Therapeutic combinations comprising a multi-specific t cell engager

Abstract

The invention relates to combination and combination therapies, particularly for the treatment of a myeloid malignancy, such as acute myeloid leukemia (AML) or a myelodysplastic syndrome (MDS). The combination therapies include (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one 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

[0001] THERAPEUTIC COMBINATIONS COMPRISING A MULTI-SPECIFIC T CELL ENGAGER

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to combination therapies comprising, separately or together, (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor- associated antigen, suitably one 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 the treatment of a malignancy, e.g., a myeloid malignancy, e.g., acute myeloid leukemia (AML) or a myelodysplastic syndrome (MDS). Also disclosed are methods of using of such combination therapies and kits comprising such combination therapies.

[0004] BACKGROUND

[0005] Acute myeloid leukemia (AML) is a complex and heterogeneous disease with poor prognosis characterized by rapid cellular proliferation, aggressive clinical course, and high mortality rates. Despite the introduction of new drugs, the overall survival rates for AML remain low, including in patients younger than 60 years of age, but particularly in elderly patients who are ineligible for stem cell transplants. A significant challenge in treating AML is the limited ability of current therapies to effectively eliminate leukemic stem cells (LSCs) while sparing healthy hematopoietic stem cells (HSCs) as LSCs exhibit resistance to chemotherapy and possess self-renewal capabilities, making them difficult to target.

[0006] The recent introduction of several new drugs has provided novel treatment options for AML. For example, amongst these new drugs are monoclonal antibodies targeting specific proteins, e.g., CD33 or CD123, on the surface of leukemia cells, either alone or conjugated with cytotoxic agents, thereby selectively killing the cancer cells. However, these therapies have shown limitations in terms of adverse effects and efficacy. In addition, immunotherapy approaches targeting AML have faced obstacles due to the absence of AML-specific target antigens and the clonal heterogeneity of tumors within and between patients. T cell engager (TCE) therapies that focus on single AML antigens have been limited by toxicities resulting from binding to healthy HSCs and myeloid cells, as well as relapse caused by leukemic clones lacking the targeted antigen and therefore escaping targeted therapies. For example, while low or absent on HSCs, CD33 and CD123 are present on healthy hematopoietic cells: CD33 on most myeloid cells, and CD123 predominantly on plasmacytoid dendritic cells, basophils, and endothelial cells, potentially leading to off-tumor toxicities by agents targeting a single tumor-associated antigen (TAA). To overcome these challenges, simultaneous targeting of multiple tumor-associated antigens (TAAs) on AML blasts and LSCs is required, aiming to enhance therapeutic efficacy without increasing toxicity. A multi-specific T cell engaging designed ankyrin repeat protein (DARPin) has been developed to simultaneously bind CD3 on T cells and three AML tumor-associated antigens (TAAs): CD70, CD33, and CD123. This DARPin, described in W02022190016, has been designed to target AML blasts and LSCs that express these TAAs. By achieving an avidity-like selectivity and efficacy, this multispecific binding protein aims to selectively kill AML cells that co-express at least two of these TAAs while sparing healthy cells expressing a single TAA. Experimental findings demonstrated that this DARPin effectively induced selective killing of AML blasts and LSCs while reducing effects on healthy cells as well as minimizing cytokine release.

[0007] Other agents used in the treatment of myeloid malignancies are, inter alia, the nucleoside analog azacitidine and the BH3-mimetic venetoclax. These therapeutic agents have demonstrated favorable efficacy and safety profiles in clinical trials, and they have been approved by the U.S. Food and Drug Administration (FDA) as a combination therapy for the treatment of newly diagnosed AML in patients who are age 75 years or older, or who have comorbidities that preclude use of intensive induction chemotherapy. Azacitidine is a hypomethylating agent that acts 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 critical role in promoting cell survival. However, while this combination therapy has shown improved overall survival rates, higher complete remission rates, and reduced rates of treatment-related mortality compared to traditional chemotherapy regimens, there remains a need for further advancements in AML therapy 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.

[0008] SUMMARY OF THE INVENTION

[0009] The present invention relates to combination therapies comprising, separately or together, (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor- associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor, for example venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, for example azacitidine or decitabine. As explained elsewhere herein, venetoclax 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. Such a combination can provide a particularly effective therapy for the treatment of cancer, particularly myeloid malignancies such as acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS).

[0010] Based on the disclosure provided herein, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following embodiments (E). Specifically, the present disclosure provides the following aspects, advantageous features and embodiments, respectively alone or in combination: EMBODIMENTS

[0011] E1. A combination comprising, separately or together, (i) a recombinant binding protein comprising (a) a binding domain specifically binding CD3, (b) a binding domain specifically binding CD33 and / or a binding domain specifically binding CD70, and (c) optionally a binding domain specifically binding CD123, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent.

[0012] E2. The combination according to E1 , wherein said recombinant binding domain is derived from an antibody or antigen binding fragment thereof, an antibody mimetic, a scaffold protein, and / or a repeat protein, such as an ankyrin repeat protein.

[0013] E3. A combination comprising, separately or together, (i) a recombinant binding protein comprising (a) a first ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent.

[0014] E4. The combination according to E3, wherein the recombinant binding protein comprises said first ankyrin repeat domain specifically binding CD3, and i. a second ankyrin repeat domain specifically binding CD123; or ii. a second ankyrin repeat domain specifically binding CD33; or iii. a second ankyrin repeat domain specifically binding CD70; or iv. a second ankyrin repeat domain specifically binding CD123 and a third ankyrin repeat domain specifically binding CD70; or v. a second ankyrin repeat domain specifically binding CD33 and a third ankyrin repeat domain specifically binding CD70; or vi. a second ankyrin repeat domain specifically binding CD123, a third ankyrin repeat domain specifically binding CD33; or vii. a second ankyrin repeat domain specifically binding CD123, a third ankyrin repeat domain specifically binding CD33 and a fourth ankyrin repeat domain specifically binding CD70.

[0015] E5. The combination according to any one of E1-E4, wherein the recombinant binding protein comprises (a) said first ankyrin repeat domain specifically binding CD3, (b) a second ankyrin repeat domain specifically binding CD123, (c) a third ankyrin repeat domain specifically binding CD33 and (d) a fourth ankyrin repeat domain specifically binding CD70. E6. The combination according to any one of E1-E5, wherein the Bcl-2 inhibitor is venetoclax, or a pharmaceutically acceptable salt thereof.

[0016] E7. The combination according to any one of E1-E6, wherein the hypomethylating agent is azacitidine or decitabine.

[0017] E8. The combination according to any one of E1-E7, wherein the hypomethylating agent is azacitidine.

[0018] E9. The combination according to any one of E1-E7, wherein the hypomethylating agent is decitabine.

[0019] E10. The combination according to any one of E1-E9, wherein the recombinant binding protein further comprises at least one, or at least two binding domains with binding specificity for human serum albumin.

[0020] E11. The combination according to E10, wherein the recombinant binding protein comprises at least one or at least two ankyrin repeat domains with binding specificity for human serum albumin.

[0021] E12. The combination according to E11 , wherein the recombinant binding protein comprises at least two ankyrin repeat domains with binding specificity for human serum albumin.

[0022] E13. The combination according to any one of E3-E12, wherein the ankyrin repeat domain specifically binding CD3 comprises an amino acid sequence at least about 80% identical with SEQ ID NO: 1 .

[0023] E14. The combination according to any one of E3-E13, wherein the ankyrin repeat domain specifically binding CD33 comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 2 or 3.

[0024] E15. The combination according to any one of E3-E14, wherein the ankyrin repeat domain specifically binding CD123 comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 4 to 7.

[0025] E16. The combination according to any one of E3-E15, wherein the ankyrin repeat domain specifically binding CD70 comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 8 to 9.

[0026] E17. The combination according to any one of E11 -E16, wherein the ankyrin repeat domain with binding specificity for human serum albumin comprises an amino acid sequence at least about 80% identical with SEQ ID NO: 10.

[0027] E18. The combination according to any one of E1-E17, wherein said recombinant binding protein comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or 100% identical with any one of SEQ ID NOs: 12 to 18. E19. The combination according to any one of E1-E18, comprising (i) a recombinant binding protein comprising an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or 100% identical with any one of SEQ ID NOs: 12 to 18, and (ii) a Bcl-2 inhibitor.

[0028] E20. The combination according to E19, wherein the Bcl-2 inhibitor is venetoclax, or a pharmaceutically acceptable salt thereof.

[0029] E21. The combination according to any one of E1-E20, comprising (i) a recombinant binding protein comprising an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or 100% identical with any one of SEQ ID NOs: 12 to 18, and (ii) a hypomethylating agent.

[0030] E22. The combination according to E21 , wherein the hypomethylating agent is azacitidine or decitabine.

[0031] E23. The combination according to E22, wherein the hypomethylating agent is azacitidine.

[0032] E24. The combination according to E22, wherein the hypomethylating agent is decitabine.

[0033] E25. The combination according to any one of E1-E24, wherein said recombinant binding protein comprises an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or 100% identical with SEQ ID NO: 12.

[0034] E26. The combination according to any one of E1-E23 and E25, comprising (i) a recombinant binding protein comprising an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or 100% identical with SEQ ID NO: 12, (ii) venetoclax, or a pharmaceutically acceptable salt thereof, and (iii) azacitidine.

[0035] E27. The combination according to any one of E1-E22, E24 and E25, comprising (i) a recombinant binding protein comprising an amino acid sequence at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98% or 100% identical with SEQ ID NO: 12, (ii) venetoclax, or a pharmaceutically acceptable salt thereof, and (iii) decitabine.

[0036] E28. The combination according to any one of E1-E27, wherein (i) said recombinant binding protein, (ii) said Bcl-2 inhibitor and (iii) said hypomethylating agent are separate.

[0037] E29. The combination according to any one of E1-E28, wherein said combination comprises one or more additional anti-proliferative agent(s), suitably anti-cancer agent(s).

[0038] E30. The combination as defined in any one of E1-E29 for use in therapy.

[0039] E31 . The combination as defined in any one of E1-E30 for use in the treatment of a malignancy. E32. The combination as defined in any one of E1 -E31 for use in the treatment of a myeloid malignancy.

[0040] E33. The combination as defined in any one of E1 -E32 for use in the treatment of a malignancy, suitably a myeloid malignancy, comprising administering to a subject in need thereof a therapeutically effective amount of the therapeutic agents comprised in the combination, wherein (i) said recombinant binding protein, (ii) said Bcl-2 inhibitor and / or (iii) said hypomethylating agent are administered separately, concurrently, or sequentially.

[0041] E34. A recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor- associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, for use in the treatment of a malignancy, suitably a myeloid malignancy, comprising administering to a subject in need thereof said recombinant binding protein separately, concurrently, or sequentially with

[0042] (i) a Bcl-2 inhibitor and / or (ii) a hypomethylating agent.

[0043] E35. A Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, for use in the treatment of a malignancy, suitably a myeloid malignancy, comprising administering to a subject in need thereof said Bcl-2 inhibitor separately, concurrently, or sequentially with (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) optionally a hypomethylating agent.

[0044] E36. A hypomethylating agent for use in the treatment of a malignancy, suitably a myeloid malignancy, comprising administering to a subject in need thereof said hypomethylating agent separately, concurrently, or sequentially with (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor- associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and

[0045] (ii) optionally a Bcl-2 inhibitor.

[0046] E37. A method for treatment of a malignancy, suitably a myeloid malignancy, comprising administering to a subject in need thereof a therapeutically effective amount of (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent. E38. The combination for use according to E33, the recombinant binding protein for use according to E34, the Bcl-2 inhibitor for use according to E35, the hypomethylating agent for use according to E36, or the method according to E37, wherein administration of said recombinant binding protein, said Bcl- 2 inhibitor and / or said hypomethylating agent is performed in any order.

[0047] E39. The combination for use according to E33 or E38, the recombinant binding protein for use according to E34 or E38, the Bcl-2 inhibitor for use according to E35 or E38, the hypomethylating agent for use according to E36 or E38, or the method according to E37 or E38, wherein (i) said recombinant binding protein, (ii) said Bcl-2 inhibitor and / or said hypomethylating agent are administered separately.

[0048] E40. The combination for use according to any one of E33 and E38-E39, the recombinant binding protein for use according to any one of E34 and E38-E39, the Bcl-2 inhibitor for use according to any one of E35 and E38-E39, the hypomethylating agent for use according to any one of E36 and E38- E39, or the method according to any one of E37-E39, wherein (i) said recombinant binding protein and (ii) said Bcl-2 inhibitor and / or said hypomethylating agent are administered concurrently.

[0049] E41. The combination for use according to any one of E33 and E38-E39, the recombinant binding protein for use according to any one of E34 and E38-E39, the Bcl-2 inhibitor for use according to any one of E35 and E38-E39, the hypomethylating agent for use according to any one of E36 and E38- E39, or the method according to any one of E37-E39, wherein (i) said recombinant binding protein and (ii) said Bcl-2 inhibitor and / or said hypomethylating agent are administered sequentially.

[0050] E42. The combination for use according to any one of E33 and E38-E41 , the recombinant binding protein for use according to any one of E34 and E38-E41 , the Bcl-2 inhibitor for use according to any one of E35 and E38-E41 , the hypomethylating agent for use according to any one of E36 and E38- E41 , or the method according to any one of E35-E41 , wherein (i) said recombinant binding protein and (ii) said Bcl-2 inhibitor and / or said hypomethylating agent are administered in jointly therapeutically effective amounts.

[0051] E43. The combination for use according to any one of E33 and E38-E42, the recombinant binding protein for use according to any one of E34 and E38-E42, the Bcl-2 inhibitor for use according to any one of E35 and E38-E43, the hypomethylating agent for use according to any one of E36 and E38- E42, or the method according to any one of E35-E42, wherein (i) said recombinant binding protein and (ii) said Bcl-2 inhibitor and / or said hypomethylating agent are administered in synergistically effective amounts.

[0052] E44. The combination for use according to any one of E33 and E38-E43, the recombinant binding protein for use according to any one of E34 and E38-E43, the Bcl-2 inhibitor for use according to any one of E35 and E38-E43, the hypomethylating agent for use according to any one of E36 and E38- E43, or the method according to any one of E37-E43, wherein said malignancy is a myeloid malignancy, which is acute myeloid leukemia (AML); a myelodysplastic syndrome (MDS); a myeloproliferative neoplasm (MPN); chronic myeloid leukemia (CML); or chronic myelomonocytic leukemia (CMML), suitably wherein the myeloid malignancy is AML, AML / MDS, or MDS.

[0053] E45. The combination for use according to E44, the recombinant binding protein for use according to E44, the Bcl-2 inhibitor for use according to E44, the hypomethylating agent for use according to E44, or the method according to E44, wherein the myeloid malignancy is AML, AML / MDS or MDS.

[0054] E46. The combination for use according to E44 or E45, the recombinant binding protein for use according to E44 or E45, the Bcl-2 inhibitor for use according to E44 or E45, the hypomethylating agent for use according to E44 or E45, or the method according to E44 or E45, wherein the myeloid malignancy is AML.

[0055] E47. The combination for use according to E44 or E45, the recombinant binding protein for use according to E44 or E45, the Bcl-2 inhibitor for use according to E44 or E45, the hypomethylating agent for use according to E44 or E45, or the method according to E44 or E45, wherein the myeloid malignancy is AML / MDS.

[0056] E48. The combination for use according to any one of E33 and E38-E47, the recombinant binding protein for use according to any one of E34 and E38-E47, the Bcl-2 inhibitor for use according to any one of E35 and E38-E47, the hypomethylating agent for use according to any one of E36 and E38- E47, or the method according to any one of E37-E47, wherein treatment is first-line treatment or second-line treatment.

[0057] E49. The combination for use according to any one of E33 and E38-E48, the recombinant binding protein for use according to any one of E34 and E38-E48, the Bcl-2 inhibitor for use according to any one of E35 and E38-E48, the hypomethylating agent for use according to any one of E36 and E38- E48, or the method according to any one of E37-E48, wherein the subject is relapsed and / or refractory.

[0058] E50. The combination for use according to any one of E33 and E38-E49, the recombinant binding protein for use according to any one of E34 and E38-E49, the Bcl-2 inhibitor for use according to any one of E35 and E38-E49, the hypomethylating agent for use according to any one of E36 and E38- E49, or the method according to any one of E37-E49, wherein the subject is newly diagnosed and / or is ineligible for standard intensive chemotherapy.

[0059] E51. The combination for use according to any one of E33 and E38-E50, the recombinant binding protein for use according to any one of E34 and E38-E50, the Bcl-2 inhibitor for use according to any one of E35 and E38-E50, the hypomethylating agent for use according to any one of E36 and E38- E50, or the method according to any one of E37-E50, wherein the subject is aged 75 years or older.

[0060] E52. The combination for use according to any one of E33 and E38-E51 , the recombinant binding protein for use according to any one of E34 and E38-E51 , the Bcl-2 inhibitor for use according to any one of E35 and E38-E51 , the hypomethylating agent for use according to any one of E36 and E38- E51 , or the method according to any one of E37-E51 , wherein the subject is ineligible for standard intensive chemotherapy, suitably wherein the subject has a comorbidity that precludes the use of standard intensive chemotherapy.

[0061] E53. The combination for use according to any one of E33 and E38-E52, the recombinant binding protein for use according to any one of E34 and E38-E52, the Bcl-2 inhibitor for use according to any one of E35 and E38-E52, the hypomethylating agent for use according to any one of E36 and E38- E52, or the method according to any one of E37-E52, further comprising monitoring the subject’s peripheral blood and / or bone marrow blast count.

[0062] E54. The combination for use according to E53, the recombinant binding protein for use according to E53, the Bcl-2 inhibitor for use according to E53, the hypomethylating agent for use according to E53, or the method according to E53, wherein the bone marrow blast count is reduced to less than about 25% and / or the bone marrow blast percentage is reduced by more than about 50% as compared to pretreatment.

[0063] E55. Use of (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one 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 a malignancy.

[0064] E56. Use of (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent for the treatment of a malignancy.

[0065] E57. A medicament comprising, separately or together, (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one 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) said recombinant binding protein and (ii) said Bcl-2 inhibitor and / or the hypomethylating agent are provided in jointly therapeutically, preferably synergistically effective amounts for the treatment of a malignancy.

[0066] E58. A pharmaceutical combination comprising, separately or together, (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent, and at least one pharmaceutically acceptable carrier or excipient.

[0067] E59. A kit comprising a combination according to any one of E1 -E33 together with instructions for simultaneous or sequential administration thereof for use in the treatment of a malignancy, suitably a myeloid malignancy, suitably acute myeloid leukemia or myelodysplastic syndrome.

[0068] E60. Use of a (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one 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 kit for use in treating disease.

[0069] Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0070] BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 : Synergistic effect of the combination treatment with the recombinant binding protein with SEQ ID NO: 12 (“Protein #1”), the Bcl-2 inhibitor venetoclax and the hypomethylating agent azacitidine. Cells from the AML cell line MOLM-13 were co-cultured 1 :1 with PBMC-derived, CTV- labelled T cells from a healthy donor (“Nr. 53”) for three days and treated with the indicated EC50 concentration of each compound vs. 2-fold incremental / decremental titrations. The number of viable MOLM-13 cells was assessed by FACS (number of cells were normalized to counting beads). A) Protein #1 only; B) azacitidine with Protein #1 ; C) venetoclax with Protein #1 ; D) azacitidine and venetoclax and E) azacitidine, venetoclax and Protein #1. DMSO: dimethylsulfoxide (vehicle control), Aza: azacitidine, Ven: venetoclax.

[0072] Figure 2: Synergistic effect of the combination treatment with Protein #1 , venetoclax and azacitidine. Cells from the AML cell line MOLM-13 were co-cultured 1 :1 with PBMC-derived, CTV-labelled T cells from three different healthy donors (Fig 2A, Fig 2B, Fig 2C) for three days and treated with a combination of EC50 concentrations of each compound vs. 2-fold incremental / decremental titrations. The number of viable MOLM-13 cells was assessed by FACS (number of cells were normalized to counting beads) and synergy scores were calculated based on the number of surviving cells versus vehicle-treated cells. Synergy scores were calculated according to the method of Chou-Talalay using the CompuSyn software. Cl: combination index, Fa: fraction affected.

[0073] Figure 3: Synergy-like effects were observed upon treatment of primary AML cells CD34+LSCs from three different donors (•, ■, ▲) with a combination of Protein #1 , venetoclax and azacitidine. Primary AML cells were purified and cultured at a 1 :1 ratio with allogeneic T cells from healthy donors in the presence of 10 pM Protein #1 or control non-binding (NB)-CD3 DARPin ± 1 pM azacitidine and 5 nM venetoclax as indicated for four days, followed by culture for 14 days and assessed for colony formation. CFU: colony forming unit. Figure 4: Protein #1 induces potent killing of AML cell lines. MOLM-13 cells were engineered by CRISPR / Cas9 to express different combinations of tumor-associated antigens (TAA). Protein #1 induced T cell activation (Fig 4A) and tumor cell killing (Fig 4B) in co-cultures of T-cells with MOLM- 13 cell lines expressing different TAA combinations as indicated in the legend. T cell activation was assessed by flow cytometry through the detection of the upregulation of CD25 on CD8+T cells, and tumor cell killing was assessed based on the cell count of remaining living cells. T-cell activation and tumor cell killing were assessed after 48 h co-incubation of T cells and tumor cells at an effector-to- target (E:T) of 5:1 .

[0074] Figure 5: Binding of Protein #1 to MOLM-13 clones and T cells. MOLM-13 cells were engineered by CRISPR / Cas9 to express different combinations of tumor-associated antigens (TAA). Tumor antigen associated (TAA)-specific binding of Protein #1 to MOLM-13 cells with knockout in one, two or three TAAs as indicated in (Fig 5A) and CD3-specific binding to pan T cells (Fig 5B) was confirmed by flow cytometry. In Fig 5B the average ± standard deviation of Protein #1 binding to T cells from three different donors is shown. Control molecules shown: NB-CD3 DARPin (non-TAA binding) and NB- TAA DARPin (non-CD3 binding). MFI: mean fluorescence intensity.

[0075] DETAILED DESCRIPTION

[0076] Definitions

[0077] Selected terms are defined below and throughout the specification. It is understood that unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one skilled in the art in the technical field of the invention.

[0078] The use of any and all examples, or exemplary language (e g., "such as") provided herein, is intended merely to better illustrate the disclosure and does not pose a limitation on the scope unless otherwise claimed.

[0079] As used herein, the articles "a" and "an” can mean “one”, but it is also consistent with the meaning of “one or more”, “at least one” and “one or more than one”.

[0080] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or", unless context clearly indicates otherwise and is to be interpreted as an inclusive “or” meaning any one or any combination.

[0081] Throughout this specification and the claims which follow, and unless the context requires otherwise, the terms “comprising” and “including” can be used interchangeably. The terms “comprising” and “including” are to be interpreted as specifying the presence of the stated features or components as referred to, but does not preclude 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 encompassed by the term “consisting of’. Consequently, the term “consisting of’ can be used in place of the terms “comprising” and “including” to provide for more specific embodiments of the invention. "About" and "approximately" shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 10% of a given value or range of values. For example, where a dosage is mentioned as “about” a particular value, it is intended to include a range around the specified value of plus or minus 10%. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term “about”.

[0082] It also is to be understood, although not always explicitly stated, that the reagents described herein are merely examples and that equivalents of such are known in the art.

[0083] The terms “combination”, “a combination” or “in combination with” refers to a treatment in which a subject, for example a human subject, is given (administered) two or more therapeutic agents. The “combinations” described herein are suitable for use in combination therapy. The combinations disclosed herein comprise, separately or together, (1)(i) a recombinant binding protein comprising (a) a binding domain specifically binding CD3, (b) a binding domain specifically binding CD33 and / or a binding domain specifically binding CD70, and optionally (c) a binding domain specifically binding CD123, and (ii) a Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof; (2)(i) a recombinant binding protein comprising (a) a binding domain specifically binding CD3, (b) a binding domain specifically binding CD33 and / or a binding domain specifically binding CD70, and optionally (c) a binding domain specifically binding CD123, and (ii) a hypomethylating agent, suitably azacitidine or decitabine, or (3)(i) a recombinant binding protein comprising (a) a binding domain specifically binding CD3, (b) a binding domain specifically binding CD33 and / or a binding domain specifically binding CD70, and optionally (c) a binding domain specifically binding CD123, (ii) a Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and (iii) a hypomethylating agent, suitably azacitidine or decitabine. More specifically, the combinations disclosed herein comprise, separately or together, (1)(i) a recombinant binding protein comprising (a) a first ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor, such as venetoclax, or a pharmaceutically acceptable salt thereof; (2) (i) a recombinant binding protein comprising (a) a first ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one 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 repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, (ii) a Bcl-2 inhibitor, such as venetoclax, or a pharmaceutically acceptable salt thereof and (iii) a hypomethylating agent, such as azacitidine or decitabine. Thereby it is not intended to imply that the therapy or the therapeutic agents must be physically mixed or administered at the same time and / or formulated for delivery together, although these methods of delivery are within the scope described herein. Accordingly, the term “combination” refers to either a fixed combination in one dosage unit form, wherein the therapeutic agents of the combination are both administered to a patient simultaneously in the form of a single entity or dosage, or a non-fixed combination wherein the therapeutic agents as described herein are administered independently either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the individual therapeutic agents in the body of the patient, especially such that that the combination partners show a cooperative, e.g., synergistic effect. Each of the components (e.g., powders or liquids) may be reconstituted or diluted to a desired dose prior to administration.

[0084] The term “pharmaceutical combination” as used herein means a product that results from the mixing or combining of more than one therapeutic agent and includes both fixed and non-fixed combinations of the therapeutic agents, and which further comprise a pharmaceutically acceptable excipient, diluent and / or carrier.

[0085] As used herein, “administer” or “administration” refers to the act of physically delivering a substance as it exists outside the body into a subject. Administration includes all forms suitable for delivering the therapeutic agents of the combination.

[0086] The term "combination therapy" refers to a therapeutic approach in which two or more distinct pharmaceutical agents, treatment modalities, or interventions are administered simultaneously, sequentially, or in a coordinated manner to treat a medical condition or disease, e.g., a cancer or malignancy as described herein. The combination of these elements is designed to produce a synergistic or complementary effect that enhances therapeutic outcomes, improves efficacy, reduces side effects, and / or targets multiple aspects of the disease simultaneously. In the context of this patent application, "combination therapy" specifically pertains to the disclosed pharmaceutical combination for use in treatment, which involves the simultaneous, concurrent or sequential administration of multiple active therapeutic agents to achieve a therapeutic effect for the treatment of the specified medical condition.

[0087] The terms “co-administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partners (therapeutic agents) to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. A therapeutic agent in a combination therapy can be administered concurrently with, prior to, or subsequent to, one or more other therapies or therapeutic agents. The therapeutic agents can be administered in any order. In general, each therapeutic agent will be administered at a dose and / or on a time schedule determined forthat agent. Such administration also encompasses the use of each type of therapeutic agent in a sequential manner, either at approximately the same time or at different times. It will further be appreciated that the additional therapeutic agent utilized in this combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that additional therapeutic agents utilized in combination be administered at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized as single-agent therapeutics. In certain embodiments, the Bcl-2 inhibitor and / or the hypomethylating agent is administered at a therapeutic or lower-than therapeutic dose relative to a single-agent dose level. In certain embodiments, the concentration of the Bcl-2 inhibitor and / or the hypomethylating agent that is required to achieve the desired therapeutic effect is lower when the Bcl-2 inhibitor and / or the hypomethylating agent is used or administered in combination with the recombinant binding protein described herein than when the Bcl-2 inhibitor and / or the hypomethylating agent is administered individually. In certain embodiments, the concentration or dosage of the Bcl-2 inhibitor and / or the hypomethylating agent that is required to achieve a therapeutic effect is lower than the therapeutic dose of the Bcl-2 inhibitor and / or the hypomethylating agent as a monotherapy, e.g., lower by 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 certain embodiments, the concentration or dosage of the recombinant binding protein described herein that is required to achieve a therapeutic effect is lower than the therapeutic dose of said recombinant binding protein as a monotherapy, e.g., lower by 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.

[0088] As used herein, the term “polypeptide” refers to a molecule comprising a chain of multiple amino acids linked via peptide bonds. Preferably, a polypeptide consists of more than eight amino acids linked via peptide bonds. The term “polypeptide” also includes multiple chains of amino acids, linked together by S-S bridges between cysteines.

[0089] As used herein, the term "protein" refers to a molecule comprising a polypeptide, wherein at least part of the polypeptide has, or is able to acquire, a defined three-dimensional arrangement by forming secondary, tertiary, and / or quaternary structures within a single polypeptide chain and / or between multiple polypeptide chains. If a protein comprises two or more polypeptide chains, the individual polypeptide chains may be linked non-covalently or covalently, e.g., by a disulfide bond between two polypeptides. A part of a protein, which individually has, or is able to acquire, a defined three- dimensional arrangement by forming secondary and / or tertiary structure, is termed "protein domain”.

[0090] As used herein, the term "binding protein" refers to a protein comprising at least one binding domain. A binding protein may also comprise two, three, four, five or more binding domains. Preferably, said binding protein is a recombinant binding protein. Furthermore, any such binding protein may comprise additional polypeptides (such as e.g., polypeptide tags, peptide linkers, fusion to other proteinaceous domains with binding specificity, cytokines, hormones, or antagonists), or chemical modifications (such as coupling to polyethylene-glycol, toxins, small molecules, antibiotics and alike) or the like.

[0091] The term “binding domain” refers to a protein domain exhibiting binding specificity for a target. Preferably, said binding domain is a recombinant binding domain. In the context of the present invention, the recombinant binding proteins bind to particular target antigens, e.g., CD3 and one or more or two or more tumor-associated antigens (TAAs), such as CD33, CD123 and CD70. It is preferred that the recombinant binding proteins “specifically bind” to their target antigen, wherein the term “specifically bind” refers to the ability of the recombinant binding protein to preferentially immunoreact with a given target, e.g., CD3, and one or more or two or more of the TAAs CD33, CD123 and CD70. The recombinant binding proteins presently provided are “multispecific”, i.e., they contain two or more binding sites, suitably three or more binding sites, suitably four binding sites which specifically bind a particular target antigen.

[0092] The term "target" or “target antigen” refers to an individual molecule such as a nucleic acid molecule, a polypeptide or protein, a carbohydrate, or any other naturally occurring molecule, including any part of such individual molecule, or to complexes of two or more of such molecules, or to a whole cell or a tissue sample, or to any non-natural compound, which the binding site of the binding domain is designed to bind to. In the context of the present invention, CD3 expressed on T-cells and one or more of the tumor-associated antigens (TAAs) CD33, CD123 and / or CD70 are the target, i.e., the recombinant binding proteins described herein bind to these target antigens.

[0093] As used herein, “tumor-associated antigen” (TAA) refers to antigens that are expressed by tumor cells and can be recognized by the immune system. TAAs can be derived from normal cellular components but are overexpressed, mutated, or aberrantly expressed in or on tumor cells, making them distinctive markers of the tumor. TAAs can be targeted by the immune system to recognize and eliminate tumor cells, although they may pose challenges due to potential cross-reactivity with healthy tissues. Preferred TAAs in the context of the present disclosure are CD123, CD33 and / or CD70, but the skilled person is also aware of other suitable TAAs, e.g., TAAs associated with myeloid malignancies such as AML.

[0094] The term “CD3" or "Cluster of Differentiation 3" refers to a multimeric protein complex composed of four distinct chains. In mammals, the complex contains a CD3y (gamma) chain, a CD36 (delta) chain, and two CD3s (epsilon) chains. These chains associate with the T-cell receptor (TCR) and the CD3 chain to generate an activation signal in T cells. CD3 is critical 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 orchestrating adaptive immune responses. The amino acid sequences of human CD3 gamma, delta, epsilon and zeta chains are shown in NCBI Ref. Seq. NP_ 000064.1 , NP_000723.1 , NP_000724.1 and NP_932170.1 , respectively. Ankyrin repeat domains specifically binding CD3 are disclosed in WO2022129428 (incorporated by reference in its entirety).

[0095] “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 inhibiting anti-tumor immune responses. Its aberrant expression has been observed in various cancers, including hematological malignancies. CD70 is expressed on a high proportion of AML blast cells and leukemic stem cells (Perna et al., Cancer Cell, 32(4), 2017, 506-519). Specific examples of human CD70 include the polypeptide with amino acid sequence with UniProt ID P32970. Ankyrin repeat domains specifically binding CD70 are disclosed in WO2022215032 (incorporated by reference in its entirety).

[0096] The term “CD33” refers to myeloid cell surface antigen CD33, which is a sialic-acid-binding immunoglobulin-like lectin (Siglec) that plays a role in mediating cell-cell interactions and in maintaining immune cells in a resting state. CD33 is highly expressed on leukemic blast cells in AML (Ehninger, et al. Blood Cancer Journal 4, e218 (2014)). The amino acid sequence of human CD33 is referenced in UniProt ID P20138. Ankyrin repeat domains specifically binding CD33 are disclosed in WO2022190010 (incorporated by reference in its entirety).

[0097] The term “CD123" refers to the interleukin-3 receptor subunit alpha. CD123 is expressed on a large proportion of AML blast cells and leukemic stem cells (Ehninger, et al. Blood Cancer Journal 4, e218 (2014)). The amino acid sequence of human CD123 is referenced in UniProt ID P26951. Ankyrin repeat domains specifically binding CD123 are disclosed in W02022190018 (incorporated by reference in its entirety).

[0098] The term “binding specificity”, “has binding specificity for a target”, “specifically binding to a target”, “binding to a target with high specificity”, “specific for a target” or “target specificity” and the like means that a binding protein or binding domain binds to a target with a lower dissociation constant (i.e. it binds with higher affinity) than it binds to an unrelated protein such as the E. coli maltose binding protein (MBP). Preferably, the dissociation constant (“KD”) for the target is at least 102; more preferably, at least 103; more preferably, at least 104; or more preferably, at least 105times lower than the corresponding dissociation constant for MBP. Methods to determine dissociation constants of protein-protein interactions, such as surface plasmon resonance (SPR) based technologies (e.g. SPR equilibrium analysis) or isothermal titration calorimetry (ITC) are known to the person skilled in the art. The measured KD values of a particular protein-protein interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of KD values are preferably made with standardized solutions of protein and a standardized buffer, such as PBS.

[0099] The term "repeat protein" refers to a protein comprising one or more repeat domains. Preferably, a repeat protein comprises one, two, three, four, five or six repeat domains. Furthermore, said repeat protein may comprise additional non-repeat protein domains, polypeptide tags and / or peptide linkers. The repeat domains can be binding domains. Patent application W02002020565 and Forrer et al., 2003 (Forrer, P., Stumpp, M.T., Binz, H.K., Pluckthun, A., 2003. FEBS Letters 539, 2-6), contain a general description of repeat protein features and repeat domain features, techniques, and applications. The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as structural units, wherein said repeat modules have structural and sequence homology. Preferably, a repeat domain further comprises an N-terminal and / or a C-terminal capping module. For clarity, a capping module can be a repeat module. Such repeat domains, repeat modules, and capping modules, sequence motives, as well as structural homology and sequence homology are known, e.g., see leucine-rich repeat domains (W02002020565), tetratricopeptide repeat domains (Main, E.R., Xiong, Y., Cocco, M.J., DAndrea, L., Regan, L., Structure 11 (5), 497- 508, 2003), and armadillo repeat domains (W02009040338). It is further known that such repeat domains are different from proteins comprising repeated amino acid sequences, where every repeated amino acid sequence is able to form an individual domain (for example FN3 domains of Fibronectin).

[0100] Suitable recombinant binding proteins comprising a recombinant binding protein comprising (a) a first ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70 are described in W02022190016 (incorporated by reference in its entirety).

[0101] The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as structural units, wherein said repeat modules have structural and sequence homology. Preferably, a repeat domain further comprises an N-terminal and / or a C-terminal capping module. For clarity, a capping module can be a repeat module. Such repeat domains, repeat modules, and capping modules, sequence motives, as well as structural homology and sequence homology are known to the practitioner in the art from examples of ankyrin repeat domains (W02002 / 020565), leucine-rich repeat domains (W02002 / 020565), tetratricopeptide repeat domains (Main, E.R., et al., Structure 11 (5), 497-508, 2003), and armadillo repeat domains (W02009 / 040338). It is further known to the practitioner in the art that such repeat domains are different from proteins comprising repeated amino acid sequences, where every repeated amino acid sequence is able to form an individual domain (for example FN3 domains of Fibronectin).

[0102] The term "ankyrin repeat domain" refers to a repeat domain comprising two or more consecutive ankyrin repeat modules as structural units. Ankyrin repeat domains may be modularly assembled into larger ankyrin repeat proteins, optionally with half-life extension domains, using standard recombinant DNA technologies (see, e.g., Forrer, P., et al., FEBS letters 539, 2-6, 2003); W02002 / 020565, WO2016 / 156596; WO2018 / 054971).

[0103] The term “designed” as used in “designed repeat protein”, “designed repeat domain” and the like refers to the property that such repeat proteins and repeat domains, respectively, are man-made and do not occur in nature. In certain embodiments, binding proteins are designed ankyrin repeat proteins and they comprise at least one designed ankyrin repeat domain.

[0104] The term "antibody" means not only intact antibody molecules, but also any fragments and variants of antibody molecules that retain immunogen-binding ability. Such fragments and variants are known in the art. Accordingly, the term "antibody" encompasses intact immunoglobulin molecules, antibody fragments such as, e.g., Fab, Fab', F(ab')2, and single chain V region fragments (scFv), bispecific antibodies, chimeric antibodies, antibody fusion polypeptides, and unconventional antibodies.

[0105] Alternative scaffolds include any polypeptides or proteins comprising a binding domain that is capable of binding a target and that is not derived from an antibody or immunoglobulin molecule. The binding domain of alternative scaffolds may comprise or may be derived from a variety of different polypeptide or protein structures. Alternative scaffolds include, but are not limited to, adnectins (monobodies), affibodies, affilins, affimers and aptamers, affitins, alphabodies, anticalins, armadillo repeat proteinbased scaffolds, atrimers, avimers, fynomers, knottins, and Kunitz domain peptides. Alternative scaffolds are described, e.g., in Yu et al., Annu Rev Anal Chem (Palo Alto Calif). 2017 June 12; 10(1): 293-320. doi: 10.1146 / annurevanchem-061516-045205.

[0106] The term “recombinant” as used in “recombinant binding protein”, “recombinant polypeptide” and the like, means that said protein or polypeptide is produced by the use of recombinant DNA technologies. For example, a recombinant DNA molecule (e.g., produced by gene synthesis) encoding a polypeptide can be cloned into a bacterial expression plasmid (e.g., pQE30), yeast expression plasmid, mammalian expression plasmid, or plant expression plasmid, or a DNA enabling in vitro expression. If, for example, such a recombinant bacterial expression plasmid is inserted into appropriate bacteria (e.g., Escherichia coli), these bacteria can produce the polypeptide(s) encoded by this recombinant DNA. The correspondingly produced polypeptide or protein is called a recombinant polypeptide or recombinant protein.

[0107] As used herein, “Bcl-2” or the “Bcl-2 protein” refers to the first member of the Bcl-2 protein family to be identified in humans, B-cell lymphoma 2. The Bcl-2 family of proteins, a collection of pro- and anti- apoptotic proteins related to Bcl-2, play an integral role in regulating the intrinsic apoptotic pathway with the anti-apoptotic members of the family (e.g., Bcl-2) typically antagonizing the pro-apoptotic members. In many cancers, Bcl-2 is overexpressed, leading to the evasion of apoptosis and enhanced cell survival.

[0108] As used herein, a “Bcl-2 inhibitor” refers to any agent, compound or molecule capable of specifically inhibiting the activity of Bcl-2, in particular an agent, compound or molecule capable of inhibiting the anti-apoptotic activity of Bcl-2. The term “inhibition”, “inhibitor” or “antagonist” includes a reduction in a certain parameter, e.g., an activity, of a given molecule or pathway. For example, inhibition of an activity of Bcl-2 by about 5%, about 10%, about 20%, about 30%, about 40% or more is included by this term. Thus, inhibition can be, but need not be, 100%.

[0109] As used herein, a “hypomethylating agent” refers to any agent, compound or molecule capable of inhibiting DNA methylation and / or promoting DNA demethylation. Such agents can act through various mechanisms, including direct inhibition of DNA methyltransferases, incorporation into DNA leading to interference with methylation processes, or other indirect mechanisms.

[0110] For determining the percent identity between two sequences (e.g., polynucleotide or polypeptide) the sequences are aligned for optimal comparison. A position in the first sequence is considered identical to the corresponding position in the second sequence when they have the same nucleotide or amino acid. The percent 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, the alignment is typically performed over 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), the alignment is carried out 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. Gaps or missing positions in a shorter sequence are considered non-identical positions. Various computer programs are available to determine sequence homology. The Needleman and Wunsch algorithm, can be used with specific parameters like using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6 to calculate percent identity between amino acid or nucleic acid sequences. One example of suitable parameters is a Blosum 62 scoring matrix, a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0111] The term “dose” refers to a specified amount of a therapeutic agent administered at one time. As used herein, the dose is the amount of the therapeutic agent (also referred to as “drug”) that elicits a therapeutic effect. The dose would, for example, be declared on a product package or in a product information leaflet.

[0112] The term “dosage” refers to the administration of a specific amount, number, and frequency of doses over a specified period of time.

[0113] The term “malignancy” or “cancer” encompasses diseases in which abnormal cells proliferate in an uncontrolled manner. Cancer encompasses solid tumors and liquid tumors, as well as primary tumors and metastases. A “tumor” comprises one or more cancerous cells. These abnormal cells can invade the surrounding tissues. Malignant cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

[0114] As used herein, the term “myeloid malignancy” refers to a clonal disease of cells derived from the myeloid lineage in 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 categorized and diagnosed according to the WHO 2008 classification, as well as the international Consensus Classification (ICC) on myeloid neoplasms and acute leukemia (Arber et al. Blood (2022) 140 (11): 1200-1228).

[0115] As used herein, the term “acute myelogenous leukemia” or “acute myeloid leukemia” (AML) refers to hematopoietic neoplasms involving myeloid cells and is a type of cancer that affects the blood and bone marrow. AML is characterized by clonal proliferation of myeloid precursors (“blasts”) with reduced differentiation capacity in the bone marrow, which eventually infiltrate the blood and other organs. In AML, the abnormal myeloid cells lead to bone marrow failure and severely compromise the production of all types of blood cells, leading to anemia, weakened immune function, decreased platelet production, as well as disruption of bone marrow function. The term “AML” includes various subtypes categorized under both the 5thEdition of the World Health Organization (WHO) classification of hematolymphoid tumors and International Consensus Classification 2022 systems. According to the WHO classification, the AML subtypes encompass AML with recurrent genetic abnormalities; AML with myelodysplasia-related changes; therapy-related myeloid neoplasms; myeloid sarcoma; myeloid proliferations related to Down syndrome; blastic plasmacytoid dendritic cell neoplasm; and AML not otherwise categorized, such as acute megakaryoblastic leukemia and acute basophilic leukemia.

[0116] Additionally, AML can be categorized according to the French-American-British (FAB) classification, encompassing the subtypes: MO (acute myeloblastic leukemia, minimally differentiated); M1 (acute myeloblastic leukemia, without maturation); M2 (acute myeloblastic leukemia, with granulocytic maturation); M3 (promyelocytic, or acute promyelocytic leukemia (APL)); M4 (acute myelomonocytic leukemia); M4eo (myelomonocytic together with bone marrow eosinophilia); M5 (acute monoblastic leukemia (M5a) or acute monocytic leukemia (M5b)); M6 (acute erythroid leukemia); or M7 (acute megakaryoblastic leukemia).

[0117] The term “myelodysplastic syndromes” (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 often shows dysplastic features such as abnormal cell morphology, cytogenetic abnormalities, and impaired differentiation. This can result in peripheral blood cytopenias and an increased risk of developing acute myeloid leukemia (AML). The majority of MDS cases exhibit clonality, with somatic genetic aberrations detectable in targeted next-generation sequencing (NGS) panels (around 90%) and conventional karyotype (approximately 50%) of cases. The absence of clonality does not preclude an MDS diagnosis if qualifying dysplasia and persistent cytopenia are present. Certain genetic abnormalities that induce persistent cytopenia are still considered MDS-defining even without dysplasia. In pediatric cases, somatic aberrations in genes like SETBP1 , ASXL1 , RUNX1 , and RAS / MAPK pathway mutations define the genomic landscape of MDS. The refractory cytopenia of childhood (RCC) represents a distinctive entity within this context. The classification of MDS encompasses various subtypes based on dysplastic lineages, cytopenias, cytoses, 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 dysplasia, MDS with single lineage dysplasia, MDS with multilineage dysplasia, MDS with excess blasts (MDS-EB), and MDS / AML. Each subtype is distinguished by specific criteria in terms of blast count, genetic mutations, cytogenetics, and other features. MDS can also be categorized according to the French-American-British (FAB) classification, encompassing the subtypes: refractory anemia (RA); refractory anemia with ring sideroblasts (RARS); refractory anemia with excess blasts (RAEB); refractory anemia with excess blasts in transformation (RAEB-T); and chronic myelomonocytic leukemia (CMML).

[0118] In ICC 2022, a new category MDS / AML (eliminating the MDS with excess blasts, grade 2 in WHO 2017) has been created for cases with cytopenic myeloid neoplasm and 10 to 19% of blasts in peripheral blood and / or bone marrow. This is to allow patients in this group to be able to participate in clinical trials for treatment of AML or MDS depending on clinical conditions. The diagnostic criteria for MDS / AML are identical to those of AML requiring over or equal to 20% blasts. In contrast, WHO 2022 continues to classify these patients as MDS-IB (increased blasts, term changed from excess blasts) grade 2 to avoid over-treatment. However, WHO indicates that MDS-IB2 can be considered as AML for therapeutic purposes if clinically indicated. As used herein, “AML”, “AML / MDS” and “MDS” refers to any of the conditions encompassed by the ICC and / or WHO and / or FAB classifications for these diseases.

[0119] As used herein, “leukemic stem cells” (LSCs) or “leukemic stem progenitor cells” (LSPCs) are a subset of leukemic cells that have stem-like properties, including self-renewal and the ability to give rise to different cell types within the leukemic clone, including blast cells. LSCs / LSPCs are believed to be responsible for the initiation, maintenance, and relapse of AML. They have the capacity for selfrenewal, enabling them to sustain the leukemic population, and they can differentiate into blast cells and other leukemic progeny. “Blast cells” are immature and abnormal cells that accumulate in the bone marrow and peripheral blood in AML. They are derived from hematopoietic stem or progenitor cells that have undergone leukemic transformation. These cells exhibit an impaired ability to differentiate into mature blood cells, leading to an accumulation of immature blasts, and blast cells often represent a significant proportion of the leukemic cell population in AML. While LSCs / LSPCs represent a relatively small proportion of primary AML blast cells, their unique properties and critical role in disease initiation, maintenance, and relapse make them an important target for treatment of AML. LSCs may be characterized as cells that are CD34+, CD45int, CD90-, Lin-. Blast cells may be detected by flow cytometric assessment, e.g., based on CD45dim, or cell morphologic assessment of cells obtained from a bone marrow biopsy of the subject, or a peripheral blood smear. The proportion of blasts is determined versus total cells in the sample. By way of further example, cell morphological assessment can be used to determine the number of morphologically identified blasts relative to the total number of cells in the field of view being examined.

[0120] The terms “subject”, “patient”, “subject in need thereof’, and “patient in need thereof’ are used interchangeably herein and refer to a living organism suffering from one or more of the diseases described herein (e.g., AML) that can be treated by administration of a combination as described herein. A subject is “in need of’ a treatment if such subject would benefit biologically, medically and / or in quality of life from such treatment.

[0121] As used herein, the terms “treat”, “treatment” and “treating” refer to the reduction or amelioration of the progression, severity and / or duration of a disorder, e.g., a proliferative disorder, or the amelioration of one or more symptoms (suitably, one or more discernible symptoms) of the disorder resulting from the administration of one or more therapies. In some embodiments, the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter, or both. In other embodiments, the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size and / or cancerous cell count.

[0122] “Standard intensive chemotherapy”, “intensive induction therapy” or “induction therapy” involves the administration of high-dose chemotherapy agents with the aim of achieving complete remission (CR) by eliminating leukemic cells in the bone marrow and restoring normal hematopoiesis. A commonly used regimen for induction chemotherapy in AML comprises “7+3” induction chemotherapy characterized by 7 days of high dose cytarabine followed by 3 days of anthracycline administration (e.g., daunorubicin or idarubicin). Another commonly used regimen for induction chemotherapy in AML comprises the administration of Vyxeos®. Eligible newly diagnosed AML patients receive standard intensive chemotherapy with the aim of inducing complete remission of AML, typically with the intention of the patient undergoing a stem cell transplant following successful chemotherapy. However, not all newly diagnosed AML patients are eligible for this standard intensive chemotherapy, which is associated with significant toxicity and side-effects, making it unsuitable for some patients. These patients are termed “ineligible for standard intensive chemotherapy”. A patient may be ineligible for standard intensive chemotherapy because, for example, they exhibit one or more comorbidities indicating they would not tolerate the toxicity of the standard intensive chemotherapy regimen, or the prognostic factors characterizing their disease indicate an unfavorable outcome of standard intensive chemotherapy. AML patients over the age of 60 are often assessed as ineligible for standard intensive chemotherapy, with other factors to be considered including the cytogenetics and / or molecular abnormalities of AML being treated.

[0123] 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 reappear (e.g., cytopenia), a different systemic treatment is prescribed. The different treatment administered after the first-line treatment is referred to herein as “second-line” therapy.

[0124] The term “relapsed” refers to a situation where patients who have had a remission of the disease after therapy have a return of disease related symptoms or progression of the disease, for example the return of leukemia cells in the bone marrow and a decrease in normal blood cells.

[0125] The term “refractory or resistant” refers to a circumstance where patients, even after intensive treatment, the disease does not respond adequately to initial treatment or fails to achieve the desired level of improvement, for example residual leukemia cells remain in the bone marrow of a patient.

[0126] The term “partial remission” refers to the response criteria as defined by European LeukemiaNet (ELN) 2022 (Dbhner et al, Blood (2022) 140 (12): 1345-1377).

[0127] The term “complete remission” refers to the response criteria as defined by European LeukemiaNet (ELN) 2022 (Dbhner et al, Blood (2022) 140 (12): 1345-1377), and includes complete remission (CR), CR without measurable residual disease (CRMRD-), CR with incomplete hematologic recovery (CR), and morphologic leukemia-free state (MLFS).

[0128] As used herein, an “anti-proliferative agent” refers to an agent that is capable of preventing or inhibiting cellular proliferation, suitably an agent that is capable of preventing proliferation of cells with abnormal growth behavior. In particular, “anti-cancer agent” refers to any agent that is capable of preventing, inhibiting and / or treating cancer growth directly or indirectly. Such agents include chemotherapeutic agents, immunotherapeutic agents, anti-angiogenic agents, radionuclides, antibody molecules, targeted anti-cancer therapies, gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapy, or oncolytic drugs, cytotoxic agents, immune-based therapies (e.g., cytokines, immunostimulants, or cell-based immune therapies) and the like.

[0129] A "therapeutically effective amount" refers to an amount that is effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agents are outweighed by the therapeutically beneficial effects. A "therapeutically effective dosage" preferably modulates a measurable parameter in a desired manner, e.g., reduction or elimination of leukemic blasts and / or a reduction in the need for blood transfusions and / or change in status from transfusion-dependence to transfusion-independence and / or normalization of blood parameters (e.g. red blood cell counts, white blood cell counts and / or platelet counts), by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.

[0130] The term “jointly therapeutically active” or “joint therapeutic effect” means that the therapeutic agents may be given separately (in a chronologically staggered manner) in such time intervals that they show a beneficial interaction (joint therapeutic effect) in the treated subject.

[0131] The term “synergistically effective” refers to the combined or simultaneous action of multiple factors, substances, or treatments, e.g., the combination of therapeutic agents described herein, which produce a greater effect or outcome than the sum of their individual effects. The term indicates that two or more agents, therapies, or interventions work together in a cooperative and amplified manner to enhance their efficacy or therapeutic impact. For example, reduction of blast cells and / or LSCs / LSPCs on exposure to the combination of the therapeutic agents disclosed herein is greater than the additive effect of the individual therapeutic agents as monotherapy. This synergistic effect can result in improved treatment outcomes, enhanced efficacy, increased response rates, and / or reduced side effects compared to using each therapeutic agent alone.

[0132] A suitable method for determining whether synergistic effects arise from a combination of therapeutic agents can be calculated, for example, using suitable methods such as Sigmoid-Emax equation (Holford, N. H. G. and Scheiner, L. B., Clin. Pharmacokinet. 6: 429-453 (1981)), the equation of Loewe additivity (Loewe, S. and Muischnek, H., Arch. Exp. 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 equation referred to above can be applied to experimental data to generate a corresponding graph to aid in assessing the effects of the combination. The corresponding graphs associated with the equations referred to above are the concentration-effect curve, isobologram curve and combination index curve, respectively. According to the Chou-Talalay method, a combination index (Cl) of <1 shows synergy, Cl=1 shows an additive effect, and a Cl>1 shows antagonism.

[0133] The term “cycle” refers to a specific period of time expressed in days or months that is repeated on a regular schedule. The cycle as disclosed herein is more preferably expressed in days. For example, the cycle can be 28 days, 30 days, 60 days, or 90 days. Most preferably, the “cycle” as referred to herein is 28 days long.

[0134] The term “drug holiday” refers to a time interval within a dosing regimen during which a therapeutic agent is not administered to a patient. The drug holiday can reduce the likelihood or degree of side- effects, can allow organ functions to recuperate, and / or can enable the patient to retain sensitivity to the therapeutic agent.

[0135] The term “dosing regimen”, as used herein, refers to the treatment plan specifically indicating the administration pattern of a therapeutic agent over a period of time. The dosing regimen defines the amount of a therapeutic agent and the number and frequency of its administrations over a specified period of time that is employed in the treatment of a disease. The dosage regimen would be explained, for example, in the Dosage and Administration Section or Posology and method of administration section of labeling for human prescription drugs.

[0136] The dosage regimen may include a “step-up dosing regimen”. As used herein, the term “step-up dosing regimen” involves initiating treatment with a lower-than-standard dosage of the therapeutic agent, and gradually increasing the dosage overtime until reaching the target therapeutic level when the “initial administration” of the therapeutic agent occurs.

[0137] "Overall survival" refers to a clinical endpoint that measures the length of time from the start of a particular treatment or intervention until the death of a patient from any cause.

[0138] "Progression-free survival" (PFS) refers to a clinical endpoint that measures the length of time from the start of a particular treatment or intervention until the disease being treated shows signs of progression or worsening, or until death from any cause, whichever occurs first.

[0139] “Dosage unit form” as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active therapeutic agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0140] As used herein, the term “transfusion independence” indicates that the subject has attained the ability to maintain adequate hemoglobin levels, platelets and other essential blood parameters without the need for regular blood transfusions.

[0141] A “pharmaceutically acceptable excipient” refers to a substance that aids the administration of an active agent to a subject by, for example, modifying the stability of an active agent or modifying the absorption by a subject upon administration. A pharmaceutically acceptable excipient typically has no significant adverse toxicological effect on the patient. Examples of pharmaceutically acceptable excipients include, for example, water, NaCI (including salt solutions), sugars, bulking agents, buffers, diluents, antioxidants, binders, fillers, emulsifiers, extenders, disintegrating agents, lubricants, coating agents, sweeteners, flavoring agents, humectants, preservatives, alcohols, oils, gelatins, solubilizers, wetting agents, carbohydrates such as amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidine, coloring agents, and the like. One of skill in the art will recognize that other pharmaceutical excipients known in the art are useful in the present invention and include those listed in for example the Handbook of Pharmaceutical Excipients, Sheskey P.J., Hancock, B.C., Moss, G.P., Goldfarb, D.J., 9th Ed., The Pharmaceutical Press, RPS Publishing (2020).

[0142] Combinations The combinations presently provided comprise, separately or together, (i) a recombinant binding protein comprising (a) a binding domain specifically binding CD3, (b) a binding domain specifically binding CD33 and / or a binding domain specifically binding CD70, and (c) optionally a binding domain specifically binding CD123, and (ii) a Bcl-2 inhibitor and / or (iii) a hypomethylating agent.

[0143] Without wishing to be bound by theory, the combination presently provided is considered particularly effective for the treatment of myeloid malignancies, suitably AML and MDS, due to the combined therapeutic effect on leukemic cells, particularly blast cells and / or LSCs.

[0144] As shown herein, the therapeutic agents of the combination or combination therapy are synergistically effective against AML cells.

[0145] In one embodiment, the recombinant binding protein comprises (a) a first binding domain specifically binding CD3, and i. (b) a second binding domain specifically binding CD33; ii. (b) a second binding domain specifically binding CD70; or iii. (b) a second binding domain specifically binding CD123 and (c) a third binding domain specifically binding CD70; or iv. (b) a second binding domain specifically binding CD33 and (c) a third binding domain specifically binding CD70; or v. (b) a second binding domain specifically binding CD123, (c) a third binding domain specifically binding CD33; or vi. (b) a second binding domain specifically binding CD123, (c) a third binding domain specifically binding CD33 and (d) a fourth binding domain specifically binding CD70.

[0146] In an embodiment, the binding protein comprises or consists of an antibody or antigen binding fragment thereof, an antibody mimetic, a scaffold protein, and / or a repeat protein, particularly an ankyrin repeat protein.

[0147] In a more specific aspect, the combination presently provided comprises, separately or together, (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor- associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and, (ii) Bcl-2 inhibitor, for example venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, for example azacitidine or decitabine.

[0148] In one embodiment, the recombinant binding protein comprises (a) a first ankyrin repeat domain specifically binding CD3, and i. (b) a second ankyrin repeat domain specifically binding CD123; or ii. (b) a second ankyrin repeat domain specifically binding CD33; or

[0149] Hi. (b) a second ankyrin repeat domain specifically binding CD70; or iv. (b) a second ankyrin repeat domain specifically binding CD123 and (c) a third ankyrin repeat domain specifically binding CD70; or v. (b) a second ankyrin repeat domain specifically binding CD33 and (c) a third ankyrin repeat domain specifically binding CD70; or vi. (b) a second ankyrin repeat domain specifically binding CD123, (c) a third ankyrin repeat domain specifically binding CD33; or vii. (b) a second ankyrin repeat domain specifically binding CD123, (c) a third ankyrin repeat domain specifically binding CD33 and (d) a fourth ankyrin repeat domain specifically binding CD70.

[0150] In a preferred embodiment, the recombinant binding protein comprises (a) a first ankyrin repeat domain specifically binding CD3, (b) a second ankyrin repeat domain specifically binding CD123, (c) a third ankyrin repeat domain specifically binding CD33 and (d) a fourth ankyrin repeat domain specifically binding CD70.

[0151] In certain embodiments, the ankyrin repeat domain specifically binding CD3 comprises an 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 with SEQ ID NO: 1 , the ankyrin repeat domain specifically binding CD33 comprises an 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 with any one of SEQ ID NOs: 2 to 3, the ankyrin repeat domain specifically binding CD123 comprises an 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

[0152] 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about

[0153] 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about

[0154] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical with any one of SEQ ID NOs: 4 to 7, and the ankyrin repeat domain specifically binding CD70 comprises an 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 with any one of SEQ ID NOs: 8 to 9.

[0155] In certain embodiments, the ankyrin repeat domain specifically binding CD3 comprises an 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 with SEQ ID NO: 1 , the ankyrin repeat domain specifically binding CD33 comprises an 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 with SEQ ID NO: 2, the ankyrin repeat domain specifically binding CD123 comprises an 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

[0156] 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about

[0157] 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about

[0158] 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical with SEQ ID NO:

[0159] 4, and the ankyrin repeat domain specifically binding CD70 comprises an 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

[0160] 100% identical with SEQ ID NO: 8. In certain embodiments, the ankyrin repeat domain specifically binding CD3 comprises an 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 with SEQ ID NO: 1 , the ankyrin repeat domain specifically binding CD33 comprises an 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 with

[0161] SEQ ID NO: 3, the ankyrin repeat domain specifically binding CD123 comprises an 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 with SEQ ID NO: 5, and the ankyrin repeat domain specifically binding CD70 comprises an 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 with SEQ ID NO: 8. In certain embodiments, the ankyrin repeat domain specifically binding CD3 comprises an 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

[0162] 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about

[0163] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0164] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical with SEQ ID NO: 1 , the ankyrin repeat domain specifically binding CD33 comprises an 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 with SEQ ID NO: 2, the ankyrin repeat domain specifically binding CD123 comprises an 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

[0165] 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about

[0166] 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about

[0167] 97%, at least about 98%, at least about 99% or 100% identical with SEQ ID NO: 7, and the ankyrin repeat domain specifically binding CD70 comprises an 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 with

[0168] SEQ ID NO: 8. In certain embodiments, the ankyrin repeat domain specifically binding CD3 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about

[0169] 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about

[0170] 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about

[0171] 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about

[0172] 98%, at least about 99% or 100% identical with SEQ ID NO: 1 , the ankyrin repeat domain specifically binding CD33 comprises an 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 with SEQ ID NO: 2, the ankyrin repeat domain specifically binding CD123 comprises an 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 with SEQ ID NO: 4, and the ankyrin repeat domain specifically binding CD70 comprises an 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 with SEQ ID NO: 8. In certain embodiments, the ankyrin repeat domain specifically binding CD3 comprises an 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 with SEQ ID NO: 1 , the ankyrin repeat domain specifically binding CD33 comprises an 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

[0173] 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about

[0174] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0175] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical with SEQ ID NO: 2, the ankyrin repeat domain specifically binding CD123 comprises an 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 with SEQ ID NO: 6, and the ankyrin repeat domain specifically binding CD70 comprises an 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 with SEQ ID NO: 8. In certain embodiments, the ankyrin repeat domain specifically binding CD3 comprises an 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

[0176] 100% identical with SEQ ID NO: 1 , the ankyrin repeat domain specifically binding CD33 comprises an 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 with SEQ ID NO: 3, the ankyrin repeat domain specifically binding CD123 comprises an 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

[0177] 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about

[0178] 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about

[0179] 97%, at least about 98%, at least about 99% or 100% identical with SEQ ID NO: 5, and the ankyrin repeat domain specifically binding CD70 comprises an 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 with

[0180] SEQ ID NO: 9. In certain embodiments, the ankyrin repeat domain specifically binding CD3 comprises an 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 with SEQ ID NO: 1 , the ankyrin repeat domain specifically binding CD33 comprises an 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 with SEQ ID NO: 2, the ankyrin repeat domain specifically binding CD123 comprises an 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

[0181] 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about

[0182] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0183] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical with SEQ ID NO: 4, and the ankyrin repeat domain specifically binding CD70 comprises an 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 with SEQ ID NO: 8.

[0184] In a particularly preferred embodiment, said recombinant binding protein comprises at least one, or at least two further ankyrin repeat domains specifically binding serum albumin, suitably human serum albumin (HSA). Suitably, the ankyrin repeat binding domain specifically binding serum albumin, suitably human serum albumin, comprises an 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

[0185] 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about

[0186] 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about

[0187] 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical with SEQ ID NO:

[0188] 10. In one embodiment, the ankyrin repeat domains comprised in the recombinant binding protein are arranged in any order from the N-terminus to the C-terminus. In one embodiment, the one or more serum albumin binding ankyrin repeat domain, the CD33-binding ankyrin repeat domain, the CD123- binding ankyrin repeat domain, the CD70-binding ankyrin repeat domain, and the CD3-binding ankyrin repeat domain are arranged in any order from the N-terminus to the C-terminus.

[0189] In one embodiment, the ankyrin repeat domains are arranged from the N-terminus to the C-terminus in the following order: (serum albumin binding domain) - (serum albumin binding domain) - (CD33- binding domain) - (CD123 binding domain) - (CD70 binding domain) - (CD3 binding domain).

[0190] In another embodiment, the ankyrin repeat domains are arranged from the N-terminus to the C- terminus in the following order: (serum albumin binding domain) - (serum albumin binding domain) - (CD70-binding domain) - (CD123 binding domain) - (CD33 binding domain) - (CD3 binding domain).

[0191] In another embodiment, the ankyrin repeat domains are arranged from the N-terminus to the C- terminus in the following order: (serum albumin binding domain) - (serum albumin binding domain) - (CD70-binding domain) - (CD33 binding domain) - (CD123 binding domain) - (CD3 binding domain).

[0192] In one embodiment, said recombinant binding protein simultaneously binds CD3, CD123, CD33, and CD70. In another embodiment, said recombinant binding protein simultaneously binds CD3, CD123, CD33, CD70, and HSA.

[0193] In some embodiments, the binding domains comprised in the recombinant binding protein are covalently linked with a peptide linker. A suitable peptide linker is provided with SEQ ID NO: 11 . Accordingly, in one embodiment, the peptide linker comprises the amino acid sequence with SEQ ID NO: 11.

[0194] In some embodiments, the recombinant binding protein comprising an ankyrin repeat domain specifically binding CD3, an ankyrin repeat domain specifically binding CD123, an ankyrin repeat domain specifically binding CD33, an ankyrin repeat domain specifically binding CD70 comprises, at least one, suitably two ankyrin repeat domains specifically binding HSA, comprises at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about

[0195] 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about

[0196] 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about

[0197] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity with any one of the amino acid sequences with SEQ ID NOs: 12 to 18. In one embodiment, said 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 with SEQ ID NO: 12. In another embodiment, said 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 with SEQ ID NO: 13. In another embodiment, said 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 with SEQ ID NO: 14. In another embodiment, said 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 with SEQ ID NO: 15. In another embodiment, said 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 with SEQ ID NO: 16. In another embodiment, said 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 with SEQ ID NO: 17. In another embodiment, said 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 with SEQ ID NO: 18.

[0198] In one preferred embodiment, the combination comprises (i) a recombinant binding protein specifically binding 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

[0199] 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about

[0200] 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about

[0201] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity with any one of the amino acid sequences with SEQ ID NOs: 12 to 18, (ii) venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) azacitidine. In another preferred embodiment, the combination comprises (i) a recombinant binding protein specifically binding 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

[0202] 100% sequence identity with any one of the amino acid sequences with SEQ ID NOs: 12 to 18, (ii) venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) decitabine.

[0203] In one preferred embodiment, the combination comprises (i) a recombinant binding protein specifically binding CD3, CD123, CD33 and CD70, said recombinant binding protein comprising an amino acid sequence with 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) venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) azacitidine.

[0204] In another preferred embodiment, the combination comprises (i) a recombinant binding protein specifically binding CD3, CD123, CD33 and CD70, said recombinant binding protein comprising an amino acid sequence with 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

[0205] 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about

[0206] 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about

[0207] 98%, at least about 99% or 100% sequence identity with SEQ ID NO: 12, (ii) venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) decitabine.

[0208] In one embodiment, the combination comprises (i) a recombinant binding protein comprising an amino acid sequence with SEQ ID NO: 12, (ii) venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) azacitidine.

[0209] In another embodiment, the combination comprises (i) a recombinant binding protein comprising an amino acid sequence with SEQ ID NO: 12, (ii) venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) decitabine.

[0210] A preferred Bcl-2 inhibitor of the combinations disclosed herein is venetoclax, described in US8,546,399 B2 (incorporated by reference). Venetoclax is a selective Bcl-2 inhibitor that binds tightly to the Bcl-2 protein and inhibits its anti-apoptotic function. Venetoclax 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 the pro-apoptotic protein, venetoclax effectively counteracts the anti-apoptotic signals transmitted by Bcl-2. This disruption releases pro-apoptotic factors, allowing them to activate the intrinsic apoptotic pathway and induce programmed cell death in cancer cells. Venetoclax is authorized in the US for use in combination with azacitidine, decitabine or low dose cytarabine for the treatment of newly diagnosed AML in adults who are aged 75 years or older or who have comorbidities that preclude use of intensive induction chemotherapy. As used herein, the term “venetoclax” (also known as ABT-199 and GDC-0199; CAS number 1257044-40-8) refers to the compound having the chemical structure shown below:

[0211] Venetoclax may be provided in any suitable form such that it effectively inhibits the Bcl-2 protein. Such forms include but are not limited to any suitable polymorphic, amorphous or crystalline forms or any isomeric ortautomeric forms. Pharmaceutically acceptable salts in accordance with the present disclosure include salts of acidic or basic groups. Venetoclax may also be provided in the form of a hydrate, anhydrate or solvate.

[0212] In preferred embodiments, the Bcl-2 inhibitor is venetoclax, or a pharmaceutically acceptable salt thereof.

[0213] In certain 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 azacytidine) and decitabine.

[0214] 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 analog of cytidine, and primarily inhibits the enzyme DNA methyltransferase (DNMT1) after its incorporation into DNA, resulting in depletion of DNA methyltransferases and subsequent DNA hypomethylation. Decitabine, similarly, incorporates into DNA and irreversibly inhibits DNA methyltransferases, particularly DNMT1 , leading to prolonged inhibition and DNA hypomethylation. Azacitidine and decitabine have the ability to restore normal gene expression by reactivating silenced genes, including tumor suppressor genes. The alteration of DNA methylation patterns and the reestablishment of appropriate gene regulation contribute to their therapeutic efficacy. These hypomethylating agents have been approved by the US FDA as monotherapy as well as in combination for the treatment of MDS and AML. In certain preferred embodiments, the hypomethylating agent is azacitidine. In other preferred embodiments, the hypomethylating agent is decitabine. Low-dose cytarabine (cytosine-p-D-arabinofuranoside) exerts its effects on rapidly dividing cells by being incorporated into DNA during the S-phase of the cell cycle. Once incorporated, it inhibits DNA synthesis by acting as a chain terminator and preventing further elongation of the DNA strand, leading to the disruption of DNA replication, and ultimately inducing apoptosis in the affected cells. Low dose cytarabine can be used instead of a hypomethylating agent. In some embodiments, instead of a hypomethylating agent, the combination comprises low dose cytarabine.

[0215] In a preferred embodiment, i) the recombinant binding protein, (ii) the Bcl-2 inhibitor and (iii) the hypomethylating agent are separate.

[0216] Also provided is a pharmaceutical combination comprising, separately or together, (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, suitably azacitidine or decitabine.

[0217] Pharmaceutical compositions

[0218] The therapeutic agents of the combinations described herein may be combined or formulated in any manner allowing the combination therapy to be administered to a subject or patient in need thereof. The combination may be formulated for single dose administration or for multiple dose administration. In certain embodiments, the therapeutic agents of the combinations may be co-formulated, i.e., formulated as a single pharmaceutical composition. For embodiments wherein the therapeutic agents are co-formulated, the combination or composition is suitable for simultaneous administration of the therapeutic agents. In preferred embodiments, the therapeutic agents of the combinations described herein are formulated as separate compositions or pharmaceutical compositions.

[0219] For embodiments wherein the therapeutic agents are formulated separately, the therapeutic agents may be administered simultaneously or separately. If the different therapeutic agents are administered separately, the therapeutic agents may be administered in any order. The interval between administration of the therapeutic agents may be any suitable time interval. Preferably, the therapeutic agents of the combination are administered within time intervals that the therapeutic agents can exert a joint therapeutic effect, suitably a synergistic effect. The administration of the different compositions may be carried out once (for a single dose administration) or repeatedly (for a multiple dose administration). In one embodiment, the therapeutic agents of the combinations are coformulated, i.e., formulated as a single pharmaceutical composition.

[0220] The recombinant binding proteins comprised in the combination disclosed herein may be formulated using any suitable pharmaceutical carrier(s), adjuvants) and / or excipient(s). Pharmaceutically acceptable excipients that may be used to formulate the recombinant binding proteins include buffers, pH stabilizers, isotonicity agents, salts, cryoprotectants and the like. The Bcl-2 inhibitor (preferably venetoclax or a pharmaceutically acceptable salt thereof) may be formulated using any suitable pharmaceutical carriers, adjuvants and / or excipients. Venetoclax is sold under the tradename “Venclexta®”. Venclexta® tablets for oral administration are supplied as tablets that contain 10, 50, or 100 mg venetoclax as the active ingredient. Each tablet also contains the following inactive ingredients: copovidone, colloidal silicon dioxide, polysorbate 80, sodium stearyl fumarate, and calcium phosphate dibasic. In addition, the 10 mg and 100 mg coated tablets also include iron oxide yellow, polyvinyl alcohol, polyethylene glycol, talc, and titanium dioxide. The 50 mg coated tablets also include iron oxide yellow, iron oxide red, iron oxide black, polyvinyl alcohol, talc, polyethylene glycol and titanium dioxide. Suitably, venetoclax is administered as Venclexta®. Accordingly, in some embodiments described herein the Bcl-2 inhibitor is Venclexta®.

[0221] The hypomethylating agent or the anti-metabolite cytarabine may be formulated using any suitable pharmaceutical carriers, adjuvants and / or excipients. Azacitidine is marketed under the tradename “Vidaza®”. Vidaza® for injection is supplied as a sterile lyophilized powder that contains mannitol. Suitably, azacitidine is administered as Vidaza®. Accordingly, in some embodiments of the described herein the hypomethylating agent is Vidaza®. Decitabine is marketed under the tradename “Dacogen®”. Dacogen® for injection is supplied as a sterile lyophilized powder that contains monobasic potassium phosphate (potassium dihydrogen phosphate) and sodium hydroxide. Suitably, decitabine is administered as Dacogen®. Accordingly, in some embodiments described herein the hypomethylating agent is Dacogen®.

[0222] In certain embodiments, the compositions are formulated for administration to a subject via any suitable route of administration including but not limited to intramuscular, intravenous, subcutaneous, and oral administration. In certain embodiments, the compositions are formulated as aqueous solutions, tablets, capsules, powders or any other suitable dosage form.

[0223] For embodiments wherein the therapeutic agents of the combination are formulated separately, i.e., as separate compositions, the separate compositions may be formulated for the same route of administration. For embodiments wherein the therapeutic agents of the combination are formulated separately, i.e., as separate compositions, the separate compositions may be formulated for different routes of administration. For example, the recombinant binding protein may be formulated for intravenous administration, and the Bcl-2 inhibitor (preferably venetoclax) may be formulated for oral administration, and the hypomethylating agent may be formulated for subcutaneous or intravenous administration. In preferred embodiments, wherein the combination includes (i) the recombinant binding protein as described herein, and (ii) venetoclax or a pharmaceutically acceptable salt thereof and / or (iii) azacitidine, the recombinant binding protein is administered intravenously, venetoclax or a pharmaceutically acceptable salt thereof is administered orally whilst azacitidine is administered subcutaneously or intravenously. In preferred embodiments, wherein the combination includes (i) the recombinant binding protein as described herein, and (ii) venetoclax or a pharmaceutically acceptable salt thereof and / or (iii) decitabine, the recombinant binding protein is administered intravenously, venetoclax or a pharmaceutically acceptable salt thereof is administered orally whilst decitabine is administered intravenously. In other embodiments, wherein the combination includes (i) the recombinant binding protein as described herein, and (ii) venetoclax or a pharmaceutically acceptable salt thereof and / or (iii) low dose cytarabine, the recombinant binding protein is administered intravenously, venetoclax or a pharmaceutically acceptable salt thereof is administered orally whilst low dose cytarabine is administered subcutaneously.

[0224] Treatment

[0225] The combinations described supra can be used in therapy, in particular in the treatment of a malignancy, particularly a myeloid malignancy, such as AML or MDS.

[0226] Accordingly, presently provided is a combination comprising, separately or together, (i) a recombinant binding protein comprising (a) a binding domain specifically binding CD3, (b) a binding domain specifically binding CD33 and / or a binding domain specifically binding CD70, and optionally (c) a binding domain specifically binding CD123, (ii) Bcl-2 inhibitor, for example venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, for example azacitidine or decitabine, for use in the treatment of a malignancy, suitably a myeloid malignancy.

[0227] In one aspect provided is a combination comprising, separately or together, (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor, suitably venetoclax or a pharmaceutically acceptable salt thereof and / or (iii) a hypomethylating agent, suitably azacitidine or decitabine, for use in the treatment of a malignancy, suitably a myeloid malignancy, in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of the therapeutic agents comprised in the combination. Suitably, said recombinant binding protein, said Bcl-2 inhibitor and / or said hypomethylating agent are administered separately, concurrently, or sequentially. Suitably, said recombinant binding protein, said Bcl-2 inhibitor and / or said hypomethylating agent are provided in synergistically effective amounts for the treatment of a malignancy.

[0228] In another aspect, provided is a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70 for use in the treatment of a malignancy, suitably a myeloid malignancy, comprising administering to a subject in need thereof said recombinant binding protein separately, concurrently, or sequentially with (i) a Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or (ii) a hypomethylating agent, suitably azacitidine or decitabine.

[0229] In another aspect, provided is a Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof for use in the treatment of a malignancy, suitably a myeloid malignancy, comprising administering to a subject in need thereof said Bcl-2 inhibitor separately, concurrently, or sequentially with (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a hypomethylating agent, suitably azacitidine or decitabine.

[0230] In yet another aspect, provided is a hypomethylating agent, suitably azacitidine or decitabine, for use in the treatment of a malignancy, suitably a myeloid malignancy, comprising administering to a subject in need thereof said hypomethylating agent separately, concurrently, or sequentially with (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor- associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof.

[0231] In another aspect, provided is a method for the treatment of a malignancy, suitably a myeloid malignancy, in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor and / or(iii) a hypomethylating agent.

[0232] In a further aspect, provided is the use of (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor, suitably venetoclax, 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 a malignancy.

[0233] In yet another aspect, provided is a medicament comprising, separately or together, a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, suitably azacitidine or decitabine, for simultaneous, sequential or separate administration and wherein said recombinant binding protein, said Bcl-2 inhibitor and / or said hypomethylating agent are provided in synergistically effective amounts for the treatment of a malignancy. The therapeutic agents of the combinations disclosed herein can be administered together in a single composition or administered 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 the different therapeutic agents may be carried out once (for a single dose administration) or repeatedly (for a multiple dose administration). The first agent and the additional agents (e.g., second, third agents) can be administered via the same administration route or via different administration routes. In a preferred embodiment described herein, the therapeutic agents are administered separately. Administration can be simultaneous or sequential. In an embodiment, administration of two or of three therapeutic agents comprised in the combinations disclosed herein are administered separately, but simultaneously. For example, the recombinant binding protein is administered at the same time (simultaneously, for example in a single injection) as the Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, but these two therapeutic agents are administered separately (i.e., the therapeutic agents are not comprised in the same dosage form). In an embodiment, administration of two or of three therapeutic agents comprised in the combinations disclosed herein are administered separately and concurrently. For example, the recombinant binding protein is administered at the same time or within a close timeframe (concurrently) as the Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, but these two therapeutic agents are administered separately. In an embodiment, administration of two or of three therapeutic agents comprised in the combinations disclosed herein are administered separately and sequentially. For example, the recombinant binding protein is administered in a specific order or sequence with a time interval between administration (sequential) of the Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, but these two therapeutic agents are administered separately (not comprised in the same dosage form). In an embodiment, the therapeutic agents are administered in jointly therapeutically effective amounts. Suitably, the therapeutic agents are administered in synergistically effective amounts. Accordingly, in one aspect, provided is a method of enhancing the efficacy of an anti-cancer therapeutic agent by using it in combination with another anti-cancer therapeutic agent, particularly a method using a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70 together with a Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or a hypomethylating agent, suitably azacitidine or decitabine, to provide enhanced efficacy not achievable by administering similar, or lower doses, of any of the therapeutic agents as a single agent.

[0234] In one embodiment, the malignancy is a myeloid malignancy. In particular embodiments, treatment is for treating myeloid malignancies, wherein a myeloid malignancy refers to any clonal disease of hematopoietic stem or progenitor cells.

[0235] In one embodiment, the myeloid malignancy is acute myeloid leukemia (AML); a myelodysplastic syndrome (MDS); a 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.

[0236] In one embodiment, the subject in need of treatment is a newly diagnosed subject. In one embodiment, the subject in need of treatment has relapsed disease and / or refractory disease.

[0237] Treatment as described herein may be first-line treatment or second-line treatment.

[0238] In one embodiment, treatment is first-line treatment in fit AML subjects.

[0239] In one embodiment, treatment is first-line treatment in unfit AML subjects. Suitably, said unfit AML patients have poor cytogenetic risk and / or have one or more mutations in TP53.

[0240] In one embodiment, treatment is post-hematopoietic stem cell transplant (HSCT). Suitably, such treatment post-HSCT is second-line treatment. Suitably, said second-line post-HSCT treatment is in azacitidine / venetoclax naive patients, i.e. in patients not having been exposed to azacitidine and / or venetoclax.

[0241] In one embodiment, treatment is first-line treatment is in high-risk MDS patients. In another embodiment, treatment is first-line treatment is in chronic myelomonocytic leukemia (CMML) patients.

[0242] The subject that is receiving treatment may be a subject who is ineligible for standard intensive chemotherapy. Suitably, such a patient may be newly diagnosed. Such a subject may be ineligible for standard intensive chemotherapy because said subject has one or more co-morbidities that preclude the use of standard intensive chemotherapy. Alternatively, the subjects described herein may be eligible for other treatments, e.g., for standard intensive chemotherapy, but the combination disclosed herein offers a novel and innovative approach to treating AML, providing improved efficacy, reduced toxicity, and potential synergistic effects not achievable with traditional monotherapies or existing treatment regimens.

[0243] The subject in need of treatment may be 60 years or older, or may be 75 years or older.

[0244] Furthermore, the subject’s peripheral blood and / or bone marrow blast count may be monitored. Suitably, treatment as described herein reduces the bone marrow blast count to less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% as compared to pre-treatment and / or reduces the bone marrow blast percentage by more than about 50%, more than about 60%, more than about 70%, more than about 80% or more than about 90% as compared to pretreatment. Suitably, the bone marrow blast cell percentage is also reduced by more than about 50% as compared with the bone marrow blast cell percentage prior to performing the method or uses described herein (“pre-treatment”). Alternatively, or in addition, transfusion independence of red blood cells or platelets, or both, for at least about 8 weeks, at least about 10 weeks, at least about 12 weeks is attained. Measurable residual disease (MRD) refers to the presence of residual leukemia cells in the body at very low levels after initial treatment, despite achieving a (physically) complete remission, e.g., as measured by levels of red blood cells, white blood cells and / or platelets or remission. These residual cells are often present at levels that are undetectable using standard diagnostic methods, such as microscopic examination of the 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 patients achieve complete remission, the persistence of MRD suggests that there is a risk of disease relapse in the future. In some embodiments, complete remission is induced. In some embodiments, partial remission is achieved. In some embodiments, a measurable residual disease (MRD) status that is negative is induced. In certain embodiments, complete remission without measurable residual disease is induced. A partial remission includes a decrease of the bone marrow blast percentage of about 5% to about 25% and a decrease of bone marrow blast percentage by at least about 50% compared to pre-treatment. The combination therapies and methods described herein may also be used to prepare a subject having a myeloid malignancy for a bone marrow transplantation. In certain embodiments, the combination therapies and methods are carried out so as to reduce the blast cell count in the bone marrow and / or peripheral blood prior to transplant. The methods and combination therapies are used to reduce the malignant blast cell count to less than about 5%. In one embodiment, the methods and combination therapies are used to reduce the malignant blast cell count to less than about 5% to prepare the subject for a bone marrow transplant.

[0245] In one embodiment, treatment as described herein increases progression-free survival (PFS). PFS may be increased by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, by at least about 90%, or by at least about 100%. Treatment according to the methods and combination therapies described herein may increase overall survival (OS). In one embodiment, OS is increased by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, by at least about 90%, or by at least about 100%. OS or PFS may be increased relative to no treatment or relative to standard of care treatment. Standard of care treatments may differ in clinical practice, for example in different countries. The treatments for myeloid malignancies are varied and include chemotherapy, radiation therapy, stem cell transplant and targeted therapies.

[0246] Dosing

[0247] Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered overtime, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0248] As demonstrated in the Examples, the combination of the therapeutic agents described herein is particularly advantageous, because they exhibit synergistic efficacy. Preferably, therefore, in all embodiments described herein, the dose at which the recombinant binding protein is administered and / or provided in the combination therapies, and the dose at which the Bcl-2 inhibitor and / or the dose at which the hypomethylating agent are administered and / or provided in the combination therapies are each selected such that the therapeutic agents exhibit a joint therapeutic effect, suitably a synergistic therapeutic effect. Suitably, whether the combination exerts a synergistic effect is determined by the Chou-Talalay method. Suitably, the dose at which the recombinant binding protein, the Bcl-2 inhibitor and / or the hypomethylating agent is administered and / or provided in the combination is chosen such that the combination of therapeutic agents exhibits a Cl of less than 1 or less than 0.5 as determined by the Chou-Talalay method.

[0249] Suitably, in certain embodiments, the dose at which the recombinant binding protein, the Bcl-2 inhibitor and / or the hypomethylating agent is administered and / or provided in the combination are each selected such that the combination exhibits a Cl of less than 1 and Fa of >0.5, as determined by the Chou-Talalay method.

[0250] The therapeutic agents described herein can be used together as provided herein. The therapeutic agents can be administered together or separately, depending on the intended dosage amount and frequency of administration, since it is contemplated that the treatments described herein may be continued 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 deemed appropriate by the treating physician.

[0251] In certain embodiments, multiple doses of the recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, are administered. In certain such embodiments, administration of each dose of said recombinant binding protein is separated by 5-20 days. In certain embodiments, administration of each dose of the recombinant binding protein is separated by 5-10 days. In certain embodiments, said recombinant binding protein is administered once weekly. In one embodiment, said 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, said recombinant binding protein is administered in a step-up dosing regimen, wherein administration of the initial dose is preceded by at least one, at least two, at least three step-up doses, optionally wherein the subsequent step-up dose comprises a higher amount of the recombinant binding protein relative to the preceding step-up dose. In one embodiment, said recombinant binding protein is administered parenterally, suitably intravenously. In another embodiment, a 28-day treatment cycle comprising said recombinant binding protein comprises a drug holiday 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.

[0252] The Bcl-2 inhibitor, suitably venetoclax or pharmaceutically acceptable salt thereof, may be dosed according to any regimen determined to be effective forthat therapeutic agent compound. In a preferred embodiment, the Bcl-2 inhibitor is venetoclax, or a pharmaceutically acceptable salt thereof. The FDA prescribing information for use of venetoclax (Venclexta®) in the treatment of AML proposes a dosing schedule with a 3-day or a 4-day step-up phase followed by a maintenance phase. Where Venclexta® is prescribed in combination with azacitidine or 20 mg / m2decitabine, a dosing schedule is recommended consisting of: 100 mg Venclexta® on day 1 ; 200 mg Venclexta® on day 2; 400 mg Venclexta® on day 3; followed by a maintenance phase of 400 mg Venclexta® daily in combination with azacitidine at 75 mg / m2(once daily on days 1 to 7 of a 28-day treatment cycle) or with decitabine at 20 mg / m2(once daily on days 1 to 5 of a 28-day treatment cycle) daily thereafter until disease progression or unacceptable toxicity is observed. In situations where Venclexta® is prescribed in combination with low-dose cytarabine, a dosing schedule is recommended consisting of: 100 mg Venclexta® on day 1 ; 200 mg Venclexta® on day 2; 400 mg Venclexta® on day 3; and 600 mg Venclexta® daily in combination with 20 mg / m2cytarabine (once daily on days 1 to 10 of a 28-day treatment cycle) thereafter until disease progression or unacceptable toxicity is observed.

[0253] In a preferred embodiment, venetoclax is administered at a dose of about 10 mg to about 800 mg (e.g., per day), more preferably at a dose of about 100 mg to about 600 mg per day. In a preferred embodiment, venetoclax is administered at a dose of about 100 mg on day 1 , a dose of about 200 mg on day 2, a dose of about 400 mg on day 3, and a dose of about 400 mg on days 4 to 28, suitably once daily of a 28-day treatment cycle until disease progression or unacceptable toxicity is observed. In an alternative embodiment, venetoclax is administered at a dose of about 100 mg on day 1 , a dose of about 200 mg on day 2, a dose of about 400 mg on day 3, and a dose of about 600 mg on days 4 to 28, suitably once daily of a 28-day treatment cycle until disease progression or unacceptable toxicity is observed. In one embodiment, venetoclax is administered orally. It is also contemplated that the dose and / or frequency and / or duration of administration of the Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, is decreased. Suitably, the 28-day treatment cycle comprising the Bcl-2 inhibitor, suitably venetoclax or a pharmaceutically acceptable salt thereof, comprises a drug holiday 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 comprised in the combinations disclosed herein act synergistically. The timepoint and the extent of dose reduction of the Bcl-2 inhibitor are factors that the attending clinician will tailor according to the patient’s needs.

[0254] In a preferred embodiment, the hypomethylating agent is azacitidine. In one embodiment disclosed herein, azacitidine is administered at a dose of about 70 to about 80 mg / m2(e.g., per day), suitably at a dose of about 75 mg / m2per day. In a preferred embodiment, azacitidine is administered at a dose of about 75 mg / m2per day once daily on days 1 to 7 of a 28-day treatment cycle. In one embodiment, azacitidine is administered intravenously. In an alternative embodiment, azacitidine is administered subcutaneously.

[0255] In another preferred embodiment, the hypomethylating agent is decitabine. In one embodiment, decitabine is administered at a dose of about 10 to about 30 mg / m2(e.g., per day), suitably at a dose of about 20 mg / m2per day. In a preferred embodiment, decitabine is administered at a dose of about 20 mg / m2per day once daily on days 1 to 5 of each 28-day treatment cycle. In one embodiment, decitabine is administered intravenously. In another embodiment, the hypomethylating agent is cytarabine. In one embodiment, cytarabine is administered at a dose of about 10 to about 30 mg / m2(e.g., per day), suitably at a dose of about 20 mg / m2per day. In a preferred embodiment, cytarabine is administered at a dose of about 20 mg / m2per day once daily on days 1 to 10 of each 28-day treatment cycle. In one embodiment, decitabine is administered subcutaneously.

[0256] Because hypomethylating agents, such as azacitidine or decitabine, can induce cytopenia, it is also conceivable that the dose of these therapeutic agents is reduced (or omitted altogether) after a first 28-day treatment cycle. For example, once the bone marrow and / or peripheral blood blast percentage is reduced to less than about 10% or to below less than about 5%, the dose of the hypomethylating agent, suitably azacitidine, decitabine or low dose cytarabine, may be reduced (or omitted altogether). The timepoint and the extent of dose reduction of the hypomethylating agent are factors that the attending clinician will tailor according to the patient’s needs.

[0257] In one embodiment, the recombinant binding protein is administered 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day prior to administration of the Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or the hypomethylating agent, suitably azacitidine. In one embodiment, the recombinant binding protein is administered 7 days, 6 days, 5 days, 4 days, 3 days, 2 days, or 1 day after administration of the Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or the hypomethylating agent, suitably azacitidine.

[0258] In some embodiments, the treatment cycle is repeated. For example, the treatment may comprise of 2 or 3 or 4 or more 28-day treatment cycles. In one embodiment, the first 28-day treatment cycle comprises the Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or the hypomethylating agent, suitably azacitidine, and the second, and optionally further, 28-day treatment cycle comprises the recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, the Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or the hypomethylating agent, suitably azacitidine. In one embodiment, the first and second 28-day treatment cycles comprises the Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or the hypomethylating agent, suitably azacitidine, and the third, and optionally further, 28-day treatment cycle comprises the recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, the Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or the hypomethylating agent, suitably azacitidine.

[0259] Further Combination Therapies In certain embodiments, the methods or combination therapies described herein are administered with one or more additional anti-cancer therapy modes, e.g., targeted anti-cancer therapies, gene therapy, viral therapy, RNA therapy, bone marrow transplantation, nanotherapy, or oncolytic drugs, cytotoxic agents, immune-based therapies (e.g., cytokines, immunostimulants, or cell-based immune therapies), surgical procedures or radiation procedures, or a combination of any of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In certain embodiments, the methods or combination therapy comprises the administration of one or more additional antiproliferative agent(s), suitably anti-cancer agent(s). Such anti-cancer agent(s) may comprise one or more agents suitable for use in treating myeloid malignancies, for example agents suitable for use in treating AML.

[0260] Alternatively, or in combination with the aforesaid combinations, the methods and combination therapies described herein can be administered in combination with one or more of: an immunomodulator (e.g., an activator of a costimulatory molecule or an inhibitor of an inhibitory molecule, e.g., an immune checkpoint molecule); a vaccine, e.g., a therapeutic cancer vaccine; or other forms of cellular immunotherapy.

[0261] Any combination and sequence of other therapeutic agents, procedures or modalities (e.g., as described herein) can be used in combination with the treatments as described herein. The methods and combination therapies described herein can be administered before other treatment methods, concurrently with other treatment methods, between cycles of such other treatments, or during remission of the disorder.

[0262] Kits

[0263] Also within the scope of the present invention is a kit comprising one or more of the therapeutic agents described herein. The kit can also include one or more other elements: instructions for use; other reagents for use with the compound(s); devices or other materials for preparing the compound for administration, such as a mixing container; pharmaceutically acceptable carriers; and devices or other materials for administration to a subject, such as a syringe.

[0264] Accordingly, the invention provides a kit comprising a combination comprising (i) a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, suitably azacitidine or decitabine together with instructions for simultaneous or sequential administration thereof for use in the treatment of a malignancy, suitably a myeloid malignancy, suitably acute myeloid leukemia and / or myelodysplastic syndrome. Suitably, the kit comprises separately (i) a pharmaceutical composition comprising a recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one 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 venetoclax, 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, together with instructions for dosing to a subject in need thereof of the pharmaceutical composition comprising the Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or dosing of the pharmaceutical composition comprising a hypomethylating agent before, simultaneously or after the dosing of the pharmaceutical composition comprising the recombinant binding protein.

[0265] Another aspect provides the use of (i) recombinant binding protein comprising (a) an ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one selected from the group consisting of CD123, CD33 and CD70, and (ii) a Bcl-2 inhibitor, suitably venetoclax, or a pharmaceutically acceptable salt thereof, and / or (iii) a hypomethylating agent, suitably azacitidine or decitabine, for the manufacture of a kit for treating a malignancy, suitably a myeloid malignancy.

[0266] INCORPORATION BY REFERENCE

[0267] All publications, patents, and accession numbers mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

[0268] EQUIVALENTS

[0269] While specific embodiments of the subject invention have been discussed, the above specification is illustrative and not restrictive. Many variations of the invention will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.

[0270] Sequence table

[0271] EXAMPLES The Examples below are set forth to aid in the understanding of the invention but are not intended to, and should not be construed to, limit the scope in any way.

[0272] Example 1

[0273] Combining multiple therapeutic agents with distinct but complementary mechanisms of action can enhance therapeutic efficacy while minimizing adverse effects. Synergy scores as a quantitative measure provide a systematic approach for evaluating the synergistic interactions between therapeutic agents beyond the additive effects of individual therapeutic agents.

[0274] A study was carried out with the recombinant binding protein with SEQ ID NO: 12 (“Protein #1”) in combination with Azacitidine (AZA) and Venetoclax (VEN) on the AML cell line, MOLM-13 (DSMZ, cat.: ACC 554).

[0275] Pan T cells were isolated from buffy coat from blood donations from three healthy volunteers using the Pan T cell Kit (Miltenyi, cat: 130-096-535) and labelled with cell trace violet (CTV) (Thermo Fisher cat.: C34557). MOLM-13 cells (1 .5x104cells / well) were co-cultured with CTV-labelled PBMCs (1.5x104cells / well) in a 1 :1 ratio in RPMI 1640 medium + 10% FBS and 10 pM human serum albumin (HSA; to provide saturation of the HSA-binding DARPins) in a 96-well U-bottom plate. Killing EC50 values for each of Protein #1 , VEN and AZA after 72 h incubation was determined by counting the number of alive MOLM-13 tumor cells in FACS Fortessa by adding precision beads to the cells (Biolegend cat.: 424902). Treated MOLM-13 cells versus control cells (vehicle-only treated) were normalized by the number of beads. The combination of Protein #1 with AZA and / or VEN was then assessed by repeating the same killing assay with EC50 concentrations of each compound (5 pM of Protein #1 , 5 nM venetoclax, 0.25 pM azacitidine) versus 2-fold incremental / decremental titrations. The effect of drug treatment was calculated as the ratio of alive cells vs vehicle-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 synergy scores using the CompuSyn software as previously described (Riether et al, Nature Medicine volume 26, pagesl 459-1467 (2020)) by assessing combination index (Cl) and fraction affected (FA) values, with Cl values <1 indicating synergism, =1 additive effect, >1 antagonism. Fa values of 0, 0.5 and 1 correspond to 0, 50% and 100% of cells killed.

[0276] As shown in Figures 1 A-E, Protein #1 in combination with VEN or in combination with both VEN and AZA synergistically eliminated MOLM-13 cells at a broad dose range. At higher effect levels, the combinations exhibited strong synergy. The Cl of the combination was low for all donors tested, indicating very strong synergy (see Figure 2). These experiments demonstrated that Protein #1 , VEN and AZA act synergistically at all dose levels tested (see Figure 1 E).

[0277] Example 2

[0278] The effect of the combination of Protein #1 with AZA / VEN at physiologically relevant EC50 concentrations was also assessed on primary AML CD34+ sorted LSCs (Riether C et al. Nat Med. 2020;26(9):1459-1467). AML samples (peripheral blood mononuclear cells or bone marrow mononuclear cells) from three separate donors were FACS-sorted for expression of CD45int / Lin_ / CD907CD34 CD38+ / _and then co-cultured at an effector to target (E:T) ratio of 1 :1 with purified allogeneic T cells derived from a healthy volunteer and treated with 10 pM Protein #1 or control non- binding-CD3 DARPin ± 1 pM azacitidine and 5 nM venetoclax for four days in a 96-well round-bottom plate in StemSpan SFEM medium (STEMCELL Technologies) containing a cytokine mix (StemSpan CC100, STEMCELL Technologies) and 10 pM HSA. After incubation, cells were transferred into cultivation medium containing cytokines and 1 .27% methylcellulose to assess colony forming capacity for each condition. Colonies were counted after 14 days additional culture by light microscopy and plotted using GraphPad Prism. The results, shown in Figure 3, demonstrate that the synergistic effect observed in the AML cell line also translates into significant reduction in the number of LSCs obtained from an AML patient compared to vehicle-treated, aza / ven or recombinant binding protein only, even when used at sub-optimal EC50 doses.

[0279] Synergy was observed when Protein #1 was used together with azacitidine and venetoclax at sub- optimal EC50 doses.

[0280] The data presented in Figures 1-3 demonstrate that the combination of a recombinant binding protein simultaneously engaging T cells by binding CD3, as well as several TAAs together with azacitidine and venetoclax exhibited a strong synergistic effect against AML cells in the presence of allogeneic T cells. This synergy is particularly advantageous since it indicates that when the therapeutic agents are used in combination, significantly lower concentrations of each agent may be used compared to the concentrations required to achieve the same effect as each agent in monotherapy alone. This effect was demonstrated for an exemplary AML cell line, as well as for primary cells derived from AML patients. The data provided here demonstrate that for all patient samples the combination therapy inhibited LSC colony formation to an extent significantly greater than either therapy alone.

[0281] Example 3

[0282] In order to demonstrate that the recombinant binding protein, is able to achieve an avidity-like effect on cells, Protein #1 was assessed in potency assays 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 shows similar dose-dependent activation of CD8 T cells (see Figure 4A) and killing of MOLM-13 cells (see Figure 4B) expressing >2 TAAs (EC50s of 7-30 pM and 10-45 pM for T cell activation and cell killing, respectively), while an approximate 10-fold lower potency was measured on cells expressing a single TAA (EC50s of 40-430 pM and 105-455 pM for T cell activation and cell killing, respectively). TAA-specific binding of Protein #1 to MOLM-13 cells and KO clones (see Figure 5A) and CD3-specific binding to pan T cells (see Figure 5B) was confirmed by flow cytometry. In Figure 5B, the average ± standard deviation (SD) of Protein #1 binding to T cells from three donors is shown. NB-CD3 DARPin (non-TAA binding) and NB- TAA DARPin (non-CD3 binding) are shown as control molecules.

[0283] These results demonstrate that potent activation of Protein #1 is achieved in the presence of at least two TAAs. Activation in the presence of two TAAs is expected to result in the selective activation of T cells to kill AML cells and LSCs while sparing HSCs. Further, such selective activation of T cells can result in significantly lower cytokine and chemokine release than is seen with single TAA targeting T- cell engagers. In addition, activation of T cells occurs despite the high heterogeneity of expression of TAAs, which may translate into deeper and more durable responses in the clinic.

Claims

CLAIMS1 . A combination comprising, separately or together, (i) a recombinant binding protein comprising (a) a first ankyrin repeat domain specifically binding CD3 and (b) at least one, at least two, or at least three further ankyrin repeat domains, wherein each of the at least one, at least two, or at least three further ankyrin repeat domains specifically binds one tumor-associated antigen, suitably one 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 said first ankyrin repeat domain specifically binding CD3, and i. a second ankyrin repeat domain specifically binding CD123; or ii. a second ankyrin repeat domain specifically binding CD33; or iii. a second ankyrin repeat domain specifically binding CD70; or iv. a second ankyrin repeat domain specifically binding CD123 and a third ankyrin repeat domain specifically binding CD70; or v. a second ankyrin repeat domain specifically binding CD33 and a third ankyrin repeat domain specifically binding CD70; or vi. a second ankyrin repeat domain specifically binding CD123, a third ankyrin repeat domain specifically binding CD33; or vii. a second ankyrin repeat domain specifically binding CD123, a third ankyrin repeat domain specifically binding CD33 and a fourth ankyrin repeat domain specifically binding CD70.

3. The combination according to claim 1 , wherein the recombinant binding protein comprises (a) said first ankyrin repeat domain specifically binding CD3, (b) a second ankyrin repeat domain specifically binding CD123, (c) a third ankyrin repeat domain specifically binding CD33 and (d) a fourth ankyrin repeat domain specifically binding 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 further ankyrin repeat domains specifically binding human serum albumin.

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

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

7. The combination according to any one of the preceding claims, wherein said recombinant binding protein comprises an amino acid sequence at least about 80% identical with any one of SEQ ID NOs: 12 to 18.

8. The combination according to any one of the preceding claims, wherein said recombinant binding protein comprises an amino acid sequence at least about 80% identical with SEQ ID NO: 12.

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

10. The combination according to any one of claims 1 to 8, comprising (i) a recombinant binding protein comprising the amino acid sequence with SEQ ID NO: 12, (ii) venetoclax, or a pharmaceutically acceptable salt thereof, and (iii) decitabine.11 . The combination according to any one of the preceding claims, wherein (i) said recombinant binding protein, (ii) said Bcl-2 inhibitor and (iii) said hypomethylating agent are separate.

12. A method for treatment of a malignancy, suitably a myeloid malignancy, in a subject, comprising administering to the subject in need thereof a combination as defined in any one of claims 1 to 11 .

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

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

15. A kit comprising a combination as defined in any one of claims 1 to 11 , together with instructions for simultaneous or sequential administration thereof for use in the treatment of a malignancy, suitably a myeloid malignancy, suitably acute myeloid leukemia or myelodysplastic syndrome.

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