain two or more binding sites, preferably three or more binding sites, preferably four binding sites, which specifically bind to a particular target antigen.

[0099] The term "target" or "target antigen" refers to a single molecule, such as a nucleic acid molecule, polypeptide or protein, carbohydrate, or any other naturally occurring molecule, including any part of such a single molecule, or a complex of two or more such molecules, or a whole cell or tissue sample, or any non-natural compound, to which the binding site of its binding domain is engineered to bind. In the context of this invention, one or more of CD3 and tumor-associated antigens (TAAs) CD33, CD123, and / or CD70 expressed on T cells are targets, i.e., the recombinant binding proteins described herein bind to these target antigens.

[0100] As used herein, tumor-associated antigens (TAAs) are antigens expressed by tumor cells and recognized by the immune system. TAAs may originate from normal cellular components but are overexpressed, mutated, or aberrantly expressed in or on tumor cells, making them unique markers of tumors. TAAs can be targeted by the immune system to recognize and eliminate tumor cells, but they can be challenging due to potential cross-reactivity with healthy tissues. Preferred TAAs in the context of this disclosure are CD123, CD33, and / or CD70, but those skilled in the art will also know of other suitable TAAs, such as those associated with myeloid malignancies (such as AML).

[0101] The term "CD3" or "differentiation cluster 3" refers to a multimeric protein complex composed of four distinct chains. In mammals, the complex contains a CD3γ (γ) chain, a CD3δ (δ) chain, and two CD3ε (ε) chains. These chains associate with the T cell receptor (TCR) and the CD3ζ chain to generate activation signals in T cells. CD3 is essential for T cell activation and the initiation of T cell effector functions, such as cytokine production, cytotoxicity, and the release of other immune mediators. It enables T cells to recognize and respond to specific antigens, thereby coordinating adaptive immune responses. The amino acid sequences of the human CD3 γ, δ, ε, and ζ chains are shown in NCBI Ref. Seq. NP_000064.1, NP_000723.1, NP_000724.1, and NP_932170.1, respectively. The ankyrin repeat domain that specifically binds to CD3 is disclosed in WO2022129428 (incorporated in its entirety by reference).

[0102] CD70 (also known as CD27L, TNFSF7 (tumor necrosis factor ligand superfamily member 7), CD27 ligand) is a cell surface protein involved in immune regulation. CD70 expression on tumor cells can contribute to immune evasion mechanisms, such as promoting immune checkpoint resistance and suppressing antitumor immune responses. Aberrant expression has been observed in various cancers, including hematologic malignancies. CD70 is expressed on most AML blast cells and leukemia stem cells (Perna et al., Cancer Cell, 32(4), 2017, 506-519). Specific examples of human CD70 include peptides with the amino acid sequence UniProt ID P32970. Anchorin repeat domains that specifically bind to CD70 are disclosed in WO2022215032 (incorporated in its entirety by reference).

[0103] The term "CD33" refers to the bone marrow cell surface antigen CD33, a sialic acid-binding immunoglobulin-like lectin (Siglec) that plays a role in mediating cell-cell interactions and maintaining the dormant state of immune cells. CD33 is highly expressed in leukemia blasts in AML (Ehninger et al., Blood Cancer Journal 4, e218 (2014)). The amino acid sequence of human CD33 is available in UniProt reference number P20138. The ankyrin repeat domain that specifically binds to CD33 is disclosed in WO2022190010 (in its entirety by reference).

[0104] The term "CD123" refers to the interleukin-3 receptor subunit α. CD123 is expressed in most AML blast cells and leukemia stem cells (Ehninger et al., Blood Cancer Journal 4, e218 (2014)). The amino acid sequence of human CD123 can be found in UniProt reference number P26951. The ankyrin repeat domain that specifically binds to CD123 is disclosed in WO2022190018 (incorporated in its entirety by reference).

[0105] The terms "binding specificity," "target-specific binding," "specifically binding to a target," "binding to a target with high specificity," "target-specific," or "target-specific" refer to a binding protein or binding domain binding to a target with a lower dissociation constant than it binds to unrelated proteins (such as E. coli maltose-binding protein (MBP)) (i.e., binding with higher affinity). Preferably, the dissociation constant of the target ("K") is... D " is at least 10 of the corresponding dissociation constant of MBP 2 More preferably at least 10 times 3 More preferably at least 10 times 4 At least 10 times or more preferably 5 Methods for determining the dissociation constant of protein-protein interactions, such as surface plasmon resonance (SPR)-based techniques (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC), are known to those skilled in the art. If measurements are performed under different conditions (e.g., salt concentration, pH), the specific protein-protein interaction measurement K0 will be determined. D The value can vary. Therefore, it is preferable to use standardized protein solutions and standardized buffers (such as PBS) for K... D Measurement of values.

[0106] The term "repetitive protein" refers to a protein containing one or more repeating domains. Preferably, a repeating protein contains one, two, three, four, five, or six repeating domains. Furthermore, the repeating protein may contain additional non-repetitive protein domains, polypeptide tags, and / or peptide linkers. The repeating domain may be a binding domain. Patent applications WO2002020565 and Forrer et al., 2003 (Forrer, P., Stumpp, MT, Binz, HK, Plückthun, A., 2003. FEBS Letters 539, 2-6) contain a general description of the characteristics of repeating proteins and repeating domains, as well as their techniques and applications. The term "repetitive domain" refers to a protein domain containing two or more consecutive repeating modules as structural units, wherein the repeating modules have structural and sequence homology. Preferably, the repeating domain also contains N-terminal and / or C-terminal capping modules. For clarity, the capping module may be a repeating module. Such repetitive domains, repetitive modules and end-cap modules, sequence motifs, and structural and sequence homology are known, for example, see leucine-rich repetitive domains (WO2002020565), thiocopeptide repetitive domains (Main, ER, Xiong, Y., Cocco, MJ, D'Andrea, L., Regan, L., Structure 11(5), 497-508, 2003), and armadillo repetitive domains (WO2009040338). It is also known that such repetitive domains differ from proteins containing repetitive amino acid sequences, where each repetitive amino acid sequence can form a single domain (e.g., the FN3 domain of fibronectin).

[0107] WO2022190016 describes a suitable recombinant binding protein comprising (a) a first ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, wherein each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binds to a tumor-associated antigen, which is suitably selected from the group consisting of CD123, CD33, and CD70 (incorporated in its entirety by reference).

[0108] The term "repetitive domain" refers to a protein domain comprising two or more consecutive repeating modules as structural units, wherein the repeating modules share structural and sequence homology. Preferably, the repetitive domain further comprises an N-terminal and / or C-terminal capping module. For clarity, the capping module may be a repeating module. Such repetitive domains, repeating modules and capping modules, sequence motifs, and structural and sequence homology are known to those skilled in the art based on the following examples: ankyrin repetitive domain (WO2002 / 020565), leucine-rich repetitive domain (WO2002 / 020565), 34-peptide repetitive domain (Main, ER et al., Structure 11(5), 497-508, 2003), and armadillo repetitive domain (WO2009 / 040338). Those skilled in the art also know that such repetitive domains differ from proteins comprising repeating amino acid sequences, wherein each repeating amino acid sequence is capable of forming a single domain (e.g., the FN3 domain of fibronectin).

[0109] The term "ankylin repeat domain" refers to a repeat domain that comprises two or more consecutive ankylin repeat modules as structural units. Ankylin repeat domains can be modularly assembled into larger ankylin repeat proteins, optionally with extended half-life domains, using standard recombinant DNA techniques (see, for example, Forrer, P. et al., FEBS letters 539, 2-6, 2003; WO2002 / 020565, WO2016 / 156596; WO2018 / 054971).

[0110] As used in "designed repeating proteins," "designed repeating domains," etc., the term "designed" means that such repeating proteins and repeating domains are artificial and do not exist in nature. In some embodiments, the binding proteins are designed ankyrin repeating proteins, and they contain at least one designed ankyrin repeating domain.

[0111] The term "antibody" refers not only to the complete antibody molecule but also to any fragment and variant of an antibody molecule that retains the ability to bind to immunogens. Such fragments and variants are known in the art. Therefore, the term "antibody" encompasses complete immunoglobulin molecules, antibody fragments (such as, for example, Fab, Fab', F(ab')2, and single-chain V region fragments (scFv)), bispecific antibodies, chimeric antibodies, antibody fusion peptides, and unconventional antibodies.

[0112] Alternative scaffolds include any polypeptide or protein containing a binding domain capable of binding to a target and not derived from an antibody or immunoglobulin molecule. The binding domain of an alternative scaffold may contain or be derived from a variety of different polypeptide or protein structures. Alternative scaffolds include, but are not limited to, adenettin (monoantibody), affinity peptides, afenimide, afenimide and aptamers, afenimide, alpha antibodies, anticarrier proteins, armadillo repeat-based scaffolds, afenimide, afenimide, fenomoglycin, knotting peptides, and Kuniz domain peptides. Alternative scaffolds are described, for example, in Yu et al., Annu Rev Anal Chem (PaloAlto Calif). 2017 June 12; 10(1): 293–320. doi:10.1146 / annurevanchem-061516-045205.

[0113] The term "recombinant" as used in terms such as "recombinant binding protein," "recombinant binding polypeptide," etc., refers to the fact that the protein or polypeptide is produced using recombinant DNA technology. For example, a recombinant DNA molecule encoding a polypeptide (e.g., produced through gene synthesis) can be cloned into a bacterial expression plasmid (e.g., pQE30), a yeast expression plasmid, a mammalian expression plasmid, or a plant expression plasmid, or into DNA capable of in vitro expression. If, for example, such a recombinant bacterial expression plasmid is inserted into a suitable bacterium (e.g., *Escherichia coli*), these bacteria can produce a polypeptide encoded by the recombinant DNA. The corresponding polypeptide or protein is called a recombinant polypeptide or recombinant protein.

[0114] As used herein, “Bcl-2” or “Bcl-2 protein” refers to the first member of the Bcl-2 protein family identified in humans, namely B-cell lymphoma 2. The Bcl-2 protein family is a collection of pro-apoptotic and anti-apoptotic proteins associated with Bcl-2, playing an indispensable role in regulating the intrinsic apoptosis pathway. Anti-apoptotic members of this family (such as Bcl-2) typically antagonize pro-apoptotic members. In many cancers, Bcl-2 is overexpressed, leading to apoptosis evasion and enhanced cell survival.

[0115] As used herein, “Bcl-2 inhibitor” means any agent, compound, or molecule capable of specifically inhibiting the activity of Bcl-2, particularly an agent, compound, or molecule capable of inhibiting the anti-apoptotic activity of Bcl-2. The terms “inhibition,” “inhibitor,” or “antagonist” include a reduction in certain parameters (e.g., activity) of a given molecule or pathway. For example, the term includes inhibiting the activity of Bcl-2 by about 5%, about 10%, about 20%, about 30%, about 40%, or more. Therefore, inhibition can be, but does not have to be, 100%.

[0116] As used herein, "hypomethylating agent" refers to any agent, compound, or molecule capable of inhibiting DNA methylation and / or promoting DNA demethylation. Such agents can act through a variety of mechanisms, including directly inhibiting DNA methyltransferases, incorporating into DNA to interfere with the methylation process, or other indirect mechanisms.

[0117] To determine the percentage identity between two sequences (e.g., polynucleotides or polypeptides), sequences are aligned for optimal comparison. Sequences are considered identical when a position in the first sequence has the same nucleotide or amino acid as the corresponding position in the second sequence. Percentage identity is calculated by dividing the number of identical positions by the total number of positions in the reference sequence and multiplying by 100. To assess similarity, alignment is typically performed along the length of the reference sequence. For example, to determine if a test sequence is at least 80% identical to SEQ ID NO: 1 (an example of a reference sequence), an alignment is performed against SEQ ID NO: 1, and the number of identical positions is compared. If at least 80% of the positions are identical, the test sequence is considered at least 80% identical to SEQ ID NO: 1. Vacancies or deletions in shorter sequences are considered dissimilar positions. Various computer programs are available for determining sequence homology. The Needleman and Wunsch algorithms can be used with specific parameters, such as a Blosum62 or PAM250 matrix, and vacancy weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6, to calculate the percentage identity between amino acid or nucleic acid sequences. An example of suitable parameters is a Blosum 62 scoring matrix, a vacancy penalty of 12, a vacancy extension penalty of 4, and a frameshift vacancy penalty of 5.

[0118] The term "dosage" refers to the specified amount of a therapeutic agent administered at one time. As used herein, dosage is the amount of a therapeutic agent (also known as a "medicine") that produces a therapeutic effect. Dosage will be stated, for example, on the product packaging or in the product information leaflet.

[0119] The term "dosage" refers to a dose that is administered in a specified amount, quantity, and frequency over a specified period of time.

[0120] The terms "malignant tumor" or "cancer" encompass a disease in which abnormal cells proliferate in an uncontrolled manner. Cancer includes solid tumors and liquid tumors, as well as primary tumors and metastases. A "tumor" contains one or more cancer cells. These abnormal cells can invade surrounding tissues. Malignant cells can spread locally or to other parts of the body via the bloodstream and lymphatic system.

[0121] As used herein, the term “myeloid malignancy” refers to a clonal disease of myeloid lineage cells derived from the bone marrow, characterized by abnormal proliferation, differentiation, and / or function. Myeloid malignancies primarily involve dysregulation of hematopoietic stem cells, myeloid progenitor cells, or mature cells such as granulocytes, monocytes, erythrocytes, or platelets. Myeloid malignancies encompass a range of diseases, including but not limited to acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), chronic myelomonocytic leukemia (CMML), and chronic myeloid leukemia (CML). Myeloid malignancies can be classified and diagnosed according to the WHO 2008 classification and the International Consensus Classification (ICC) for myeloid tumors and acute leukemia (Arber et al. Blood (2022) 140 (11): 1200–1228).

[0122] As used herein, the term “acute myeloid leukemia” or “acute myeloid leukemia” (AML) refers to a hematopoietic tumor involving myeloid cells and is a type of cancer affecting the blood and bone marrow. AML is characterized by the clonal proliferation of myeloid precursors (“blasts”), a reduced ability to differentiate in the bone marrow, and ultimately infiltration of the blood and other organs. In AML, the abnormal myeloid cells lead to bone marrow failure and severely impair the production of all types of blood cells, resulting in anemia, weakened immune function, reduced platelet production, and bone marrow dysfunction. The term “AML” includes various subtypes classified under the World Health Organization (WHO) Classification of Hematologic and Lymphatic System Tumors, 5th Revision and the International Consensus Classification 2022 system. According to the WHO classification, AML subtypes encompass AML with relapsed genetic abnormalities; AML with changes associated with myelodysplastic dysplasia; therapy-related myeloid tumors; myeloid sarcomas; myeloid hyperplasia associated with Down syndrome; blastic plasmacytoid dendritic cell tumors; and AML not classified in other ways, such as acute megakaryocytic leukemia and acute basophilic leukemia.

[0123] In addition, AML can be classified according to the French-American-British (FAB) classification, covering the following subtypes: M0 (acute myeloblastic leukemia, minimally differentiated); M1 (acute myeloblastic leukemia, immature); M2 (acute myeloblastic leukemia, with mature granulocytes); M3 (promyelocyte or acute promyelocyte leukemia (APL)); M4 (acute myelomonocytic leukemia); M4eo (monocytosis and eosinophilia); M5 (acute monoblastic leukemia (M5a) or acute monocytic leukemia (M5b)); M6 (acute erythroblastic leukemia); and M7 (acute megakaryoblastic leukemia).

[0124] The term "myelodysplastic syndrome (MDS)" refers to a group of heterogeneous bone marrow disorders of clonal origin characterized by abnormal production and maturation of blood cells. The bone marrow of individuals with MDS typically displays features of developmental abnormalities, such as abnormal cell morphology, cytogenetic abnormalities, and impaired differentiation. This can lead to peripheral blood cytopenia and an increased risk of developing acute myeloid leukemia (AML). Most MDS cases exhibit clonal characteristics, with somatic genetic abnormalities detectable in both targeted next-generation sequencing (NGS) groups (approximately 90%) and conventional karyotypes (approximately 50%). The absence of clonal characteristics does not rule out an MDS diagnosis if a qualifying developmental abnormality and persistent cytopenia are present. Certain genetic abnormalities that induce persistent cytopenia are considered diagnostic for MDS even in the absence of developmental abnormalities. In pediatric cases, somatic abnormalities in genes such as SETBP1, ASXL1, and RUNX1, and mutations in the RAS / MAPK pathway define the genomic profile of MDS. Childhood refractory cytopenia (RCC) represents a unique entity within this context. MDS classifications encompass various subtypes based on developmental abnormality lineage, cytopenia, cytosis, BM and peripheral blood (PB) blast counts, cytogenetics, and mutations. These subtypes include MDS with mutated SF3B1 (MDS-SF3B1), MDS with del(5q) (MDS-del(5q)), MDS without developmental abnormalities, MDS with single lineage abnormalities, MDS with multiple lineage abnormalities, MDS with excess blasts (MDS-EB), and MDS / AML. Each subtype is distinguished by specified criteria in terms of blast count, genetic mutations, cytogenetics, and other characteristics. MDS can also be classified according to the French-American-British (FAB) classification, covering subtypes such as: refractory anemia (RA); refractory anemia with ringed sideroblasts (RARS); refractory anemia with blastosis (RAEB); refractory anemia with blastosis in transformation (RAEB-T); and chronic myeloid monocytic leukemia (CMML).

[0125] In ICC 2022, a new category of MDS / AML was created for cases with cytopenic myeloid neoplasms and blasts in peripheral blood and / or bone marrow of 10% to 19% (eliminating WHO 2017's MDS with Excess Blast Cells grade 2). This was to allow patients in this group to participate in clinical trials for the treatment of AML or MDS based on clinical conditions. The diagnostic criteria for MDS / AML are the same as those for AML requiring blasts of 20% or more. Instead, WHO 2022 continues to classify these patients as MDS-IB (Increased Blast Cells, a term changed from Excess Blast Cells) grade 2 to avoid overtreatment. However, the WHO indicates that MDS-IB2 can be considered AML for therapeutic purposes if clinically indicated.

[0126] As used in this article, “AML,” “AML / MDS,” and “MDS” refer to any condition covered by the ICC and / or WHO and / or FAB classifications of these diseases.

[0127] As used in this article, “leukemia stem cells” (LSCs) or “leukemia stem-progenitor cells” (LSPCs) are a subset of leukemia cells that possess stem cell-like properties, including self-renewal and the ability to generate different cell types, including blast cells, within the leukemia clone. LSCs / LSPCs are considered the cause of AML initiation, maintenance, and relapse. They possess the ability to self-renew, enabling them to maintain the leukemia population, and they can differentiate into blast cells and other leukemia progeny. “Blast cells” are immature and abnormal cells that accumulate in the bone marrow and peripheral blood of AML patients. They originate from hematopoietic stem cells or progenitor cells that have undergone leukemia transformation. These cells exhibit impaired ability to differentiate into mature blood cells, leading to the accumulation of immature blast cells, and blast cells typically represent a significant proportion of the leukemia cell population in AML. Although LSCs / LSPCs represent a relatively small proportion of primary AML blast cells, their unique properties and crucial role in disease initiation, maintenance, and relapse make them important targets for AML treatment. LSCs can be characterized by CD34 + CD45 int CD90 - Lin - The cells. The blast cells can be assessed by flow cytometry (e.g., based on CD45). 暗 This can be detected by cellular morphology assessment obtained from a bone marrow biopsy or peripheral blood smear of the subject. The proportion of blast cells is determined relative to the total number of cells in the sample. As a further example, cellular morphology assessment can be used to determine the number of morphologically identified blast cells relative to the total number of cells in the examined field of view.

[0128] The terms “subject,” “patient,” “subject in need,” and “patient in need” are used interchangeably herein and refer to a living individual suffering from one or more of the diseases described herein (e.g., AML) that can be treated with the combination therapy described herein. Such a subject “needs” such treatment if the subject would benefit biologically, medically, and / or in terms of quality of life from the treatment.

[0129] As used herein, the terms “treat,” “treatment,” and “treating” refer to a reduction or improvement in the progression, severity, and / or duration of a condition (e.g., a proliferative disease) caused by the application of one or more therapies, or an improvement in one or more symptoms of the condition (suitably, one or more identifiable symptoms). In some embodiments, the term “treat / treatment / treating” refers to physically inhibiting the progression of a proliferative disease by, for example, stabilizing identifiable symptoms, physiologically inhibiting the progression of a proliferative disease by, for example, stabilizing physical parameters, or both. In other embodiments, the term “treatment” refers to reducing / decreasing or stabilizing tumor size and / or cancer cell count.

[0130] "Standard intensive chemotherapy," "intensive induction therapy," or "induction therapy" involves administering high doses of chemotherapy agents with the aim of achieving complete remission (CR) by eliminating leukemia cells in the bone marrow and restoring normal hematopoietic processes. Common induction chemotherapy regimens in AML include "7+3" induction chemotherapy, characterized by 7 days of high-dose cytarabine followed by 3 days of anthracycline antibiotics (e.g., daunorubicin or idarubicin). Another commonly used induction chemotherapy regimen for AML includes administration of Vyxeos. ® Newly diagnosed AML patients who meet the eligibility criteria receive standard intensive chemotherapy with the aim of inducing complete remission of AML, often in preparation for stem cell transplantation following successful chemotherapy. However, not all newly diagnosed AML patients are suitable for this standard intensive chemotherapy, as significant toxicities and side effects make it unsuitable for some. These patients are referred to as "unsuitable for standard intensive chemotherapy." Patients may be unsuitable for standard intensive chemotherapy because, for example, they exhibit one or more comorbidities that indicate they will not tolerate the toxicities of the standard intensive chemotherapy regimen, or prognostic factors characterizing their disease suggest unfavorable outcomes with standard intensive chemotherapy. AML patients over 60 years of age are generally assessed as unsuitable for standard intensive chemotherapy, with other factors to consider including cytogenetic and / or molecular abnormalities of the AML being treated.

[0131] The term "first-line" refers to the first systemic therapy used to treat a patient. If first-line therapy fails and the disease progresses or symptoms recur (e.g., cytopenia), a different systemic therapy is prescribed. Different treatments administered after first-line therapy are referred to in this document as "second-line" therapy.

[0132] The term "relapse" refers to the recurrence of disease-related symptoms or disease progression in patients who have achieved remission after treatment, such as the recurrence of a decrease in leukemia cells and normal blood cells in the bone marrow.

[0133] The term "refractory or resistant" refers to a situation where the disease does not respond adequately to the initial treatment or fails to achieve the expected level of improvement, even after intensive therapy, for example, residual leukemia cells remain in the patient's bone marrow.

[0134] The term “partial remission” refers to the response criteria as defined by the European Leukemia Network (ELN) in 2022 (Döhner et al., Blood (2022) 140 (12): 1345–1377).

[0135] The term "complete remission" refers to the response criteria defined by the European Leukemia Network (ELN) 2022 (Döhner et al., Blood (2022) 140 (12): 1345–1377), and includes complete remission (CR) and CR with no measurable residual disease (CR). MRD− CR with incomplete hematologic recovery (CR) i ) and morphological leukemia-free status (MLFS).

[0136] As used herein, "antiproliferative agent" refers to an agent that can prevent or inhibit cell proliferation, appropriately an agent that can prevent the proliferation of cells exhibiting abnormal growth behavior. Specifically, "anticancer agent" refers to any agent that can directly or indirectly prevent, inhibit, and / or treat cancer growth. Such agents include chemotherapeutic agents, immunotherapeutic agents, anti-angiogenic agents, radionuclides, antibody molecules, targeted anticancer therapies, gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapy or oncolytic drugs, cytotoxic agents, and immunotherapies (e.g., cytokines, immunostimulants, or cell-based immunotherapies).

[0137] "Therapeutic effective dose" refers to the amount that effectively achieves the desired therapeutic outcome within the necessary dosage and time period. Therapeutic effective dose can vary depending on factors such as an individual's disease state, age, sex, and weight. Therapeutic effective dose is also the amount by which the beneficial therapeutic effect of the therapeutic agent outweighs any toxic or harmful effects. The "therapeutic effective dose" preferably adjusts measurable parameters relative to an untreated subject in a desired manner by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, for example, by reducing or eliminating leukemia blasts and / or reducing the need for transfusions and / or the change in state from transfusion dependence to transfusion independence and / or the normalization of blood parameters (e.g., red blood cell count, white blood cell count, and / or platelet count).

[0138] The terms “combined therapeutic activity” or “combined therapeutic effect” refer to the ability of therapeutic agents to be administered at such intervals (in a time-staggered manner) so that they exhibit a beneficial interaction (combined therapeutic effect) in the treated subjects.

[0139] The term "synergistic efficacy" refers to the combined or synergistic effect of multiple factors, substances, or treatments (such as combinations of therapeutic agents described herein) that produce a greater effect or outcome than the sum of their individual effects. This term indicates that two or more agents, therapies, or interventions work together in a synergistic and amplifying manner to enhance their efficacy or therapeutic effect. For example, exposure to a combination of therapeutic agents disclosed herein results in a reduction in blast cells and / or LSC / LSPC greater than the additive effect of the individual therapeutic agents as monotherapy. This synergistic effect can lead to improved treatment outcomes, enhanced efficacy, increased response rates, and / or reduced side effects compared to using each therapeutic agent alone.

[0140] Appropriate methods for determining whether a synergistic effect is produced by the combination of therapeutic agents can be, for example, calculated using suitable methods such as the Sigmoid-Emax equation (Holford, NHG and Scheiner, LB, Clin. Pharmacokinet. 6: 429-453 (1981)), the Loewe additivity equation (Loewe, S. and Muischnek, H., Arch. Experimental Pathol Pharmacol. 114: 313-326 (1926)) or the Chou-Talalay method (Chou, TC. Cancer Res. 2010;70(2): 440-446; Chou, TC. Pharmacol Rev. 2006;58(3):621-681). Each of the equations mentioned above can be applied to experimental data to produce corresponding graphs to help assess the effects of the combination. The corresponding graphs associated with the equations mentioned above are concentration-response curves, equivalence curves, and combination exponential curves, respectively. According to the Chou-Talalay method, a combination index (CI) < 1 indicates a synergistic effect, CI = 1 indicates a cumulative effect, and CI > 1 indicates an antagonistic effect.

[0141] The term "cycle" refers to a specified period of time, expressed in days or months, that repeats according to a regular schedule. The cycles disclosed herein are more preferably expressed in days. For example, a cycle can be 28 days, 30 days, 60 days, or 90 days. Most preferably, the "cycle" referred to herein is 28 days long.

[0142] The term "withdrawal period" refers to a time interval within a dosing regimen during which the therapeutic agent is not administered to the patient. Withdrawal periods can reduce the likelihood or severity of side effects, allow organ function to recover, and / or enable the patient to maintain sensitivity to the therapeutic agent.

[0143] As used herein, the term "dosing regimen" refers to a treatment plan that specifies the pattern of administration of a therapeutic agent over a period of time. A dosing regimen defines the amount of a therapeutic agent used to treat a disease and the number and frequency of administrations within a specified time period. Dosing regimens will be explained, for example, in the dosage and administration section or the dosing and administration section of the package insert for human prescription drugs.

[0144] Dosing regimens may include escalation dosing regimens. As used herein, the term "escalation dosing regimen" refers to starting treatment with a lower-than-standard dose of the therapeutic agent and gradually increasing the dose over time until the target therapeutic level is reached when the "initial administration" of the therapeutic agent occurs.

[0145] "Overall survival" is a clinical endpoint that measures the length of time from the start of a particular treatment or intervention until the patient dies from any cause.

[0146] "Progression-free survival" (PFS) is a clinical endpoint that measures the length of time from the start of a particular treatment or intervention until the treated disease shows signs of progression or worsening or until death from any cause (whichever comes first).

[0147] As used in this article, the dosage unit refers to a physical discrete unit suitable as a unit dose for a subject to be treated; each unit contains a predetermined amount of active therapeutic agent, which is calculated to combine with the desired drug carrier to produce the desired therapeutic effect.

[0148] As used in this article, the term “transfusion independence” means that a subject has acquired the ability to maintain adequate levels of hemoglobin, platelets, and other essential blood parameters without requiring regular transfusions.

[0149] "Pharmaceutically acceptable excipients" are substances that facilitate administration of an active agent to a subject by, for example, altering the stability of the active agent or altering its absorption by the subject after administration. Pharmaceutically acceptable excipients generally do not have significant adverse toxicity to patients. Examples of pharmaceutically acceptable excipients include, for example, water, NaCl (including salt solutions), sugars, swelling agents, buffers, diluents, antioxidants, binders, fillers, emulsifiers, extenders, disintegrants, lubricants, coating agents, sweeteners, flavoring agents, humectants, preservatives, alcohols, oils, gelatin, solubilizers, wetting agents, carbohydrates (such as amylose or starch), fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone, colorants, etc. Those skilled in the art will recognize that other pharmaceutical excipients known in the art can be used in this invention, including, for example, those listed in Handbook of Pharmaceutical Excipients, Sheskey PJ, Hancock, BC, Moss, GP, Goldfarb, DJ, 9th Edition, The Pharmaceutical Press, RPS Publishing (2020).

[0150] combination

[0151] The combinations provided herein may be separately or together comprising: (i) a recombinant binding protein comprising (a) a binding domain that specifically binds to CD3, (b) a binding domain that specifically binds to CD33 and / or a binding domain that specifically binds to CD70, and (c) a binding domain that optionally specifically binds to CD123, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent.

[0152] Without being bound by theory, the combination presented in this article is considered particularly effective for the treatment of myeloid malignancies, appropriately AML and MDS, due to its combined therapeutic effects on leukemia cells, especially blast cells and / or LSCs.

[0153] As shown in this article, combination or combination therapy agents have synergistic efficacy against AML cells.

[0154] In one embodiment, the recombinant binding protein comprises (a) a first binding domain that specifically binds to CD3, and

[0155] i.(b) Specifically binds to the second binding domain of CD33;

[0156] ii.(b) Specifically binds to the second binding domain of CD70; or

[0157] iii. (b) specifically binding to the second binding domain of CD123 and (c) specifically binding to the third binding domain of CD70; or

[0158] iv. (b) specifically binds to the second binding domain of CD33 and (c) specifically binds to the third binding domain of CD70; or

[0159] v. (b) specifically binds to the second binding domain of CD123, (c) specifically binds to the third binding domain of CD33; or

[0160] vi. (b) specifically binds to the second binding domain of CD123, (c) specifically binds to the third binding domain of CD33, and (d) specifically binds to the fourth binding domain of CD70.

[0161] In one embodiment, the binding protein comprises or consists of: an antibody or its antigen-binding fragment, an antibody mimic, a scaffold protein, and / or a repeating protein, particularly an ankylosing spicule repeating protein.

[0162] More specifically, the combinations provided herein may comprise, separately or together: (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of which specifically binds to a tumor-associated antigen suitably selected from the group consisting of CD123, CD33, and CD70; and (ii) a Bcl-2 inhibitor, such as veneclade or a pharmaceutically acceptable salt thereof; and / or (iii) a hypomethylating agent, such as azacitidine or decitabine.

[0163] In one embodiment, the recombinant binding protein comprises (a) a first ankyrin repeat domain that specifically binds to CD3, and

[0164] i.(b) Specifically binds to the second ankyrin repeat domain of CD123; or

[0165] ii.(b) Specifically binds to the second ankyrin repeat domain of CD33; or

[0166] iii.(b) Specifically binds to the second ankyrin repeat domain of CD70; or

[0167] iv. (b) specifically binds to the second ankyrin repeat domain of CD123 and (c) specifically binds to the third ankyrin repeat domain of CD70; or

[0168] v. (b) specifically binds to the second ankyrin repeat domain of CD33 and (c) specifically binds to the third ankyrin repeat domain of CD70; or

[0169] vi. (b) specifically binds to the second ankyrin repeat domain of CD123, (c) specifically binds to the third ankyrin repeat domain of CD33; or

[0170] vii. (b) specifically binds to the second ankyrin repeat domain of CD123, (c) specifically binds to the third ankyrin repeat domain of CD33, and (d) specifically binds to the fourth ankyrin repeat domain of CD70.

[0171] In a preferred embodiment, the recombinant binding protein comprises (a) a first ankyrin repeat domain that specifically binds to CD3, (b) a second ankyrin repeat domain that specifically binds to CD123, (c) a third ankyrin repeat domain that specifically binds to CD33, and (d) a fourth ankyrin repeat domain that specifically binds to CD70.

[0172] In some embodiments, the ankyrin repeat domain that specifically binds to CD3 comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the same amino acid sequence as SEQ ID NO: 1, and the ankyrin repeat domain that specifically binds to CD33 comprises the same amino acid sequence as SEQ ID NO: 1. The amino acid sequence of any one of 2 to 3 is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical, and the ankyrin repeat domain that specifically binds to CD123 contains the same amino acid sequence as SEQ ID NO: The amino acid sequence of any one of 4 to 7 is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical, and the ankyrin repeat domain that specifically binds to CD70 contains the same amino acid sequence as SEQ ID NO: The amino acid sequence of any one of 8 to 9 is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical.

[0173] In some embodiments, the ankyrin repeat domain that specifically binds to CD3 comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the same amino acid sequence as SEQ ID NO: 1, and the ankyrin repeat domain that specifically binds to CD33 comprises the same amino acid sequence as SEQ ID NO: 1. 2. At least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical amino acid sequences, specifically binding to the CD123 ankyrin repeat domain containing the same amino acid sequence as SEQ ID NO: 4. The amino acid sequence is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 8, and the ankyrin repeat domain that specifically binds to CD70 contains the amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 8.In some embodiments, the ankyrin repeat domain that specifically binds to CD3 comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the same amino acid sequence as SEQ ID NO: 1, and the ankyrin repeat domain that specifically binds to CD33 comprises the same amino acid sequence as SEQ ID NO: 1. 3. At least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical amino acid sequences, specifically binding to the CD123 ankyrin repeat domain containing the same amino acid sequence as SEQ ID NO: 5. At least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical amino acid sequence to SEQ ID NO: 8, and the ankyrin repeat domain that specifically binds to CD70 contains at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical amino acid sequence to SEQ ID NO: 8.In some embodiments, the CD3-specifically binding ankyrin repeat domain comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the same amino acid sequence as SEQ ID NO: 1, and the CD33-specifically binding ankyrin repeat domain comprises the same amino acid sequence as SEQ ID NO: 1. NO:2 has at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical amino acid sequences, specifically binding to the CD123 ankyrin repeat domain containing the same amino acid sequence as SEQ ID NO: 7. The amino acid sequence is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 8, and the ankyrin repeat domain that specifically binds to CD70 contains the amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 8.In some embodiments, the ankyrin repeat domain that specifically binds to CD3 comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the same amino acid sequence as SEQ ID NO: 1, and the ankyrin repeat domain that specifically binds to CD33 comprises the same amino acid sequence as SEQ ID NO: 1. 2. At least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical amino acid sequences, specifically binding to the CD123 ankyrin repeat domain containing the same amino acid sequence as SEQ ID NO: 4. The amino acid sequence is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 8, and the ankyrin repeat domain that specifically binds to CD70 contains the amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 8.In some embodiments, the ankyrin repeat domain that specifically binds to CD3 comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the same amino acid sequence as SEQ ID NO: 1, and the ankyrin repeat domain that specifically binds to CD33 comprises the same amino acid sequence as SEQ ID NO: 1. 2. At least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical amino acid sequences, specifically binding to the CD123 ankyrin repeat domain containing the same amino acid sequence as SEQ ID. NO:6 contains at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the same amino acid sequence as SEQ ID NO: 8, and the ankyrin repeat domain that specifically binds to CD70 contains at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the same amino acid sequence as SEQ ID NO: 8.In some embodiments, the ankyrin repeat domain that specifically binds to CD3 comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the same amino acid sequence as SEQ ID NO: 1, and the ankyrin repeat domain that specifically binds to CD33 comprises the same amino acid sequence as SEQ ID NO: 1. 3. At least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical amino acid sequences, specifically binding to the CD123 ankyrin repeat domain containing the same amino acid sequence as SEQ ID NO: 5. The amino acid sequence is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 9, and the ankyrin repeat domain that specifically binds to CD70 contains the amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 9.In some embodiments, the ankyrin repeat domain that specifically binds to CD3 comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% of the same amino acid sequence as SEQ ID NO: 1, and the ankyrin repeat domain that specifically binds to CD33 comprises the same amino acid sequence as SEQ ID NO: 1. 2. At least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical amino acid sequences, specifically binding to the CD123 ankyrin repeat domain containing the same amino acid sequence as SEQ ID NO: 4. The amino acid sequence is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 8, and the ankyrin repeat domain that specifically binds to CD70 contains the amino acid sequence at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to that of SEQ ID NO: 8.

[0174] In a particularly preferred embodiment, the recombinant binding protein comprises at least one or at least two additional ankyrin repeat domains that specifically bind serum albumin (suitably human serum albumin (HSA)). Suitably, the ankyrin repeat binding domains that specifically bind serum albumin (suitably human serum albumin) comprise an amino acid sequence that is at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the amino acid sequence in SEQ ID NO: 10.

[0175] In one embodiment, the ankyrin repeat domains contained in the recombinant binding protein are arranged in any order from the N-terminus to the C-terminus. In one embodiment, one or more serum albumin-binding ankyrin repeat domains, CD33-binding ankyrin repeat domains, CD123-binding ankyrin repeat domains, CD70-binding ankyrin repeat domains, and CD3-binding ankyrin repeat domains are arranged in any order from the N-terminus to the C-terminus.

[0176] In one embodiment, the ankyrin repeating domains are arranged in the following order from the N-terminus to the C-terminus: (serum albumin binding domain) - (serum albumin binding domain) - (CD33 binding domain) - (CD123 binding domain) - (CD70 binding domain) - (CD3 binding domain).

[0177] In another embodiment, the ankyrin repeating domains are arranged in the following order from the N-terminus to the C-terminus: (serum albumin binding domain) - (serum albumin binding domain) - (CD70 binding domain) - (CD123 binding domain) - (CD33 binding domain) - (CD3 binding domain).

[0178] In another embodiment, the ankyrin repeating domains are arranged in the following order from the N-terminus to the C-terminus: (serum albumin binding domain) - (serum albumin binding domain) - (CD70 binding domain) - (CD33 binding domain) - (CD123 binding domain) - (CD3 binding domain).

[0179] In one embodiment, the recombinant binding protein simultaneously binds CD3, CD123, CD33, and CD70. In another embodiment, the recombinant binding protein simultaneously binds CD3, CD123, CD33, CD70, and HSA.

[0180] In some embodiments, the binding domain contained in the recombinant binding protein is covalently linked to a peptide linker. A suitable peptide linker is provided with SEQ ID NO: 11. Therefore, in one embodiment, the peptide linker comprises the amino acid sequence having SEQ ID NO: 11.

[0181] In some embodiments, the recombinant binding protein comprising an ankyrin repeat domain that specifically binds CD3, an ankyrin repeat domain that specifically binds CD123, an ankyrin repeat domain that specifically binds CD33, and an ankyrin repeat domain that specifically binds CD70 comprises at least one, suitably two, ankyrin repeat domains that specifically bind HSA, and comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with any of the amino acid sequences having SEQ ID NO: 12 to 18. In one embodiment, the recombinant binding protein comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with the amino acid sequence having SEQ ID NO: 12. In another embodiment, the recombinant binding protein comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with the amino acid sequence having SEQ ID NO: 13. In another embodiment, the recombinant binding protein comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with the amino acid sequence having SEQ ID NO: 14.In another embodiment, the recombinant binding protein comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with the amino acid sequence having SEQ ID NO: 15. In another embodiment, the recombinant binding protein comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with the amino acid sequence having SEQ ID NO: 16. In another embodiment, the recombinant binding protein comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with the amino acid sequence having SEQ ID NO: 17. In another embodiment, the recombinant binding protein comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with the amino acid sequence having SEQ ID NO: 18.

[0182] In a preferred embodiment, the combination comprises (i) a recombinant binding protein that specifically binds to CD3, CD123, CD33, and CD70, said recombinant binding protein comprising at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with any of the amino acid sequences of SEQ ID NO: 12 to 18, (ii) Veneclare or a pharmaceutically acceptable salt thereof, and / or (iii) azacitidine.

[0183] In another preferred embodiment, the combination comprises (i) a recombinant binding protein that specifically binds to CD3, CD123, CD33, and CD70, said recombinant binding protein comprising at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with any of the amino acid sequences of SEQ ID NO: 12 to 18, (ii) Venecella or a pharmaceutically acceptable salt thereof, and / or (iii) decitabine.

[0184] In a preferred embodiment, the combination comprises (i) a recombinant binding protein that specifically binds to CD3, CD123, CD33, and CD70, said recombinant binding protein comprising an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 12, (ii) Veneclare or a pharmaceutically acceptable salt thereof, and / or (iii) azacitidine.

[0185] In another preferred embodiment, the combination comprises (i) a recombinant binding protein that specifically binds to CD3, CD123, CD33, and CD70, said recombinant binding protein comprising an amino acid sequence having at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity with SEQ ID NO: 12, (ii) Venecella or a pharmaceutically acceptable salt thereof, and / or (iii) decitabine.

[0186] In one embodiment, the combination comprises (i) a recombinant binding protein having the amino acid sequence of SEQ ID NO: 12, (ii) Veneclare or a pharmaceutically acceptable salt thereof, and / or (iii) azacitidine.

[0187] In another embodiment, the combination comprises (i) a recombinant binding protein comprising the amino acid sequence having SEQ ID NO: 12, (ii) Venecla or a pharmaceutically acceptable salt thereof, and / or (iii) decitabine.

[0188] The preferred Bcl-2 inhibitor disclosed herein is veneclade, described in US 8,546,399 B2 (incorporated by reference). Veneclade is a selective Bcl-2 inhibitor that binds tightly to the Bcl-2 protein and inhibits its anti-apoptotic function. Veneclade works by occupying the hydrophobic binding groove of Bcl-2, preventing its interaction with pro-apoptotic proteins such as Bim. By inhibiting the interaction between Bcl-2 and pro-apoptotic proteins, veneclade effectively counteracts the anti-apoptotic signaling transmitted by Bcl-2. This disruption releases pro-apoptotic factors, allowing them to activate intrinsic apoptotic pathways and induce programmed cell death in cancer cells. Veneclade is authorized in the United States in combination with azacitidine, decitabine, or low-dose cytarabine for the treatment of newly diagnosed AML in adults aged 75 years or older or with comorbidities excluded from intensive induction chemotherapy. As used herein, the term "Veneclax" (also known as ABT-199 and GDC-0199; CAS number 1257044-40-8) refers to compounds having the chemical structures shown below:

[0189]

[0190] Veneclare is provided in any suitable form that allows it to effectively inhibit the Bcl-2 protein. Such forms include, but are not limited to, any suitable polymorph, amorphous, or crystalline form, or any isomer or tautomeric form. Pharmaceutically acceptable salts according to this disclosure include salts with acidic or basic groups. Veneclare may also be provided in hydrated, anhydrous, or solvated forms.

[0191] In a preferred embodiment, the Bcl-2 inhibitor is Veneclare or a pharmaceutically acceptable salt thereof.

[0192] In some embodiments, the combinations described herein comprise one or more hypomethylating agents. Hypomethylating agents inhibit normal methylation of DNA and / or RNA. Examples of hypomethylating agents are azacitidine (or azithromycin) and decitabine.

[0193] Both azacitidine and decitabine are nucleoside analogues that interfere with nucleic acid and protein synthesis upon incorporation into RNA and DNA. Azacitidine is a pyrimidine nucleoside analogue of cytidine and, upon incorporation into DNA, primarily inhibits the enzyme DNA methyltransferase 1 (DNMT1), leading to DNA methyltransferase depletion and subsequent DNA hypomethylation. Decitabine similarly incorporates into DNA and irreversibly inhibits DNA methyltransferases, particularly DNMT1, resulting in prolonged inhibition and DNA hypomethylation. Azacitidine and decitabine have the ability to restore normal gene expression by reactivating silenced genes, including tumor suppressor genes. Alterations in DNA methylation patterns and the restoration of appropriate gene regulation contribute to their therapeutic efficacy. These hypomethylating agents have been approved by the US FDA as monotherapy and in combination for the treatment of MDS and AML. In some preferred embodiments, the hypomethylating agent is azacitidine. In other preferred embodiments, the hypomethylating agent is decitabine.

[0194] Low-dose cytarabine (cytosine-β-D-arabinofuranoside) exerts its effect on rapidly dividing cells by incorporating into DNA during the S phase of the cell cycle. Once incorporated, it inhibits DNA synthesis by acting as a strand terminator and preventing further elongation of the DNA strand, leading to disruption of DNA replication and ultimately inducing apoptosis in affected cells. Low-dose cytarabine can be used in place of hypomethylating agents. In some embodiments, the combination comprises low-dose cytarabine instead of a hypomethylating agent.

[0195] In a preferred embodiment, i) the recombinant binding protein, (ii) the Bcl-2 inhibitor, and (iii) the hypomethylating agent are separate.

[0196] Also provided is a pharmaceutical combination comprising, separately or together: (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binding to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70; and (ii) a Bcl-2 inhibitor, suitably veneclade or a pharmaceutically acceptable salt thereof; and / or (iii) a hypomethylating agent, suitably azacitidine or decitabine.

[0197] Pharmaceutical Composition

[0198] The therapeutic agents described herein can be combined or formulated in any manner that allows for administration of combination therapy to a subject or patient in need. The combination can be formulated for single-dose administration or for multiple-dose administration. In some embodiments, the therapeutic agents in the combination can be co-formulated, i.e., formulated as a single pharmaceutical composition. For embodiments in which the therapeutic agents are co-formulated, the combination or composition is suitable for simultaneous administration of the therapeutic agents. In a preferred embodiment, the therapeutic agents in the combination described herein are formulated as separate compositions or pharmaceutical compositions.

[0199] In embodiments where the therapeutic agents are formulated separately, the therapeutic agents can be administered simultaneously or separately. If different therapeutic agents are administered separately, the therapeutic agents can be administered in any order. The interval between administrations of the therapeutic agents can be any suitable time interval. Preferably, the combined therapeutic agents are administered within time intervals during which the therapeutic agents can exert a combined therapeutic effect (suitable synergistic effect). The administration of different compositions can be performed once (for single-dose administration) or repeatedly (for multiple-dose administration). In one embodiment, the combined therapeutic agents are co-formulated, i.e., formulated as a single pharmaceutical composition.

[0200] The recombinant binding proteins included in the combinations disclosed herein can be formulated using any suitable drug carrier, adjuvant, and / or excipient. Pharmaceutically acceptable excipients that can be used to formulate recombinant binding proteins include buffers, pH stabilizers, isotonic agents, salts, cryoprotectants, etc.

[0201] Bcl-2 inhibitors (preferably veneclax or a pharmaceutically acceptable salt thereof) can be formulated using any suitable drug carrier, adjuvant, and / or excipient. ® "For sale. Venclexta for oral administration." ®Tablets are provided in tablet form containing 10 mg, 50 mg, or 100 mg of Venclexta as the active ingredient. Each tablet also contains the following inactive ingredients: copovidone, colloidal silica, polysorbate 80, sodium stearate fumarate, and calcium hydrogen phosphate. In addition, the 10 mg and 100 mg coated tablets also contain iron oxide yellow, polyvinyl alcohol, polyethylene glycol, talc, and titanium dioxide. The 50 mg coated tablets also contain iron oxide yellow, iron oxide red, iron oxide black, polyvinyl alcohol, talc, polyethylene glycol, and titanium dioxide. Suitably, Venclexta is used as Venclexta. ® Administration. Therefore, in some embodiments described herein, the Bcl-2 inhibitor is Venclexta. ® .

[0202] Hypomethylating agents or antimetabolites such as cytarabine can be formulated using any suitable drug carrier, adjuvant, and / or excipient. Azacitidine is marketed under the trade name "Vidaza". ® "For Sale. Vidaza for Injection" ® It is provided in the form of a sterile lyophilized powder containing mannitol. Suitablely, azacitidine is used as Vidaza ® Application. Therefore, in some embodiments described herein, the hypomethylating agent is Vidaza. ® Diazepam is marketed under the brand name "Dacogen". ® "For Sale. Dacogen for Injection" ® It is provided in sterile lyophilized powder form, containing potassium dihydrogen phosphate (monopotassium phosphate) and sodium hydroxide. Preferably, decitabine is used as a Dacogen. ® Application. Therefore, in some embodiments described herein, the hypomethylating agent is Dacogen. ® .

[0203] In some embodiments, the composition is formulated for administration to a subject via any suitable route of administration, including but not limited to intramuscular, intravenous, subcutaneous, and oral administration. In some embodiments, the composition is formulated as an aqueous solution, tablet, capsule, powder, or any other suitable dosage form.

[0204] In embodiments where the combined therapeutic agents are formulated separately (i.e., as separate compositions), the separate compositions can be formulated for the same route of administration. In embodiments where the combined therapeutic agents are formulated separately (i.e., as separate compositions), the separate compositions can be formulated for different routes of administration. For example, the recombinant binding protein can be formulated for intravenous administration, and the Bcl-2 inhibitor (preferably veneclax) can be formulated for oral administration, and the hypomethylating agent can be formulated for subcutaneous or intravenous administration. In a preferred embodiment, the combination comprises (i) the recombinant binding protein as described herein, and (ii) veneclax or a pharmaceutically acceptable salt thereof and / or (iii) azacitidine, wherein the recombinant binding protein is administered intravenously, veneclax or a pharmaceutically acceptable salt thereof is administered orally, and azacitidine is administered subcutaneously or intravenously. In a preferred embodiment, the combination comprises (i) a recombinant binding protein as described herein, and (ii) veneclax or a pharmaceutically acceptable salt thereof, and / or (iii) decitabine, wherein the recombinant binding protein is administered intravenously, veneclax or a pharmaceutically acceptable salt thereof is administered orally, and decitabine is administered intravenously. In other embodiments, the combination comprises (i) a recombinant binding protein as described herein, and (ii) veneclax or a pharmaceutically acceptable salt thereof, and / or (iii) low-dose cytarabine, wherein the recombinant binding protein is administered intravenously, veneclax or a pharmaceutically acceptable salt thereof is administered orally, and low-dose cytarabine is administered subcutaneously.

[0205] treat

[0206] The above combination can be used in therapies, particularly for the treatment of malignant tumors, especially myeloid malignancies such as AML or MDS.

[0207] Therefore, this article provides a combination comprising, separately or together: (i) a recombinant binding protein comprising (a) a binding domain specifically binding to CD3, (b) a binding domain specifically binding to CD33 and / or a binding domain specifically binding to CD70, and optionally (c) a binding domain specifically binding to CD123, (ii) a Bcl-2 inhibitor, suitably veneclade or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, such as azacitidine or decitabine, for the treatment of malignant tumors, suitably myeloid malignancies.

[0208] In one aspect, a combination is provided, comprising separately or together (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binding to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor, suitably veneclax or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, suitably azacitidine or decitabine, the combination being used to treat a subject with a malignant tumor, suitably myeloid malignant tumor, comprising administering to a subject in need a therapeutically effective amount of the therapeutic agent contained in the combination. Suitably, the recombinant binding protein and the Bcl-2 inhibitor and / or the hypomethylating agent are administered separately, simultaneously, or sequentially. Suitably, the recombinant binding protein, the Bcl-2 inhibitor, and / or the hypomethylating agent are provided in synergistically effective amounts for the treatment of malignant tumors.

[0209] In another aspect, a recombinant binding protein is provided comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, wherein each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binds to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, the recombinant binding protein being used to treat malignant tumors, suitably myeloid malignant tumors, comprising administering the recombinant binding protein separately, simultaneously, or sequentially to a subject in need of it, along with (i) a Bcl-2 inhibitor, suitably veneclade or a pharmaceutically acceptable salt thereof, and / or (ii) a hypomethylating agent, suitably azacitidine or decitabine.

[0210] In another aspect, a Bcl-2 inhibitor, suitably veneclade or a pharmaceutically acceptable salt thereof, is provided for the treatment of malignant tumors, suitably myeloid malignancies, comprising administering the Bcl-2 inhibitor separately, simultaneously or sequentially to a subject in need of it: (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two or at least three additional ankyrin repeat domains, each of the at least one, at least two or at least three additional ankyrin repeat domains specifically binding to a tumor-associated antigen, suitably selected from the group consisting of CD123, CD33 and CD70, and (ii) a hypomethylating agent, suitably azacitidine or decitabine.

[0211] In another aspect, a hypomethylating agent, suitably azacitidine or decitabine, is provided for the treatment of malignant tumors, suitably myeloid malignancies, comprising administering the hypomethylating agent separately, simultaneously or sequentially to a subject in need of it: (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two or at least three additional ankyrin repeat domains, each of the at least one, at least two or at least three additional ankyrin repeat domains specifically binding to a tumor-associated antigen, suitably selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor, suitably veneclade or a pharmaceutically acceptable salt thereof.

[0212] In another aspect, a method is provided for treating a subject with a malignant tumor, suitably a myeloid malignant tumor, the method comprising administering to the subject in need a therapeutically effective amount of: (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, wherein each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binds to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent.

[0213] In another aspect, the following is provided: (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of which specifically binds to a tumor-associated antigen suitably selected from the group consisting of CD123, CD33, and CD70; and (ii) a Bcl-2 inhibitor, suitably veneclax or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, suitably azacitidine or decitabine, for the manufacture of a medicament for the treatment of malignant tumors.

[0214] In another aspect, a medicament is provided comprising, separately or together: a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, wherein each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binds to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70; and (ii) a Bcl-2 inhibitor, suitably veneclax or a pharmaceutically acceptable salt thereof; and / or (iii) a hypomethylating agent, suitably azacitidine or decitabine, the medicament being administered concurrently, sequentially, or separately, wherein the recombinant binding protein, the Bcl-2 inhibitor, and / or the hypomethylating agent are provided in a synergistically effective amount for treating malignant tumors.

[0215] The therapeutic agents disclosed herein can be administered together in a single composition or separately in two or more different compositions (e.g., compositions or dosage forms as described herein). The administration of the therapeutic agents can be in any order. The administration of different therapeutic agents can be performed once (for single-dose administration) or repeatedly (for multiple-dose administration). The first agent and subsequent agents (e.g., second, third agents) can be administered via the same route of administration or via different routes of administration. In the preferred embodiments described herein, the therapeutic agents are administered separately. Administration can be simultaneous or sequential. In one embodiment, the administration of two or three therapeutic agents contained in the combinations disclosed herein is separate but simultaneous. For example, a recombinant binding protein and a Bcl-2 inhibitor (suitably veneclade or a pharmaceutically acceptable salt thereof) are administered at the same time (simultaneously, e.g., in a single injection), but the two therapeutic agents are administered separately (i.e., the therapeutic agents are not contained in the same dosage form). In one embodiment, the administration of two or three therapeutic agents contained in the combinations disclosed herein is separate and simultaneous. For example, the recombinant binding protein and a Bcl-2 inhibitor (suitably veneclade or a pharmaceutically acceptable salt thereof) are administered at the same time or within a close time frame (simultaneously), but the two therapeutic agents are administered separately. In one embodiment, the administration of two or three therapeutic agents included in the combinations disclosed herein is separate and sequential. For example, the recombinant binding protein is administered in a specified order or sequence, with time intervals between the administration of the Bcl-2 inhibitor (suitably veneclade or a pharmaceutically acceptable salt thereof), but the two therapeutic agents are administered separately (not included in the same dosage form). In one embodiment, the therapeutic agents are administered at a combined therapeutically effective amount. Suitably, the therapeutic agents are administered at a synergistically effective amount. Therefore, in one aspect, a method is provided to enhance the efficacy of an anticancer therapeutic agent by combining it with another anticancer therapeutic agent, particularly using a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, wherein each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binds to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and a Bcl-2 inhibitor, suitably veneclax or a pharmaceutically acceptable salt thereof, and / or a hypomethylating agent, suitably azacitidine or decitabine, to provide enhanced efficacy that cannot be achieved by administering any therapeutic agent at a similar or lower dose as a single agent.

[0216] In one implementation, the malignant tumor is a myeloid malignant tumor. In a particular implementation, the treatment is for treating myeloid malignant tumors, wherein a myeloid malignant tumor refers to any clonal disease of hematopoietic stem cells or progenitor cells.

[0217] In one embodiment, the myeloid malignancy is acute myeloid leukemia (AML); myelodysplastic syndrome (MDS); myeloproliferative neoplasm (MPN); chronic myeloid leukemia (CML); or chronic myelomonocytic leukemia (CMML). In a preferred embodiment, the myeloid malignancy is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or AML / MDS. In one embodiment, the myeloid malignancy is MDS. In one embodiment, the myeloid malignancy is AML. In one embodiment, the myeloid malignancy is AML / MDS.

[0218] In one implementation, the subject requiring treatment is a newly diagnosed subject. In another implementation, the subject requiring treatment suffers from relapsed and / or refractory disease.

[0219] The treatments described in this article can be first-line or second-line treatments.

[0220] In one implementation, the treatment is first-line therapy in suitable AML subjects.

[0221] In one implementation, the treatment is first-line therapy in unsuitable AML subjects. Suitablely, the unsuitable AML patients have a poor cytogenetic risk and / or have one or more mutations in TP53.

[0222] In one implementation, the treatment is performed after hematopoietic stem cell transplantation (HSCT). Suitably, this post-HSCT treatment is second-line therapy. Suitably, the second-line post-HSCT treatment is performed in patients who have not received azacitidine / veneclade treatment, i.e., in patients who have not been exposed to azacitidine and / or veneclade.

[0223] In one implementation, the treatment is first-line therapy for patients with high-risk MDS. In another implementation, the treatment is first-line therapy for patients with chronic myeloid monocytic leukemia (CMML).

[0224] Subjects receiving treatment may be those unsuitable for standard intensive chemotherapy. Appropriately, such patients may be newly diagnosed. These subjects may be unsuitable for standard intensive chemotherapy because they have one or more comorbidities that preclude its use. Alternatively, the subjects described herein may be suitable for other treatments, such as standard intensive chemotherapy, but the combinations disclosed herein provide a novel and innovative approach to treating AML, offering improved efficacy, reduced toxicity, and potential synergistic effects that cannot be achieved with conventional monotherapy or existing treatment regimens.

[0225] Subjects requiring treatment can be 60 years of age or older, or 75 years of age or older.

[0226] In addition, peripheral blood and / or myeloblast counts of the subject may be monitored. Suitablely, the treatment described herein reduces the myeloblast count to less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% compared to pre-treatment levels, and / or reduces the percentage of myeloblasts by more than about 50%, more than about 60%, more than about 70%, more than about 80%, or more than about 90% compared to pre-treatment levels. Suitablely, the percentage of myeloblasts also reduces by more than about 50% compared to the percentage of myeloblasts prior to the methods or uses described herein (“pre-treatment”). Alternatively or in addition, transfusion independence of red blood cells or platelets, or both, is achieved for at least about 8 weeks, at least about 10 weeks, or at least about 12 weeks.

[0227] Measurable residual disease (MRD) refers to the presence of residual leukemia cells in the body at very low levels, such as those measured by red blood cell, white blood cell, and / or platelet levels or remission, despite achieving (physically) complete remission following initial treatment. These residual cells are often present at levels undetectable using standard diagnostic methods, such as microscopy of bone marrow or peripheral blood. The presence of MRD in AML is significant because it indicates that not all leukemia cells have been eradicated by initial treatment. Even when a patient achieves complete remission, the persistence of MRD indicates a risk of future disease relapse. In some implementations, complete remission is induced. In some implementations, partial remission is achieved. In some implementations, a negative measurable residual disease (MRD) state is induced. In some implementations, complete remission without measurable residual disease is induced. Partial remission includes a reduction of approximately 5% to approximately 25% in the percentage of myeloblasts compared to pre-treatment levels, and a reduction of at least approximately 50% in the percentage of myeloblasts. The combination therapies and methods described herein can also be used to prepare subjects with myeloid malignancies for bone marrow transplantation. In some implementations, combination therapies and methods are employed to reduce the number of blast cells in the bone marrow and / or peripheral blood prior to transplantation. The methods and combination therapies are used to reduce the malignant blast cell count to less than about 5%. In one implementation, the methods and combination therapies are used to reduce the malignant blast cell count to less than about 5% to prepare the subject for bone marrow transplantation.

[0228] In one implementation, the treatment described herein increases progression-free survival (PFS). PFS may increase by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. Treatment according to the methods and combination therapies described herein may increase overall survival (OS). In one implementation, OS increases by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%. OS or PFS may be increased relative to no treatment or relative to standard treatment regimens. Standard treatment regimens may vary in clinical practice, for example, in different countries. Treatments for myeloid malignancies are diverse and include chemotherapy, radiotherapy, stem cell transplantation, and targeted therapy.

[0229] Dosage

[0230] Adjust the dosing regimen to provide the best desired response (e.g., therapeutic response). For example, a single bolus injection may be administered, several separate doses may be administered over time, or the dose may be proportionally reduced or increased depending on the urgency of the treatment situation.

[0231] As demonstrated in the examples, the combinations of therapeutic agents described herein are particularly advantageous because they exhibit synergistic efficacy. Preferably, therefore, in all embodiments described herein, the dose of the recombinant binding protein administered and / or provided in the combination therapy, and the dose of the Bcl-2 inhibitor administered and / or provided in the combination therapy, and / or the dose of the hypomethylating agent, are each selected to cause the therapeutic agents to exhibit a combined therapeutic effect, suitably a synergistic therapeutic effect. Suitably, whether the combination exerts a synergistic effect is determined by the Chou-Talalay method. Suitably, the doses of the recombinant binding protein, the Bcl-2 inhibitor, and / or the hypomethylating agent administered and / or provided in the combination are selected to cause the combination of therapeutic agents to exhibit a CI of less than 1 or less than 0.5, as determined by the Chou-Talalay method.

[0232] Suitablely, in some embodiments, the dosage of the recombinant binding protein, Bcl-2 inhibitor, and / or hypomethylating agent administered and / or provided in the combination is selected such that the combination exhibits a CI of less than 1 and a Fa of >0.5, as determined by the Chou-Talalay method.

[0233] The therapeutic agents described herein may be used together as provided herein. The therapeutic agents may be administered together or separately, depending on the intended dosage and frequency of administration, as the treatment described herein is expected to last for 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks or more than 4 weeks, as the treating physician deems it appropriate.

[0234] In some embodiments, multiple doses of the recombinant binding protein are administered, comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, wherein each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binds to a tumor-associated antigen suitably selected from the group consisting of CD123, CD33, and CD70. In some such embodiments, the recombinant binding protein is administered at intervals of 5 to 20 days between doses. In some embodiments, the recombinant binding protein is administered at intervals of 5 to 10 days between doses. In some embodiments, the recombinant binding protein is administered once weekly. In one embodiment, the recombinant binding protein is administered at a dose of about 0.2 mg to about 150 mg or any dose in between. In one embodiment, the recombinant binding protein is administered in an escalating dosing regimen, wherein at least one, at least two, or at least three escalating doses precede the administration of the initial dose, optionally wherein subsequent escalating doses contain a higher amount of the recombinant binding protein relative to the previous escalating dose. In one embodiment, the recombinant binding protein is administered parenterally or, where appropriate, intravenously. In another embodiment, the 28-day treatment cycle comprising the recombinant binding protein includes a withdrawal period of at least about 5 days, at least about 6 days, at least about 1 week, at least about 10 days, or at least about 2 weeks.

[0235] Bcl-2 inhibitors (suitably Venecella or a pharmaceutically acceptable salt thereof) can be administered according to any regimen in which the therapeutic compound has been proven effective. In a preferred embodiment, the Bcl-2 inhibitor is Venecella or a pharmaceutically acceptable salt thereof. Regarding Venecella (Venclexta...) ® The FDA prescribing information for the use of Venclexta in the treatment of AML suggests a dosing regimen: an escalating 3- or 4-day phase followed by a maintenance phase. ® With azacitidine or 20 mg / m 2 When prescribing decitabine combination therapy, the recommended dosing regimen is as follows: 100 mg Venclexta on day 1. ® Day 2: 200mg Venclexta ® Day 3: 400mg Venclexta ® This was followed by 400mg Venclexta daily. ® With 75mg / m 2 Azacitidine combination (28-day treatment cycle, once daily from day 1 to day 7) or with 20 mg / m² 2The maintenance phase of decitabine combination (28-day treatment cycle, once daily from day 1 to day 5) continues until disease progression or unacceptable toxicity is observed. In Venclexta ® When prescribed in combination with low-dose cytarabine, the recommended dosing regimen is as follows: 100 mg Venclexta on day 1. ® Day 2: 200mg Venclexta ® Day 3: 400mg Venclexta ® ; and 600mg Venclexta daily ® With 20mg / m 2 Cytarabine combination (28-day treatment cycle, once daily from day 1 to day 10) until disease progression or unacceptable toxicity is observed.

[0236] In a preferred embodiment, veneclade is administered at a dose of about 10 mg to about 800 mg (e.g., daily), more preferably at a dose of about 100 mg to about 600 mg / day. In a preferred embodiment, veneclade is administered at a dose of about 100 mg on day 1, about 200 mg on day 2, about 400 mg on day 3, and about 400 mg from day 4 to day 28, suitably once daily, for a treatment period of 28 days until disease progression or unacceptable toxicity is observed. In an alternative embodiment, veneclade is administered at a dose of about 100 mg on day 1, about 200 mg on day 2, about 400 mg on day 3, and about 600 mg from day 4 to day 28, suitably once daily, for a treatment period of 28 days until disease progression or unacceptable toxicity is observed. In one embodiment, veneclade is administered orally. A reduction in the dose and / or frequency and / or duration of administration of a Bcl-2 inhibitor (suitably veneclade or a pharmaceutically acceptable salt thereof) is also anticipated. Suitably, a 28-day treatment cycle comprising a Bcl-2 inhibitor (suitably veneclade or a pharmaceutically acceptable salt thereof) includes a withdrawal period of at least about 5 days, at least about 6 days, at least about 1 week, at least about 10 days, or at least about 2 weeks. As shown in the examples, the individual therapeutic agents included in the combinations disclosed herein work synergistically. The timing and extent of dose reduction of the Bcl-2 inhibitor are factors that the attending clinician will adjust according to the patient's needs.

[0237] In a preferred embodiment, the hypomethylating agent is azacitidine. In one embodiment disclosed herein, azacitidine is used at a concentration of about 70 to about 80 mg / m³. 2 (For example, daily) a suitable dose of approximately 75 mg / m². 2Azacitidine is administered at a dose of approximately 75 mg / m² once daily from day 1 to day 7 of a 28-day treatment cycle. 2 Azacitidine is administered at a dose per day. In one embodiment, azacitidine is administered intravenously. In an alternative embodiment, azacitidine is administered subcutaneously.

[0238] In another preferred embodiment, the hypomethylating agent is decitabine. In one embodiment, decitabine is used at a concentration of about 10 mg to about 30 mg / m². 2 (For example, daily) a suitable dose of approximately 20 mg / m². 2 The dosage is administered at a rate of approximately 20 mg / m² once daily, from day 1 to day 5 of each 28-day treatment cycle. 2 / day dose administration. In one implementation, decitabine is administered intravenously.

[0239] In another embodiment, the hypomethylating agent is cytarabine. In one embodiment, cytarabine is used at a concentration of about 10 to about 30 mg / m³. 2 (For example, daily) a suitable dose of approximately 20 mg / m². 2 Administered at a dose of approximately 20 mg / m² once daily, from day 1 to day 10 of each 28-day treatment cycle. 2 / day dose administration. In one implementation, decitabine is administered subcutaneously.

[0240] Because hypomethylating agents (such as azacitidine or decitabine) can induce cytopenia, it is also conceivable to reduce (or omit) the dose of these agents after the first 28-day treatment cycle. For example, once the percentage of blasts in bone marrow and / or peripheral blood decreases to less than about 10% or less than about 5%, the dose of the hypomethylating agent (appropriate azacitidine, decitabine, or low-dose cytarabine) can be reduced (or omitted entirely). The timing and extent of dose reduction for hypomethylating agents are factors that the attending clinician will adjust according to the patient's needs.

[0241] In one embodiment, the recombinant binding protein is administered 7, 6, 5, 4, 3, 2, or 1 day prior to the administration of a Bcl-2 inhibitor (suitably veneclade) or a pharmaceutically acceptable salt and / or hypomethylating agent (suitably azacitidine). In another embodiment, the recombinant binding protein is administered 7, 6, 5, 4, 3, 2, or 1 day after the administration of a Bcl-2 inhibitor (suitably veneclade) or a pharmaceutically acceptable salt and / or hypomethylating agent (suitably azacitidine).

[0242] In some embodiments, treatment cycles are repeated. For example, treatment may include two, three, four, or more 28-day treatment cycles. In one embodiment, the first 28-day treatment cycle comprises a Bcl-2 inhibitor (suitably veneclade or a pharmaceutically acceptable salt thereof) and / or a hypomethylating agent (suitably azacitidine), and the second and optionally additional 28-day treatment cycles comprise a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binding to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, a Bcl-2 inhibitor, suitably veneclade or a pharmaceutically acceptable salt thereof, and / or a hypomethylating agent, suitably azacitidine. In one embodiment, the first and second 28-day treatment cycles comprise a Bcl-2 inhibitor (suitably veneclade or a pharmaceutically acceptable salt thereof) and / or a hypomethylating agent (suitably azacitidine), and the third and optionally additional 28-day treatment cycles comprise a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binding to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and a Bcl-2 inhibitor, suitably veneclade or a pharmaceutically acceptable salt thereof, and / or a hypomethylating agent, suitably azacitidine.

[0243] Other combination therapies

[0244] In some embodiments, the methods or combination therapies described herein are administered in conjunction with one or more additional modalities of cancer therapy, such as targeted cancer therapy, gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapy, or oncolytic agents, cytotoxic agents, immunotherapy (e.g., cytokines, immunostimulants, or cell-based immunotherapy), surgery, or radiation therapy, or a combination of any of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the methods or combination therapies include the administration of one or more additional antiproliferative agents, suitably anticancer agents. Such anticancer agents may comprise one or more agents suitable for treating myeloid malignancies, such as agents suitable for treating AML.

[0245] Alternatively, or in combination with the foregoing, the methods and combination therapies described herein may be administered in combination with one or more of the following: immunomodulators (e.g., activators of co-stimulatory molecules or inhibitors of inhibitory molecules (e.g., immune checkpoint molecules); vaccines, such as cancer vaccines; or other forms of cellular immunotherapy.

[0246] Any combination and sequence of other therapeutic agents, procedures, or methods (e.g., as described herein) may be used in conjunction with the treatments described herein. The methods and combinations of therapies described herein may be administered before, concurrently with, between cycles of such other treatments, or during remission.

[0247] Reagent test kit

[0248] Kits comprising one or more of the therapeutic agents described herein are also within the scope of this invention. The kit may also include one or more other elements: instructions for use; other reagents for use with the compound; apparatus or other materials, such as mixing containers, for preparing the compound for administration; pharmaceutically acceptable carriers; and apparatus or other materials, such as syringes, for administration to a subject.

[0249] Therefore, the present invention provides a kit comprising (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, wherein each of the at least one, at least two, or at least three additional ankyrin repeat domains specifically binds to a tumor-associated antigen, suitably a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor, suitably veneclax or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, suitably azacitidine or decitabine, and instructions for concurrent or sequential administration thereof, the kit being intended for the treatment of malignant tumors, suitably myeloid malignancies, suitably acute myeloid leukemia and / or myelodysplastic syndromes. Suitably, the kit comprises: (i) a pharmaceutical composition comprising a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of which specifically binds to a tumor-associated antigen suitably selected from the group consisting of CD123, CD33, and CD70, and a pharmaceutically acceptable carrier; and (ii) a pharmaceutical composition comprising a Bcl-2 inhibitor, suitably veneclax or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; and / or (iii) a pharmaceutical composition comprising a hypomethylating agent and a pharmaceutically acceptable carrier; and instructions for administering the pharmaceutical composition comprising a Bcl-2 inhibitor (suitably veneclax or a pharmaceutically acceptable salt thereof) and / or the pharmaceutical composition comprising a hypomethylating agent to a subject in need before, concurrently with, or after administration of the pharmaceutical composition comprising the recombinant binding protein.

[0250] Another aspect provides (i) a recombinant binding protein comprising (a) an ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of which specifically binds to a tumor-associated antigen suitably selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor, suitably veneclax or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, suitably azacitidine or decitabine, for the manufacture of a kit for the treatment of malignant tumors (suitably myeloid malignancies).

[0251] Incorporated by reference

[0252] All publications, patents and registration numbers mentioned in this article are incorporated herein by reference in their entirety, as if each individual publication or patent were specifically and individually cited.

[0253] Equivalent solution

[0254] While specific embodiments of the subject invention have been discussed, the foregoing description is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon reading this description and the following claims. The full scope of the invention should be determined by reference to the full scope of the claims and their equivalents, as well as the description and such variations.

[0255] sequence table

[0256]

[0257]

[0258]

[0259]

[0260] Example

[0261] The following embodiments are illustrated to aid in understanding the invention, but are not intended and should not be construed as limiting the scope in any way.

[0262] Example 1

[0263] Combining multiple therapeutic agents with different but complementary mechanisms of action can enhance therapeutic efficacy while minimizing adverse effects. Synergy scoring, as a quantitative measure, provides a systematic approach to assess synergistic interactions between therapeutic agents that go beyond the additive effects of individual agents.

[0264] The recombinant binding protein (“protein #1”) with SEQ ID NO: 12 was studied in combination with azacitidine (AZA) and veneclade (VEN) on the AML cell line MOLM-13 (DSMZ, catalog number: ACC 554).

[0265] Pan-T cells were isolated from the buffy coat of donated blood from three healthy volunteers using a pan-T cell kit (Miltenyi, catalog number: 130-096-535) and labeled with cell tracer violet (CTV) (Thermo Fisher catalog number: C34557). MOLM-13 cells (1.5 x 10⁻⁶ cells) were... 4 Cells / well) and CTV-labeled PBMCs (1.5x10⁻¹²) 4 Cells / well were co-cultured in 96-well U-shaped plates at a 1:1 ratio in RPMI 1640 medium + 10% FBS and 10 µM human serum albumin (HSA; providing saturation for HSA binding to DARPin). The killing EC50 values ​​for each of proteins #1, VEN, and AZA after 72 hours of incubation were determined by counting the number of viable MOLM-13 tumor cells in FACS Fortessa by adding precision beads (Biolegend catalog number: 424902) to the cells. Treated MOLM-13 cells were normalized to control cells (mediator-treated only) by the number of beads. The combination of protein #1 with AZA and / or VEN was then evaluated by repeating the same killing assay using the EC50 concentration of each compound (5 pM protein #1, 5 nM veneclade, 0.25 µM azacitidine) with 2-fold increment / decrement titrations. The effect of drug treatment was calculated as the ratio of viable cells to mediator-treated cells using GraphPad Prism. The Chou-Talalay method (Chou TC. Pharmacol Rev. 2006;58(3):621-681; Chou TC. Cancer Res.2010; 70(2):440-446) was used to calculate a synergistic effect score by evaluating combination index (CI) and effect score (FA) values ​​using CompuSyn software as previously described (Riether et al., Nature Medicine, Vol. 26, pp. 1459–1467 (2020)), where a CI value <1 indicates a synergistic effect, =1 indicates an additive effect, and >1 indicates an antagonistic effect. Fa values ​​of 0, 0.5, and 1 correspond to 0%, 50%, and 100% cell killing, respectively.

[0266] like Figure 1 A to Figure 1 As shown in E, protein #1, in combination with VEN or with both VEN and AZA, synergistically eliminated MOLM-13 cells over a wide dose range. At higher levels of action, the combination exhibited a strong synergistic effect. For all tested donors, the combination showed low CI, indicating a very strong synergistic effect (see [reference]). Figure 2These experiments demonstrated that proteins #1, VEN, and AZA exhibited a synergistic effect at all tested dose levels (see [link to study]). Figure 1 E).

[0267] Example 2

[0268] The effect of the combination of protein #1 and AZA / VEN on the LSC of primary AML CD34+ sorting at physiologically relevant EC50 concentrations was also evaluated (Riether C et al. Nat Med. 2020; 26(9):1459-1467). AML samples (peripheral blood mononuclear cells or bone marrow mononuclear cells) from three different donors were subjected to FACS sorting to obtain CD45. int / Lin - / CD90 - / CD34 + / CD38 + / - Cells expressed the protein and were then co-cultured with purified allogeneic T cells derived from healthy volunteers at a 1:1 effector to target (E:T) ratio. Cells were treated for 4 days in 96-well round-bottom plates with either 10 pM protein #1 or control unbound CD3 DARPin ± 1 µM azacitidine and 5 nM veneclade in StemSpan SFEM medium (STEMCELL Technologies) containing a cytokine mixture (StemSpan CC100, STEMCELL Technologies) and 10 µM HSA. After incubation, cells were transferred to medium containing cytokines and 1.27% methylcellulose to assess colony-forming capacity under each condition. After an additional 14 days of culture, colonies were counted using an optical microscope and plotted using GraphPad Prism. Figure 3 The results shown demonstrate that the synergistic effect observed in AML cell lines compared to the mediator treatment group, the aza / ven group, or the recombinant binding protein group alone also translates into a significant reduction in the number of LSCs obtained from AML patients, even when administered at the suboptimal EC50 dose.

[0269] A synergistic effect was observed when protein #1 was used in combination with suboptimal EC50 doses of azacitidine and veneclade.

[0270] Figures 1 to 3The data presented here demonstrate that, in the presence of allogeneic T cells, the combination of azacitidine and veneclade with recombinant binding proteins that bind CD3 and several TAA-synchronizing T cells exhibits a strong synergistic effect on AML cells. This synergistic effect is particularly advantageous because it shows that when the therapeutic agents are used in combination, significantly lower concentrations of each agent can be used compared to the concentrations required to achieve the same effect when each agent is used alone in monotherapy. This effect has been demonstrated for exemplary AML cell lines and for primary cells derived from AML patients. The data presented in this paper demonstrate that, for all patient samples, the combination therapy significantly inhibits LSC colony formation to a greater extent than either therapy alone.

[0271] Example 3

[0272] To demonstrate that recombinant binding proteins can exert affinity-like effects on cells, protein #1 was evaluated in a power assay in the presence of healthy allogeneic T cells and engineered MOLM-13 tumor cell lines expressing one, two, or three TAAs. MOLM-13 knockout cells were generated using CRISPR-Cas9 technology. Figure 4 This demonstrates similar dose-dependent activation of CD8 T cells (see [link]). Figure 4 A) and killing of MOLM-13 cells expressing ≥2 TAAs (see A) Figure 4 B) showed EC50 values ​​of 7 pM to 30 pM for T cell activation and 10 pM to 45 pM for cell killing, while approximately 10-fold lower potency was measured on cells expressing a single TAA (EC50 values ​​of 40 pM to 430 pM for T cell activation and 105 pM to 455 pM for cell killing). Flow cytometry confirmed the specific binding of protein #1 to TAA in MOLM-13 cells and the KO clone (see [link to relevant documentation]). Figure 5 A) and specific binding to CD3 on pan-T cells (see A) Figure 5 B). In Figure 5 In B, the mean ± standard deviation (SD) of protein #1 bound to T cells from three donors is shown. NB-CD3 DARPin (non-TAA bound) and NB-TAA DARPin (non-CD3 bound) are shown as control molecules.

[0273] These results demonstrate that efficient activation of protein #1 is achieved in the presence of at least two TAAs. Activation in the presence of two TAAs is expected to lead to selective activation of T cells, killing AML cells and LSCs while preserving HSCs. Furthermore, this selective activation of T cells results in significantly lower cytokine and chemokine release compared to that seen when targeting T cell adaptors with a single TAA. Moreover, T cell activation occurs despite the high heterogeneity of TAA expression, which may translate into a deeper and more durable response in clinical settings.

Claims

1. A combination comprising, separately or together: (i) a recombinant binding protein comprising (a) a first ankyrin repeat domain that specifically binds to CD3 and (b) at least one, at least two, or at least three additional ankyrin repeat domains, each of said at least one, at least two, or at least three additional ankyrin repeat domains specifically binding to a tumor-associated antigen, said tumor-associated antigen suitably being a tumor-associated antigen selected from the group consisting of CD123, CD33, and CD70, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent.

2. The combination according to claim 1, wherein the recombinant binding protein comprises the first ankyrin repeat domain that specifically binds to CD3, and i. Specifically binds to the second ankyrin repeat domain of CD123; or ii. Specifically binds to the second ankyrin repeat domain of CD33; or iii. Specifically binds to the second ankyrin repeat domain of CD70; or iv. Specifically binds to the second ankyrin repeat domain of CD123 and specifically binds to the third ankyrin repeat domain of CD70; or v. Specifically binds to the second ankyrin repeat domain of CD33 and specifically binds to the third ankyrin repeat domain of CD70; or vi. Specifically binds to the second ankyrin repeat domain of CD123 and specifically binds to the third ankyrin repeat domain of CD33; or vii. Specifically binds the second ankyrin repeat domain of CD123, the third ankyrin repeat domain of CD33, and the fourth ankyrin repeat domain of CD70.

3. The combination according to claim 1, wherein the recombinant binding protein comprises (a) the first ankyrin repeat domain that specifically binds CD3, (b) the second ankyrin repeat domain that specifically binds CD123, (c) the third ankyrin repeat domain that specifically binds CD33, and (d) the fourth ankyrin repeat domain that specifically binds CD70.

4. The combination according to any one of the preceding claims, wherein the recombinant binding protein comprises at least one or at least two additional ankyrin repeat domains that specifically bind to human serum albumin.

5. The combination according to any one of the preceding claims, wherein the Bcl-2 inhibitor is Veneclare or a pharmaceutically acceptable salt thereof.

6. The combination according to any one of the preceding claims, wherein the hypomethylating agent is azacitidine or decitabine.

7. The combination according to any one of the preceding claims, wherein the recombinant binding protein comprises at least about 80% of the same amino acid sequence as any one of SEQ ID NO:12 to 18.

8. The combination according to any one of the preceding claims, wherein the recombinant binding protein comprises at least about 80% of the same amino acid sequence as SEQ ID NO:

12.

9. The combination according to any one of the preceding claims, wherein the combination comprises (i) a recombinant binding protein comprising the amino acid sequence having SEQ ID NO: 12, (ii) Veneclare or a pharmaceutically acceptable salt thereof, and (iii) azacitidine.

10. The combination according to any one of claims 1 to 8, wherein the combination comprises (i) a recombinant binding protein comprising the amino acid sequence having SEQ ID NO: 12, (ii) Venecla or a pharmaceutically acceptable salt thereof, and (iii) decitabine.

11. The combination according to any one of the preceding claims, wherein (i) the recombinant binding protein, (ii) the Bcl-2 inhibitor and (iii) the hypomethylating agent are separate.

12. A method for treating a subject with a malignant tumor, suitably a myeloid malignant tumor, the method comprising administering to the subject to whom the need arises a combination as defined in any one of claims 1 to 11.

13. The method of claim 12, wherein (i) the recombinant binding protein and (ii) the Bcl-2 inhibitor and / or (iii) the hypomethylating agent are administered separately, simultaneously and / or sequentially.

14. The method according to claim 12 or claim 13, wherein the myeloid malignancy is acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), or AML / MDS.

15. A kit comprising a combination as defined in any one of claims 1 to 11 and instructions for simultaneous or sequential administration thereto, the kit being used to treat malignant tumors, suitably myeloid malignancies, suitably acute myeloid leukemia, or myelodysplastic syndrome.

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