Multispecific binding proteins

Abstract

The present disclosure relates to multispecific binding proteins comprising binding domains with binding specificity a stem cell marker, an immunoregulatory protein and an immune cell-associated antigen. In addition, the disclosure relates to nucleic acids encoding such multispecific binding proteins, pharmaceutical compositions comprising such multispecific binding proteins or nucleic acids, and the use of such multispecific binding proteins, nucleic acids or pharmaceutical compositions in methods of conditioning a subject prior to receiving a stem cell transplant.

Description

[0001] MULTISPECIFIC BINDING PROTEINS

[0002] FIELD OF THE INVENTION

[0003] The present disclosure relates to multispecific binding proteins that specifically bind to (i) a stem cell marker, (ii) an immunoregulatory protein and (iii) an immune cell-associated antigen. More specifically, the disclosure relates to multispecific binding proteins which are useful for the pretreatment of a subject prior to receiving a stem cell transplantation. Such a subject may be suffering from a condition in which a stem cell transplantation is considered beneficial, for example a hematologic disease or a hematological malignancy, such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML).

[0004] BACKGROUND

[0005] Stem cells play a pivotal role in tissue maintenance and regeneration by possessing the unique ability to self-renew and differentiate into specialized cells. Hematopoietic stem cells (HSCs) are a type of multipotent stem cell found in the bone marrow, where they give rise to all types of blood cells in the body, including red blood cells, white blood cells, and platelets.

[0006] Hematopoietic stem cell transplantation (HSCT) involves infusing active hematopoietic stem cells (HSCs) collected from various sources, such as bone marrow, peripheral blood, or umbilical cord blood, to restore normal blood cell production in individuals with damaged or defective bone marrow or immune systems. This procedure serves as a critical therapy for eliminating bone marrow-related diseases like leukemia or correcting congenital immunodeficiencies.

[0007] The success of HSCT heavily relies on the preparative or conditioning regimen to clear bone marrow spaces. Prevailing conditioning methodologies, such as irradiation (e.g., total body irradiation) and DNA alkylating / modifying agents are non-targeted. These aggressive conditioning regimens effectively decimate the recipient's immune and other cells, and also trigger detrimental repercussions across numerous organ systems resulting possibly in organ damage and secondary malignancies and frequently precipitate life-threatening complications. The severe toxicity of these conditioning regimens severely limits the clinical applicability of bone marrow transplantation as they are contraindicated for large groups of patients. By advancing safer and more targeted conditioning protocols, the safety of transplantation could significantly improve, making this powerful form of cell therapy accessible to a broader range of patients.

[0008] Therapeutic modalities that selectively target the endogenous hematopoietic stem cell population, thereby sparing cells and tissues, such as platelets, white blood cells, and red blood cells may provide improved conditioning regimens. The depletion of host HSCs and facilitating the integration of donor HSCs has been shown experimentally in animal models using monoclonal anti-CD117 antibodies, including in combination with blockade of CD47 (Chhabra et al, Sci Transl Med. 2016 Aug 10;8(351):351 ra105), signifying the potential for targeted conditioning regimens using biologic agents. The cell surface marker CD117 can be used to identify specific hematopoietic progenitor cells residing in the bone marrow. CD117, also recognized as c-kit or stem cell factor (SCF) receptor, is pivotal in governing diverse cellular processes, including cell survival, proliferation, and differentiation, particularly HSCs. High expression levels of CD117 are observed in HSCs, multipotent progenitors (MPPs), and common myeloid progenitors (CMPs), serving as a marker for these cell types. During hematopoiesis, CD117 is present on early progenitor cells, orchestrating their self-renewal and guiding their differentiation into distinct blood cell lineages.

[0009] CD47, localized on the cell surface of most cells, acts as a key regulator of phagocytosis by engaging with multiple ligands. Its widespread expression encompasses various cell types and tissues, playing a crucial role as a cellular ligand for SIRPa, predominantly found on myeloid cells, including macrophages, granulocytes, myeloid dendritic cells, and mast cells. Of particular importance is its role in inhibiting phagocytosis through the CD47-SIRPa signaling complex, serving as a “don’t-eat-me” signal for phagocytes. However, CD47 is expressed on the surface of most healthy cells, including red blood cells, and targeting CD47 is associated with numerous side effects, including depletion of red blood cells.

[0010] To fully harness the curative potential of HSCT, conditioning regimens with sufficient potency but devoid of undesirable toxicity are required. There is a need for novel, non-myeloablative, compositions and methodologies capable of conditioning the recipient's tissues, especially bone marrow, while mitigating deleterious toxicity and reducing the incidence of severe adverse reactions.

[0011] SUMMARY OF THE INVENTION

[0012] The present invention is directed to multispecific binding proteins. More particularly, the present invention is directed to a multispecific binding protein that specifically binds to (i) a stem cell marker, (ii) an immunoregulatory protein and (iii) an immune cell-associated antigen. Such multispecific binding proteins may comprise one or more ankyrin repeat domains. Such multispecific binding proteins may be used in the targeted depletion of hematopoietic stem cells through activation of innate immune cells, particularly in the context of hematopoietic stem cell transplantation (HSCT).

[0013] 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:

[0014] E1 . A multispecific binding protein comprising (1) a first binding agent with binding specificity for an immunoregulatory protein, (2) a second binding agent with binding specificity for the first binding agent, and (3) a third binding agent with binding specificity for a stem cell-associated antigen, wherein binding of said second binding agent and said third binding agent is mutually exclusive. E2. The multispecific binding protein of E1 , wherein said second binding agent reversibly binds said first binding agent.

[0015] E3. The multispecific binding protein of E1 or E2, wherein said first binding agent is released from binding to the second binding agent upon binding of the third binding agent to said stem cell- associated antigen.

[0016] E4. A multispecific binding protein comprising (1) a first binding agent with binding specificity for an immunoregulatory protein, (2) a second binding agent with a first binding region with binding specificity for a first target, wherein said first target is the first binding agent and a second binding region with binding specificity for a second target, wherein said second target is a stem cell- associated antigen, and wherein binding of said second binding agent to the first and second targets is mutually exclusive.

[0017] E5. The multispecific binding protein of E4, wherein the first binding region of said second binding agent reversibly binds said first binding agent, and wherein said first binding region is released upon binding of the second binding region to said stem cell-associated antigen.

[0018] E6. The multispecific binding protein of E4 or E5, wherein said first binding agent is released from binding to the first target of the second binding agent upon binding of the second target to said stem cell-associated antigen.

[0019] E7. The multispecific binding protein of any one of E1 to E6, comprising a further binding agent with binding specificity for an immune cell-associated antigen.

[0020] E8. The multispecific binding protein of any one of E1 to E7, comprising a further binding agent with binding specificity for a stem cell-associated antigen.

[0021] E9. The multispecific binding protein of any one of E1 to E8, wherein said immunoregulatory protein is CD47.

[0022] E10. The multispecific binding protein of any one of E7 to E9, wherein said immune cell is an innate immune cell, suitably an NK cell, a monocyte or a macrophage.

[0023] E11. The multispecific binding protein of any one of E7 to E10, wherein said immune cell-associated antigen is CD16a.

[0024] E12. The multispecific binding protein of any one of E8 to E12, wherein said second binding agent and said further binding agent with binding specificity for a stem cell-associated antigen bind to the same stem cell-associated antigen, or bind to a different stem cell-associated antigen.

[0025] E13. The multispecific binding protein of any one of E8 to E12, wherein said second binding agent and said further binding agent with binding specificity for a stem cell-associated antigen bind to the same epitope on the stem cell-associated antigen.

[0026] E14. The multispecific binding protein of any one of E8 to E12, wherein said second binding agent and said further binding agent with binding specificity for a stem cell-associated antigen bind to a different epitope on the stem cell-associated antigen.

[0027] E15. The multispecific binding protein of any one of E1 to E14, wherein the stem cell is a hematopoietic stem cell and / or a cancer stem cell. E16. The multispecific binding protein of any one of E1 to E15, wherein said stem cell-associated antigen is CD117.

[0028] E17. The multispecific binding protein of any one of E1 to E17, wherein said binding agents are an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

[0029] E18. The multispecific binding protein of E17, wherein said designed repeat domain is a designed ankyrin repeat domain.

[0030] E19. The multispecific binding protein of any one of E1 to E18, wherein said first binding agent with binding specificity for an immunoregulatory protein comprises an ankyrin repeat domain with binding specificity for CD47.

[0031] E20. The multispecific binding protein of any one of E1 to E19, wherein said first binding agent comprises an ankyrin repeat domain with binding specificity for CD47, comprising an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 8.

[0032] E21 . The multispecific binding protein of any one of E9 to E20, wherein said multispecific binding protein binds CD47 in PBS with a dissociation constant (KD) of or below about 107M.

[0033] E22. The multispecific binding protein of any one of E9 to E21 , wherein said multispecific binding protein with binding specificity for CD47 is capable of blocking the interaction of CD47 with signal- regulatory protein alpha (SIRPa).

[0034] E23. The multispecific binding protein of any one of E4 to E22, wherein said second binding agent comprises an ankyrin repeat domain with (a) a first binding specificity for a CD47-binding ankyrin repeat domain and (b) a second binding specificity for CD117, wherein binding to the first and second targets is mutually exclusive.

[0035] E24. The multispecific binding protein of any one of E4 to E23, wherein said second binding agent comprises an ankyrin repeat domain comprising an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 12 to 16.

[0036] E25. The multispecific binding protein of any one of E4 to E24, wherein said second binding agent binds to said first target with a dissociation constant (KD) of or below about 105M and / or to said second target with a dissociation constant (KD) of or below about 105M.

[0037] E26. The multispecific binding protein of any one of E7 to E25, wherein said further binding agent with binding specificity for an immune cell-associated antigen comprises an ankyrin repeat domain with binding specificity for CD16a.

[0038] E27. The multispecific binding protein of any one of E7 to E26, wherein said further binding agent with binding specificity for an immune cell-associated antigen comprises an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 9.

[0039] E28. The multispecific binding protein of any one of E7 to E27, wherein said multispecific binding protein binds CD16a in PBS with a dissociation constant (KD) of or below about 107M. E29. The multispecific binding protein of any one of E7 to E28, wherein said multispecific binding protein with binding specificity for an immune cell-associated antigen is capable of stimulating effector cells, suitably innate immune cells.

[0040] E30. The multispecific binding protein of any one of E8 to E29, wherein said further binding agent with binding specificity for a stem cell-associated antigen comprises an ankyrin repeat domain with binding specificity for CD117.

[0041] E31 . The multispecific binding protein of any one of E8 to E30, wherein said further binding agent with binding specificity for a stem cell-associated antigen comprises an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 10, 11 , or 26 to 29.

[0042] E32. The multispecific binding protein of any one of E8 to E31 , wherein said multispecific binding protein binds CD117 in PBS with a dissociation constant (KD) of or below about 107M.

[0043] E33. The multispecific binding protein of any one of E8 to E32, wherein said further binding agent with binding specificity for a stem cell-associated antigen comprises an ankyrin repeat domain with binding specificity for CD117, wherein said ankyrin repeat domain with binding specificity for CD117 competes with ligand binding.

[0044] E34. The multispecific binding protein of any one of E1 to E33, wherein the multispecific binding protein conditionally blocks CD47 signaling.

[0045] E35. The multispecific binding protein of any one of E1 to E34, wherein the multispecific binding protein blocks stem cell factor (SCF) signaling.

[0046] E36. The multispecific binding protein of any one of E1 to E35, wherein said binding agents can be arranged in any order.

[0047] E37. The multispecific binding protein of any one of E1 to E36, wherein said binding agents are covalently linked with a peptide linker.

[0048] E38. The multispecific binding protein of E37, wherein said peptide linker is a proline-threonine-rich peptide linker.

[0049] E39. The multispecific binding protein of E37 or E38, wherein the amino acid sequence of said peptide linker has a length from 1 to 50 amino acids.

[0050] E40. The multispecific binding protein of any one of E1 to E39, wherein any of said ankyrin repeat domains additionally comprises a G, a S or a GS at the N-terminus.

[0051] E41. The multispecific binding protein of any one of E1 to E40, wherein said multispecific binding protein further comprises a half-life extending moiety.

[0052] E42. The multispecific binding protein of E41 , wherein said half-life extending moiety is a binding agent that specifically binds to human serum albumin.

[0053] E43. The multispecific binding protein of E42, wherein said binding agent that specifically binds to human serum albumin comprises a designed ankyrin repeat domain with binding specificity for human serum albumin. E44. The multispecific binding protein of E43, wherein said ankyrin repeat domain with binding specificity for human serum albumin comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 17 to 19.

[0054] E45. The multispecific binding protein of E43 or E44, wherein said ankyrin repeat domain with binding specificity for human serum albumin is located at the C-terminus or at the N-terminus of the multispecific recombinant binding protein.

[0055] E46. The multispecific binding protein of any one of E1 to E45, wherein said multispecific recombinant binding protein comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 1 to 7.

[0056] E47. A nucleic acid encoding the recombinant binding protein of any one of E1 to E46.

[0057] E48. A vector comprising the nucleic acid of E47.

[0058] E49. The vector of E48, wherein the vector is a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector.

[0059] E50. A cell comprising the nucleic acid of E47 or the vector of any one of E48 to E49.

[0060] E51. A method of producing a multispecific binding protein, the method comprising culturing the cell of E50 and collecting the multispecific binding protein from the cell and / or the culture medium.

[0061] E52. A pharmaceutical composition comprising the multispecific binding protein of any one of E1 to E46, the nucleic acid of E47, the vector of any one of E48 to E49, or the cell of E50, and a pharmaceutically acceptable carrier and / or diluent.

[0062] E53. Use of the multispecific binding protein of any one of E1 to E46, the nucleic acid of E47, the vector of any one of E48 to E49, the cell of E50, or the pharmaceutical composition of E52 in the manufacture of a medicament.

[0063] E54. The multispecific binding protein of any one of E1 to E46, the nucleic acid of E47, the vector of any one of E48 to E49, the cell of E50, or the pharmaceutical composition of E52 for use as a medicament.

[0064] E55. A method of treatment in a subject in need thereof, the method comprising administering to said subject an effective amount of the multispecific binding protein of any one of E1 to E46, the nucleic acid of E47, the vector of any one of E48 to E49, the cell of E50, or the pharmaceutical composition of E52.

[0065] E56. A method of selectively depleting or ablating a hematopoietic stem cell (HSC) or progenitor cell population in a subject in need thereof, the method comprising administering to said subject an effective amount of the multispecific binding protein of any one of E1 to E46, the nucleic acid of E47, the vector of any one of E48 to E49, the cell of E50, or the pharmaceutical composition of E52.

[0066] E57. A method of engrafting stem cells in a subject, the method comprising: (a) administering to the subject an effective amount of the multispecific binding protein of any one of E1 to E46, the nucleic acid of E47, the vector of any one of E48 to E49, the cell of E50, or the pharmaceutical composition of E52, thereby selectively depleting or ablating hematopoietic stem cell or progenitor cell population in a target tissue of the subject; and (b) administering a stem cell population to the target tissue of the subject, wherein the administered stem cell population engrafts in the target tissue of the subject.

[0067] E59. The multispecific binding protein for use according to E54, the nucleic acid for use according to E54, the vector for use according to E54, the cell for use according to E54, or the pharmaceutical composition for use according to E54 or the methods of any one of E55 to E57, wherein the subject has a malignant, pre-malignant or non-malignant disorder.

[0068] BRIEF DESCRIPTION OF THE DRAWINGS

[0069] Figure 1 : An exemplary multispecific binding protein comprising HSA-binding agent (a-HSA), CD16a binding agent (a-CD16a), CD117 binding agent (a-CD117), 2-in-1 binding domain comprising a first binding region for CD117 (dotted) and a second binding region for the CD47-binding agent (“mask”, striped), and CD47 binding agent (a-CD47). (A) The CD117-binding agent (a-CD117) binds CD117 on a target cell, for example a hematopoietic stem cell (HSC). (B) This brings the second CD117 binding agent comprised in the 2-in-1 binding domain into close proximity to a (further) CD117 target. (C) Due to constant opening-closing of the 2-in-1 binding domain between the mask and the CD47- binding agent (indicated by the arrows), the CD117 binding region comprised in the 2-in-1 binding domain can bind to CD117. (D) On target cells, the CD117-binding agent comprised in the 2-in-1 binding domain binds CD117, and thus the CD47-binding agent cannot bind to the mask. (E) The free CD47-binding agent can now inhibit the “do-not-eat-me” signal on the HSC as the CD47-SIRPa interaction is blocked, while the CD16a-binding agent can recruit an effector cell, for example a macrophage. (F) Inhibition of the “do-not-eat-me” signal and the effector cell recruitment leads to potent elimination of target cells, for example HSCs.

[0070] Figure 2: NK cell degranulation assay. DARPin proteins at different concentrations (10 nM (hatched), 1 nM (striped), 0.1 nM (dotted) and 0 nM (black) with binding specificity for CD117 and CD16a were tested for their capacity to mediate degranulation (% of CD107a) of isolated primary NK cells (gated as live CD56+) in the presence of Kasumi-1 cells.

[0071] Figure 3: DARPin dependent cellular phagocytosis assay (DDCP). DARPin proteins at different concentrations (10 nM (hatched), 1 nM (striped), 0.1 nM (dotted) and 0 nM (black) with binding specificity for CD117 and CD16a were tested for their capacity to induce M0 macrophages (C11 b+) to phagocytose cell-tracker labelled Kasumi-1 target cells. Shown are % of double positive CD11 b+Cell Trace+ cells.

[0072] Figure 4: Results of a competitive binding ELISA showing the mutual binding exclusivity property of several 2-in-1 domains. (A) A schematic view of the experimental setup is shown. (B) Each 2-in-1 DARPin, fixed at a concentration corresponding to the BC90 against CD117 target, was exposed to a titration of competitor, i.e., a CD47-binding DARPin of SEQ ID NO: 8. Samples and dilutions of competitor were incubated for 1 h before readout. Signal detection was made through a flag tag present on the 2-in-1 domain and using an anti-flag-tag antibody. A reduction in signal is observed in a competitor dose-dependent manner. The reduction of binding between the 2-in-1 domains (fixed at the BC90) and the CD117 target resulting from the competitor presence is represented by the triangles in the left plots. Curves in the right plots shows the reduction of the signal in function of the competitor concentration.

[0073] Figure 5: Cell binding assay. Multispecific binding proteins MSC #1 (A), MSC #2 (B), MSC #3 (C), MSC #4 (D), MSC #5 (E), MSC #6 (F) to MSC# 7 (G) as well as controls (H) were added to CHO-k1 hCD117, CHO-k1 hCD47, and Kasumi-1 cells. Shown are the measured median fluorescent intensity (MFI). Curves represent a fitted four-parameters sigmoid model.

[0074] Figure 6: Cell binding competition assay. (A) A schematic view of the experimental setup is shown: DARPin proteins (1) are added to cells, followed by the addition of (2) biotinylated CD47-bindign ankyrin repeat domain, which is (3) detected by Strep-AF647. If the tested construct results in CD117- dependent release of the masking moiety, the CD47-binding ankyrin repeat domain can bind to CD47 on CD117+cells. There is therefore competition for binding between the construct and the biotinylated CD47-binding ankyrin repeat domain in a dose dependent manner (left figure). If the tested construct does not allow CD117-dependent release of the masking moiety, the CD47-binding ankyrin repeat domain cannot bind to CD47 on CD117+cells. There is therefore no competition for binding between the construct and the biotinylated CD47-binding ankyrin repeat domain (right figure). (B-l) Multispecific binding proteins MSC #1 to #7 as well as controls were added to to CHO-k1 hCD47, and Kasumi-1 cells. Shown are the measured median fluorescent intensity (MFI). Curves represent a fitted four-parameters sigmoid model.

[0075] Figure 7: SIRPIa competition on cells. Titrated amounts of (A) multispecific binding proteins as well as (B) control proteins were added to CD47 presenting ligand cells and Jurkat-SIRPIa signalling cells).

[0076] Figure 8: DARPin dependent cellular cytotoxicity assay (DDCC).(A) Multispecific binding proteins were tested in a DDCC reporter assay on Jurkat (CD477CD16+) reporter cells only (left column) as well as Kasumi-1 (CD477CD117+) target cells. Y-Axis: Relative Light Units Luminescence (RLU). (B) . NK cell mediated cytotoxicity assay of CD34+ mobilized cells from healthy human donors. Shown is specific lysis obtained upon 24 h coculture of PBMC-purified NK cells and CD34+ CD47+ CD117+ mobilized cells from healthy donors at an E:T ratio 5:1 in the presence of MSC #2 and control DARPins. Representative example of five independent experiments.

[0077] Figure 9: DARPin-dependent cellular phagocytosis assay (DDCP). (A-B) Multispecific binding proteins were tested for their capacity to induce MO macrophages (C11 b+) to phagocytose cell-tracker labelled (A) CHO-hCD47 (CD477CD117 ) and (B) Kasumi-1 (CD477CD117+) target cells. The multispecific binding protein was applied at two different concentrations (1 nM, 0.2 nM) to CHO- hCD47 target cells and at four different concentrations (1 nM, 0.2 nM, 0.04 nM, and 0.008 nM) to Kasumi-1 target cells. Shown are % of double positive CD11 b+Cell Trace+ cells. (C) Phagocytosis of pHrodo-labelled cKit-expressing AML cell line Kasumi-1 mediated by human monocyte-derived macrophages. Shown is the AUC obtained upon 48 h co-culture in presence of increasing concentrations of MSC #2, anti-CD117 antibody, magrolimab (anti-CD47 antibody) or a combination of anti-CD117 and anti-CD47 antibodies. Representative example of at least three independent experiments. (D-E) MSC #2-induced phagocytosis of pHrodo-labelled CD117- Raji (D) and CD117+ Kasumi-1 (E) cell lines at ascending concentrations of MSC #2 mediated by human monocyte-derived macrophages. The graphs show the signal obtained upon 48-h co-culture with the indicated amounts of DARPins. Representative example of at least three independent experiments.

[0078] Figure 10: Pharmacokinetic study in a mouse model showing the serum concentrations of MSC #2 (A), MSC #5 (B), MSC #6 (C) and MSC #7 (D) as a function of time after a single intravenous administration into mice (1 mg / kg). The traces indicate roughly mono-exponential elimination of the compounds.

[0079] Figure 11: In vivo experiment in a humanised mouse model. (A) Flow cytometry analysis of hCD45+cells in peripheral blood before and after treatment with multispecific binding protein #2, anti-CD117 mAb (lgG1), or a combination of anti-CD117 mAb (lgG1) and anti-CD47 (lgG4) mAb. Displayed data are calculated as in the following equation: %hCD45+=(100*%hCD45+) / (%mCD45++%hCD45+); (Mann-Whitney test ****p<0.0001 , ***p=0.007). (B-C) Flow cytometry assessment of human CD117 - cell populations in the bone marrow of hCD34 humanized NSG mice treated with multispecific binding protein #2, or anti-CD117 mAb vs vehicle. (B) Frequency of human hCD34+hCD117+with HSC signature (hCD45dimCD45RA- HLADR+) (Kruskal-Wallis test ***p=0.004). (C) Frequency of bone marrow hCD34_hCD117+cell population of total hCD45+cells. (Kruskal-Wallis test **p=0.002; ***p=0.0004).

[0080] DETAILED DESCRIPTION OF THE INVENTION

[0081] The cornerstone of treatment for hematologic and leukemic diseases often involves the transplantation of hematopoietic stem cells (HSCs) harvested from diverse sources, such as bone marrow, peripheral blood, or umbilical cord blood. HSCs, responsible for the generation of the entire spectrum of blood cells, encompass both myeloid (including monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, and dendritic cells) and lymphoid lineages (such as T lymphocytes, B lymphocytes and natural killer (NK) cells). Hematopoietic stem cells can be identified through various cell surface markers, including CD34, CD38, CD90, CD133, CD105, CD45, and CD117. Characterized by their multipotency and self-renewal capacity, HSCs can proliferate exponentially, generating a vast number of descendant HSCs. This phenomenon underpins hematopoietic stem cell transplantation (HSCT), where a limited number of transplanted HSCs is adequate to reconstitute the hematopoietic system. However, conditioning regimens are required before the actual transplantation of allogeneic (from a donor) or autologous (from the recipient) cells. The conditioning regimen ablates the recipient's immune system using radiation or chemotherapy and to eradicate the malignant cell population and minimize the risk of new immune HSC rejection. This process usually leads to partial or complete bone marrow ablation, impacting the patient's capacity to produce new blood cells. The transplantation commences as harvested stem cells are infused into the recipient's bloodstream, swiftly migrating to the bone marrow space. There, they supplant the damaged hematopoietic system, resuming the normal production of blood cells. While allogeneic HSCT presents potential complications such as infections and graft-versus-host disease, advancements in this modality have broadened its application beyond cancer treatment. Its efficacy has extended to addressing inborn errors of metabolism and autoimmune diseases, marking a significant shift in its therapeutic landscape.

[0082] The present disclosure is directed to multispecific binding proteins that can be used in potent but safer conditioning regimens.

[0083] Disclosed herein are multispecific binding proteins comprising (1) a first binding agent with binding specificity for an immunoregulatory protein, (2) a second binding agent with binding specificity for the first binding agent, and (3) a third binding agent with binding specificity for a stem cell-associated antigen, wherein binding of said second binding agent and said third binding agent is mutually exclusive. In one embodiment, said second binding agent is reversibly bound by said first binding agent. While the first binding agent is bound by the second binding agent, the first binding agent cannot bind the immunoregulatory protein (the first binding agent is “masked”). Upon binding of the third binding agent to the stem cell-associated antigen, said first binding agent is released from binding to the second binding agent (the first binding agent is “unmasked”). The first binding agent can then bind the immunoregulatory protein. In some embodiments, the multispecific binding protein further comprises a further binding agent with binding specificity for an immune cell-associated antigen. In some embodiments, the multispecific binding protein further comprises a further binding agent with binding specificity for a stem cell-associated antigen.

[0084] The disclosure is also directed to multispecific binding proteins comprising (1) a first binding agent with binding specificity for an immunoregulatory protein, (2) a second binding agent with a first binding region with binding specificity for a first target, wherein said first target is the first binding agent and a second binding region with binding specificity for a second target, wherein said second target is a stem cell-associated antigen, and wherein binding of said second binding agent to the first and second targets is mutually exclusive. Suitably, the first binding agent is reversibly bound by the first binding region of the second binding agent. While the first binding agent is bound by the first binding region of the second binding agent, the first binding agent cannot bind the immunoregulatory protein (the first binding agent is “masked”). Upon binding of the second binding region to said stem cell- associated antigen, the first binding agent is released from the first binding region of the second binding agent (the first binding agent is “unmasked”). The first binding agent can then bind the immunoregulatory protein, i.e., binding to the immunoregulatory protein is “conditional” and thus can only occur in the presence of stem cell-associated antigen. In some embodiments, the multispecific binding protein further comprises a further binding agent with binding specificity for an immune cell- associated antigen. In some embodiments, the multispecific binding protein further comprises a further binding agent with binding specificity for a stem cell-associated antigen.

[0085] In one embodiment, said second binding agent and said further binding agent with binding specificity for a stem cell-associated antigen bind to the same antigen. In one embodiment, said second binding agent and said further binding agent with binding specificity for a stem cell-associated antigen bind to a different antigen.

[0086] In one embodiment, said binding agents with binding specificity for a stem cell-associated antigen bind to the same epitope on the stem cell-associated antigen. In another embodiment, said binding agents with binding specificity for a stem cell-associated antigen bind to a different epitope on the stem cell-associated antigen. Suitably, said stem cell-associated antigen is CD117. In one embodiment, the one or both of the binding agents with binding specificity for a stem cell-associated antigen compete(s) with SCF for binding to CD117. In some embodiments, the stem cell is a hematopoietic stem cell. In some embodiments, the stem cell is a cancer stem cell (CSC), e.g., a leukemic stem cell.

[0087] In one embodiment, the immunoregulatory protein is CD47.

[0088] In one embodiment, said immune cell is an innate immune cell. Suitably, said innate immune cell is an NK cell, a monocyte or a macrophage. Suitably, said immune cell-associated antigen is CD16a.

[0089] The activating receptor CD16a, also referred to as FcyRIIIA, is present on the surface of innate immune cells, including natural killer (NK) cells, monocytes and macrophages. NK cells are cytotoxic and produce immune-signaling molecules such as IFN-y and TNF-a. Upon engagement, CD16a initiates a potent signaling cascade in NK cells, culminating in cytokine production and the execution of cytotoxic activities through a mechanism known as antibody-dependent cellular cytotoxicity (ADCC). In macrophages, CD16a serves as a critical receptor involved in antibody-dependent cellular phagocytosis (ADCP). Upon binding of CD16a receptors on monocytes and macrophages, a cascade of intracellular signaling events is initiated, triggering the activation of phagocytic mechanisms. This activation leads to the engulfment of the target cell and their subsequent degradation within the macrophage. CD47 blockade and activation of CD16a synergize to enhance phagocytosis by macrophages and monocytes. CD47 blockade removes the "don't-eat-me" signal expressed on target cells, making these more susceptible to phagocytosis. Meanwhile, CD16a acts as an activating receptor, initiating phagocytic and / or cytotoxic processes. When both mechanisms are engaged, CD47 blockade eliminates the inhibitory signal, while CD16a activation amplifies the phagocytic and / or cytotoxic response, leading to increased clearance of target cells by enhancing the ability of phagocytes to recognize and engulf these cells and by enhancing cytotoxicity.

[0090] The multispecific binding proteins described herein may be employed in a conditioning regimen, whereby endogenous hematopoietic stem cells are ablated selectively to facilitate the engraftment of exogenous hematopoietic stem cells, while avoiding systemic toxicities associated with conventional conditioning agents.

[0091] Stem cells, e.g., haematopoetic stem cells, are selectively targeted by the multispecific binding proteins disclosed herein. The binding agent with binding specificity for a stem-cell associated antigen, e.g., CD117, anchors the multispecific protein to the stem cell, e.g., a hematopoietic stem cell and / or leukemic stem cell. In one embodiment, binding of the multispecific binding protein to CD117 blocks stem cell factor (SCF) signaling. Upon binding of the multispecific binding protein, the first binding agent with binding specificity for an immunoregulatory protein is unmasked. Once unmasked, the multispecific binding protein can bind the immunoregulatory protein. Suitably, the immunoregulatory protein is CD47. Upon binding to CD47, the interaction between CD47 and its target ligands, e.g., SIRPa, is blocked, and the “don’t-eat-me” signal is suppressed. Suppression of this signal sensitizes the cell, suitably hematopoietic stem cell and / or cancer stem cell, to detection by cells of the innate immune system, e.g., monocytes, macrophages and / or NK cells. In the absence of a stem-cell associated antigen, e.g., CD117, the immunoregulatory protein is masked, thereby avoiding blocking CD47 on non-target cells. Unmasking only occurs in the presence of a stem cell marker, suitably CD117. Suitably, the multispecific binding proteins comprise a further binding agent with binding specificity for an immune cell. In one embodiment, said binding agent binds to CD16a. Binding of the multispecific binding protein to CD16a engages macrophages and / or NK cells via the activating FcyRllla receptor, while simultaneously avoiding triggering inhibitory FcyRllb receptor. The conditional CD47 blockade on CD117-positive target cells allows the use of strong receptor activation of innate immune cells while avoiding systemic toxicity associated with untargeted anti-CD47 agents. In some embodiments, said first, second, third and / or further binding agents are an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain. In a preferred embodiment, the binding agents comprise designed repeat domains, suitably ankyrin repeat domains. Designed ankyrin repeat protein libraries (W02002 / 020565; Binz et al., Nat. Biotechnol. 22, 575-582, 2004; Stumpp et al., Drug Discov. Today 13, 695-701 , 2008) can be used for the selection of targetspecific designed ankyrin repeat domains that bind to their target with high affinity and / or high avidity. Such target-specific designed ankyrin repeat domains in turn can be used as valuable components of multispecific binding proteins for the treatment of diseases. Designed ankyrin repeat proteins are a class of binding molecules which have the potential to overcome limitations of monoclonal antibodies, hence allowing novel therapeutic approaches. Such ankyrin repeat proteins may comprise a single designed ankyrin repeat domain or may comprise a combination of two or more designed ankyrin repeat domains with the same or different target specificities (Stumpp et al., Drug Discov. Today 13, 695-701 , 2008; U.S. Patent No. 9,458,211). Ankyrin repeat proteins comprising only a single designed ankyrin repeat domain are small proteins (14 kDa) which can be selected to bind a given target protein with high affinity and specificity. These characteristics, and the possibility of combining two or more designed ankyrin repeat domains in one protein, make designed ankyrin repeat proteins ideal agonistic, antagonistic and / or inhibitory drug candidates.

[0092] In another aspect, provided is a multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for CD47, (2) a second ankyrin repeat domain with binding specificity for the CD47-binding ankyrin repeat domain, (3) a third ankyrin repeat domain with binding specificity for CD16a, wherein binding of said second ankyrin repeat domain and said third ankyrin repeat domain is mutually exclusive, and (4) a fourth ankyrin repeat domain with binding specificity for CD117. In a more specific embodiment, the multispecific binding protein may comprise, instead of the second ankyrin repeat domain with binding specificity for the CD47-binding ankyrin repeat domain and the fourth ankyrin repeat domain with binding specificity for CD117, a single ankyrin repeat domain with a first binding specificity for the CD47-binding ankyrin repeat domain and a second binding specificity for CD117, wherein binding to CD47-binding ankyrin repeat domain and CD117 is mutually exclusive.

[0093] Accordingly, disclosed herein are multispecific binding proteins comprising (1) a first ankyrin repeat domain with binding specificity for CD47, (2) a second ankyrin repeat domain with a first binding specificity for the CD47-binding ankyrin repeat domain and a second binding specificity for CD117, wherein binding to the CD47-binding ankyrin repeat domain and CD117 is mutually exclusive, (3) a third ankyrin repeat domain with binding specificity for CD16a, optionally (4) a fourth ankyrin repeat domain with binding specificity for CD117.

[0094] In one aspect, provided is a multispecific binding proteins comprising (1) a first ankyrin repeat domain with binding specificity for CD47, (2) a second ankyrin repeat domain with a first binding specificity for the CD47-binding ankyrin repeat domain and a second binding specificity for CD117, wherein binding to the CD47-binding ankyrin repeat domain and CD117 is mutually exclusive, (3) a third ankyrin repeat domain with binding specificity for CD16a, and (4) a fourth ankyrin repeat domain with binding specificity for CD117.

[0095] Definitions

[0096] 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.

[0097] 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.

[0098] 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.

[0099] As used herein, the articles "a" and "an” can mean “one”, but their use is also consistent with the meaning of “one or more”, “at least one” and “one or more than one”.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] “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”. It is also 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.

[0104] The term “binding agent” or “binding moiety” refers to any molecule capable of binding a target molecule. Binding agents include, for example, antibodies, antibody fragments, aptamers, peptides (e.g., Williams et al., J Biol Chem 266:5182-5190 (1991)), alternative scaffolds, antibody mimics, repeat proteins, e.g., designed ankyrin repeat proteins, receptor proteins and any other naturally occurring interaction partners of the target molecule, and can comprise natural proteins and proteins modified or genetically engineered, e.g., to include non-natural residues and / or to lack natural residues.

[0105] As used herein, the term “multispecific binding protein”, refers to a binding protein with the ability to bind specifically to multiple targets or antigens. A multispecific binding protein can recognize and bind to distinct epitopes or molecules, suitably simultaneously, thereby allowing it to interact with and potentially modulate multiple biological targets. Preferably, the multispecific binding proteins are recombinant multispecific binding proteins. The multispecific binding proteins described herein bind to at least one marker present on stem cells and / or malignant cells and at least one immunoregulatory molecule. Suitably, the multispecific binding proteins are also directed to an activating molecule present on effector cells that include, but are not limited to, NK cells, monocytes and / or macrophages. Any combination of said markers, immunoregulatory molecules and activating molecules are comprised within the present disclosure.

[0106] An “immunoregulatory molecule” refers to a type of molecule, e.g., a protein, which plays a role in modulating or regulating the immune response within the body, e.g., by controlling the activation, proliferation, differentiation, and / or function of immune cells.

[0107] By a “stem cell-associated antigen” we include the meaning that a protein is (specifically) expressed by the stem cell, and may be associated with the cell such that one or more region of the ligand is present on an outer face of the cell surface. For example, the ligand may be inserted into the cell plasma membrane (i.e., orientated as a transmembrane protein) with one or more region presented on the extracellular surface. Alternatively, the entire ligand may be outside the cell with covalent and / or ionic interactions localizing it to a specific region or regions of the cell surface. The stem cell may be a cancer stem cell and / or a normal (non-cancer) stem cell.

[0108] As used herein, the term “target” 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. Preferably, a target is a naturally occurring or nonnatural polypeptide or protein, or a polypeptide or protein containing chemical modifications, for example, naturally occurring or non-natural phosphorylation, acetylation, or methylation.

[0109] “CD16a”, also known as FcyRllla (Fc gamma receptor Illa), is a glycoprotein receptor primarily found on the surface of certain immune cells, notably natural killer (NK) cells, macrophages, and monocytes. This receptor plays a pivotal role in mediating the effector functions of these immune cells upon engagement with the Fc portion of immunoglobulin G (IgG) antibodies. CD16a is involved in antibody-dependent cellular cytotoxicity (ADCC) (also referred to herein as DARPin-dependent cellular cytotoxicity (DDCC), an immune mechanism where immune cells, upon binding to IgG-coated target cells, exert cytotoxicity, leading to the elimination of the targeted cells. The engagement of CD16a with the Fc region of IgG antibodies triggers cellular activation, leading to various immune responses such as target cell lysis, cytokine release, and phagocytosis (antibody-dependent cellular phagocytosis or “ADCP”, also referred to herein as DARPin-dependent cellular phagocytosis (DDCP)), depending on the cell type expressing CD16a. CD16a is therefore an activating molecule present on effector cells, and binding agents that can activate such effector cells are therefore “capable of stimulating effector cells”. CD16a is an example of an “immune cell-associated antigen”. The amino acid sequence of human CD16a (hCD16a) is referenced as UniProt Ref. No. P08637.

[0110] Ankyrin repeat domains specifically binding CD16a are disclosed in WO2024 / 251628 (incorporated by reference).

[0111] The term “CD47” refers to a transmembrane polypeptide, which belongs to the immunoglobulin superfamily. Human CD47 (Cluster of Differentiation 47) is identified by UniProt Ref. No. Q08722 and is also known as integrin associated protein (IAP), OA3 or MER6. CD47 partners with membrane integrins and also binds ligands including signal-regulatory protein alpha (SIRPalpha). CD47 functions as a marker of “self and transmits a "don't-eat-me" signal by binding to SIRPalpha expressed by myeloid cells, macrophages, dendritic cells and neutrophils. In this context, the role of CD47 is to prevent the engulfment (phagocytosis) of healthy cells by these immune cells. CD47 is therefore an “immunoregulatory molecule”. Ankyrin repeat domains specifically binding CD47 are disclosed in WO2024 / 251695 (incorporated by reference).

[0112] The term “CD117”, also known as c-Kit or stem cell factor receptor (SCFR), refers to a transmembrane glycoprotein receptor belonging to the receptor tyrosine kinase (RTK) family identified with UniProt Ref. No. P10721-2. It is encoded by the KIT gene. CD117 plays a pivotal role in various biological processes, particularly in hematopoiesis (the formation of blood cells) and in the regulation of proliferation, survival, and differentiation of hematopoietic stem cells and progenitor cells. This receptor is characterized by its extracellular ligand-binding domain, a transmembrane region, and an intracellular tyrosine kinase domain. Upon binding to its ligand, stem cell factor (SCF), CD117 undergoes dimerization and autophosphorylation, initiating downstream signaling cascades that regulate cell growth, differentiation, and survival. CD117 is primarily expressed on hematopoietic stem cells and some cancer stem cells. It is therefore a “stem-cell associated antigen”. Ankyrin repeat domains specifically binding CD117 are disclosed in EP24150551.0 (incorporated by reference).

[0113] The term “mutual exclusivity” or “mutually exclusive” e.g., as used in “mutually exclusive binding” refers to the property of the binding agent to bind a first target and a second target only in a substantially non-simultaneous manner. In such a mutually exclusive binding, binding of said first target influences binding of said second target (and / or vice versa) such that simultaneous binding of said first and second targets is substantially prevented. Underlying causes for this effect include steric hindrance and / or an overlap of paratopes. In the context of the present disclosure, a binding interaction is considered to be mutually exclusive if, when bound to one of said first and second targets, said binding agent cannot bind more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45% or more than 50% of the molar equivalent of the other target. The extent of such mutual exclusivity can be measured by competitive binding assays in which binding of the first target by the binding agent is compared to binding of the second target.

[0114] Such a competitive binding assay may be adapted from standard competitive binding assays used in the field of antibodies, as known by the person skilled in the art. An example of a competitive binding assay is shown in Example 5.

[0115] 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 of cysteines.

[0116] Furthermore, the term “peptide” also encompasses peptides modified by, e.g., glycosylation, and proteins comprising two or more polypeptide chains, each of length of 4 to 600 amino acids long, cross-linked by, e.g., disulfide bonds, such as, e.g., insulin and immunoglobulins.

[0117] 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”.

[0118] The term “recombinant” as used in “recombinant protein”, “recombinant polypeptide” and the like, means that said protein or polypeptide is produced by the use of recombinant DNA technologies known to the practitioner skilled in the art. 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, QIAgen), 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”.

[0119] 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 (e.g., DM1), small molecules, antibiotics and alike) known to the person skilled in the art.

[0120] The term “binding domain” refers to a protein domain with binding specificity for a target. Preferably, said binding domain is a recombinant binding domain. Patent application W02002 / 020565 and Forrer et al., (FEBS Letters 539, 2-6, 2003)), contain a general description of repeat protein features and repeat domain features, techniques and applications. 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.

[0121] 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 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 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).

[0122] 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). Suitably, an ankyrin repeat domain comprises an N-terminal capping module, a C-terminal capping module, and at least one internal repeat module. Capping modules are located at the N-and / or C-terminal end of an ankyrin repeat domain, typically forming tight tertiary interactions (i.e., tertiary structure interactions) with the ankyrin repeat module(s) in between, thereby providing a cap that shields the hydrophobic core of the ankyrin repeat domain at the side from exposure to the solvent. The N-and / or C-terminal capping modules may be derived from a capping unit or other structural unit found in a naturally occurring repeat protein adjacent to a repeat unit. Examples of capping sequences are described in International Patent Publication Nos. WO 2002 / 020565 and WO 2012 / 069655, in U.S. Patent Publication No. US2013 / 0296221 , and by Interlandi et al., J Mol Biol. 2008 Jan 18;375(3):837-54.

[0123] The term “construct” as used herein refers to a binding protein comprising one or more designed ankyrin repeat domain and optionally a peptide linker and / or tag sequence. An example of a peptide linker is provided in SEQ ID NO: 21 and an example of a tag sequence is provided in SEQ ID NO: 20.

[0124] 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. The multispecific binding proteins disclosed herein are designed repeat proteins and they comprise at least one designed ankyrin repeat domain. Preferably, the designed repeat domain is a designed ankyrin repeat domain. All of the amino acid sequences described herein may be substituted by one or more amino acids, suitably up to 15, up to 14, up to 13, up to 12, up to 11 , up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 substitution or deletion is made in any of the amino acid sequences described herein. Suitably, the amino acid substitution^) or deletions) are all made in framework residues. Suitably, the amino acid substitution(s) or deletions) are all made in non-randomized positions. The location of randomized positions in a designed ankyrin repeat domain is disclosed, e.g., in Binz et al., Nature Biotech. 22(5): 575-582 (2004). For the purposes of the present disclosure, a substitution in framework residues shall apply to all embodiments irrespective of whether such substitution is explicitly described.

[0125] The term “framework residue(s)” refers to amino acid residues of a repeat domain, which contribute to the folding topology, i.e. which contribute to the folding of said ankyrin repeat domain or which contribute to the interaction with a neighboring module. Such contribution may be the interaction with other residues in the repeat domain, or the influence on the polypeptide backbone conformation as found in a-helices or p-sheets, or the participation in amino acid stretches forming linear polypeptides or loops. Such framework and target interaction residues may be identified by analysis of the structural data obtained by physicochemical methods, such as X-ray crystallography, NMR and / or CD spectroscopy, or by comparison with known and related structural information.

[0126] The multispecific binding protein may be a variant of a multispecific binding protein described herein, provided such a variant retains binding specificity for the ligands of the parent multispecific binding protein. Variants may be made using the methods of protein engineering and site-directed mutagenesis known in the art using the recombinant polynucleotides (see example, see Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2001 , Cold Spring Harbor Laboratory Press, which is incorporated herein by reference). “Variants” of the multispecific binding protein includes insertions, deletions and substitutions, either conservative or non-conservative. Included are variants of the sequence of the multispecific binding protein where such variations do not substantially alter the activity and / or binding specificity for the ligands of the multispecific binding protein.

[0127] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), betabranched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0128] In addition, the second last position of any ankyrin repeat domain described herein can be “A” or“L”, and / or the last position can be “A” or“N”. Furthermore, each ankyrin repeat domain described herein may optionally comprise a “G,” an “S,” or a “GS” sequence at its N-terminus.

[0129] 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 about 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 about 80% of the positions are identical, the test sequence is considered at least about 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 such as 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.

[0130] Individual binding domains may be linked covalently with a peptide linker. Suitably, the linker is of sufficient length to enable the domains to fold in such a way as to permit binding of the multispecific binding protein to its target(s). A suitable linker may comprise from 1 to 50 amino acids, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 amino acids, suitably from 6 to 38 amino acids. Examples of peptide linkers are glycine-serine-linkers and proline-threonine rich linkers of variable lengths. An example of a suitable PT-rich linker in the context of the present disclosure comprises the amino acid sequence of SEQ ID NO: 21 or of SEQ ID NO: 25.

[0131] The terms “binding specificity”, “has binding specificity for a target”, “specifically binding to a target”, “binding to a target with high specificity”, “specific for a target”, “target specificity”, or “specifically binds” 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. Binding of any molecule to another is governed by two forces, namely the association rate (kon) and the dissociation rate (kOff). The affinity of any binder [B] to a target [T] can then be expressed by the equilibrium dissociation constant KD, which is the quotient of kotf / kon. kon is a second-order rate constant of the binding reaction, with the unit whereas the dissociation reaction koff is a first-order rate constant with the unit s~1. From this it becomes clear that the association reaction depends on the concentration of the reactants, whereas the dissociation is independent of the concentration, following a simple exponential decay function.

[0132] A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure. For example, as exemplified herein, the binding affinity of a particular binding moiety to a drug molecule target can be expressed as the KD value, which refers to the dissociation constant of the binding moiety and the drug molecule target. KD is the ratio of the rate of dissociation, also called the “off-rate (koff)”, to the association rate, or “on-rate (kon)”. Thus, KD equals k0ff / k0nand is expressed as a molar concentration (M), and the smaller the KD, the stronger the affinity of binding.

[0133] KD values can be determined using any suitable method. One exemplary method for measuring KD is surface plasmon resonance (SPR) (see, e.g., Nguyen et al. Sensors (Basel). 2015 May 5;

[0134] 15(5):10481 -510). KD value may be measured by SPR using a biosensor system such as a BIACORE® system. BIAcore kinetic analysis comprises, e.g., analyzing the binding and dissociation of an antigen from chips with immobilized molecules (e.g., molecules comprising epitope binding domains), on their surface. Another method for determining the KD of a protein is by using Bio-Layer Interferometry (see, e.g., Shah et al. J Vis Exp. 2014; (84): 51383). A KD value may be measured using OCTET® technology (Octet QKe system, ForteBio). Alternatively, or in addition, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Id.) can also be used. Any method suitable for assessing the binding affinity between two binding partners is encompassed herein.

[0135] The term “biparatopic binding agent” refers to a binding agent, suitably a binding protein, directed against two different epitopes located on the same target protein, e.g., located on human CD117. For example, a biparatopic binding protein targeting CD117 may comprise at least a first binding domain targeting a first epitope on CD117 and a second binding domain targeting a different epitope on CD117. Suitably, said first and second binding domains compete with SCF binding to CD117. Suitably, only one binding domain competes with SCF binding to CD117.

[0136] The term “bivalent binding agent” refers to a binding agent, suitably a binding protein, directed against two identical epitopes located on the same target protein, e.g., located on human CD117. For example, a bivalent binding protein targeting CD117 may comprise at least a first binding domain targeting a first epitope on CD117 and a second binding domain targeting the same epitope on CD117. Suitably, said first and second binding domains compete with SCF binding to CD117. Suitably, said first and second binding domains do not compete with SCF binding to CD117.

[0137] There are a number of known competition binding assays that can be used to assess competition of the binding agent with a ligand for binding to a particular protein, in this case, CD117 (e.g., human CD117 protein). Suitably, such an assay involves the use of cells bearing the ligand for a particular protein, labelled ligand and the binding protein to be tested for competition. Competitive inhibition is measured by determining the amount of label bound to the cells in the presence of the test protein. Binding proteins identified by competition assay (competing binding proteins) include those binding to the same epitope as the ligand and those binding to an adjacent epitope sufficiently proximal to the epitope bound by the ligand for steric hindrance to occur. An example of a competitive binding assay is shown in Example 3.

[0138] As used herein, the terms “increase” or “enhance” (and grammatical variations thereof) describe an elevation of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control.

[0139] As used herein, the terms “reduce” and “decrease” (and grammatical variations thereof), describe, for example, a decrease of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97%o, 98%o, 99%, or 100% as compared to a control. In particular embodiments, the reduction results in no or essentially no (i.e., an insignificant amount, e.g., less than about 10% or 5%) detectable activity or amount.

[0140] As used herein, the term “stem cell” refers to an undifferentiated biological cell, which can self-renew while also differentiating into more specialized cells. One type of stem cell is the hematopoietic stem cell (HSC) which gives rise to blood cells of myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (T-cells, B-cells, NK-cells). Hematopoietic stem cells give rise to both mature blood cells and a distinct intermediate stage called “progenitor cells”. These progenitor cells, also known as precursor cells, are partially differentiated and are committed to a specific lineage, but they still retain the capability to further differentiate into various mature blood cell types.

[0141] As used herein, the term “cancer stem cell” (CSC) is referring to cancer cells, found within e.g., tumors or hematological cancers, that possess characteristics associated with normal stem cells, specifically the ability to give rise to all cell types found in a particular cancer sample. CSCs may generate tumors through self-renewal as well as differentiation into multiple cell types. Such cells are proposed to persist in tumors as a distinct population and cause relapse and metastasis by giving rise to new tumors.

[0142] As used herein, the terms “bone marrow transplantation” or “stem cell transplantation” refer to the transplantation of stem cells to a recipient. The stem cells do not necessarily have to be derived from bone marrow but could also be derived from other sources such as umbilical cord blood. As used herein, the term “HSCT” is referring to a transplantation of hematopoietic stem cells (HSCs) to a recipient, wherein the stem cells usually are collected from bone marrow, peripheral blood, or umbilical cord blood.

[0143] As used herein, the term “first transplantation” is referring to a transplantation given to a subject for the treatment of a condition, who has not received such transplantation before, or who has not received such transplantation for the treatment of said condition before. The term "re-transplantation" is considering a transplantation given to a subject who has already received such transplantation one or several times before.

[0144] As used herein, the term “recipient” in the context of transplantation is referring to the subject receiving the transplantation, in contrast to the “donor”, which is the subject donating the material (cells) to be transplanted. In an allogeneic setting, the recipient and the donor are different individuals, in an autologous setting, the recipient and the donor is the same individual. In a syngeneic setting, the donor and recipient are different individuals but are genetically identical. Xenogeneic setting means the donor is from another species than the recipient.

[0145] As used herein, the term “pre-treatment” refers to a treatment that is performed prior to another treatment or therapy. This is also referred to herein as a “conditioning regimen” or “conditioning”. Its primary purpose is twofold: Firstly, the conditioning regimen aims to eliminate or suppress the recipient's existing bone marrow or immune system, especially in cases of malignancies like leukemia or lymphoma. By doing so, it creates space within the recipient's body for donor stem cells to engraft. Secondly, the conditioning regimen helps reduce the recipient's immune response, making it more receptive to the donor stem cells. This step lowers the chances of rejection, where the recipient's immune system might attack the stem cells as foreign entities. In a particular embodiment, pretreatment is performed prior to stem cell transplantation.

[0146] The terms “subject”, “patient”, “subject in need thereof, and “patient in need thereof are used interchangeably herein and refer to a human suffering from one or more of the diseases described herein (e.g., cancer). A subject is “in need of a treatment if such subject would benefit biologically, medically and / or in quality of life from such treatment.

[0147] The term “dose” refers to a specified amount of a therapeutic agent (drug), e.g. the multispecific binding protein described herein, administered to the subject in need thereof on the particular treatment day. The dose would, for example, be declared on a product package or in a product information leaflet.

[0148] 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, e.g. the multispecific binding protein disclosed herein.

[0149] The terms “cancer” and “cancerous” are used herein to refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancer encompasses solid tumors and liquid tumors, as well as primary tumors and metastases. A “tumor” comprises one or more cancerous cells. Solid tumors typically also comprise tumor stroma.

[0150] The term “malignant cell” includes a cell which is capable of, or exhibits, uncontrolled cellular division and / or proliferation and / or the ability to metastasize and / or invade tissues in a body. Such cells may comprise cancerous tumors and are frequently resistant to many anti-proliferative therapies.

[0151] Throughout the present disclosure, the order or orientation with regard to the “first” and “second” entity, as used for instance in first and second targets, first and second repeat domains, first and second binding specificities, etc. is not meant to specify one specific order or orientation and encompasses the alternative, unless specifically defined. In other words, any reference to such a first and second entity also encompasses the alternative order where the first entity corresponds to the second one and the second entity corresponds to the first one, unless specifically defined otherwise. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description herein. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.

[0152] CD47-binding ankyrin repeat domains

[0153] CD47, also known as integrin-associated protein, is a transmembrane protein ubiquitously expressed on the surface of various cells in the body. It plays a crucial role in immune regulation and cell signaling by interacting with its receptor, signal-regulatory protein alpha (SIRPa), which is predominantly found on macrophages and other phagocytic cells. CD47 acts as a "don't eat me" signal, inhibiting phagocytosis by sending a signal that prevents these immune cells from engulfing cells expressing CD47.

[0154] The generation and characterization of recombinant binding proteins comprising ankyrin repeat domains with binding specificity for CD47 are described in patent application with number WO2024 / 251695, which is incorporated by reference in its entirety. These binding proteins have been shown to block the interaction of CD47 / SIRPa, thereby disrupting the "don’t eat me signal".

[0155] In one embodiment, said ankyrin repeat domain with binding specificity for CD47 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 to SEQ ID NO: 8. In one embodiment, said ankyrin repeat domain with binding specificity for CD47 comprises or consists of the amino acid sequence of SEQ ID NO: 8. In one embodiment, said ankyrin repeat domain with binding specificity for CD47 binds human soluble CD47 in PBS with a dissociation constant (KD) of or below about 10-7M, or of or below about 10-8M, or of or below about 10-9M, or of or below about 1 O-10M, or of or below about 10-11M. Suitably, the dissociation constant is determined by surface plasmon resonance (SPR), suitably in PBS, e.g. as described in Example 1 .

[0156] In one embodiment, said ankyrin repeat domain with binding specificity for CD47 is capable of blocking the interaction of CD47 with signal-regulatory protein alpha (SIRPa). In one embodiment, said ankyrin repeat domain with binding specificity for CD47 blocks or reduces the interaction of CD47 with signal-regulatory protein alpha (SIRPa). A suitable assay to assess whether said ankyrin repeat domain with binding specificity for CD47 is capable of blocking the interaction of CD47 with signal- regulatory protein alpha (SIRPa) is described in Example 1. In one embodiment, said ankyrin repeat domain with binding specificity for CD47 inhibits ligand binding with an IC50 of at most about 500 nM or less, at most about 400 nM or less, at most about 300 nM or less, at most about 200 nM or less, at most about 100 nM or less, at most about 90 nM or less, at most about 80 nM or less, at most about 70 nM or less, at most about 60 nM or less, at most about 50 nM or less. In one embodiment, said ankyrin repeat domain with binding specificity for CD47 reduces the interaction of CD47 with signal- regulatory protein alpha (SIRPa) by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% compared to a binding protein, suitably an ankyrin repeat domain, which does not have binding specificity for CD47.

[0157] CD16a-binding ankyrin repeat domains

[0158] CD16a, also known as FcyRllla, is a receptor expressed primarily on immune cells such as natural killer (NK) cells, macrophages, and neutrophils. This receptor plays a pivotal role in antibodydependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Upon engagement, CD16a triggers signaling cascades within immune cells, leading to the activation of various effector functions. For NK cells, it initiates cytotoxic activities against target cells, releasing cytotoxic granules and inducing cell death. In macrophages and neutrophils, CD16a activation stimulates phagocytosis, enabling these cells to engulf and eliminate target cells.

[0159] The generation and characterization of recombinant binding proteins comprising ankyrin repeat domains with binding specificity for CD16a are described in patent application with number WO2024 / 251628, which is incorporated by reference in its entirety. These binding proteins have been shown to stimulate effector cells.

[0160] In one embodiment, said ankyrin repeat domain with binding specificity for CD16a 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 to SEQ ID NO: 9. In one embodiment, said ankyrin repeat domain with binding specificity for CD16a comprises or consists of the amino acid sequence of SEQ ID NO: 9. In one embodiment, said ankyrin repeat domain with binding specificity for CD16a binds human soluble CD16a in PBS with a dissociation constant (KD) of or below about 10-7M, or of or below about 10-8M, or of or below about 10-9M, or of or below about 10-10M, or of or below about 10-11M. Suitably, the dissociation constant is determined by surface plasmon resonance (SPR), suitably in PBS, e.g. as described in Example 2.

[0161] In one embodiment, said ankyrin repeat domain with binding specificity for CD16a is capable of stimulating effector cells, suitably innate immune cells, such as NK cells, monocytes and macrophages. A suitable assay to assess whether said ankyrin repeat domain with binding specificity for CD16a is capable of stimulating effector cells is described in Example 4. In one embodiment, said ankyrin repeat domain with binding specificity for CD16a stimulates effector cells with an EC50 of at most about 200 pM or less, at most about 150 pM or less, at most about 100 pM or less, at most about 50 pM or less.

[0162] CD117-binding ankyrin repeat proteins CD117, also known as c-kit or stem cell factor receptor (SCFR), is a transmembrane tyrosine kinase receptor protein encoded by the KIT gene. It is primarily expressed on the surface of hematopoietic stem cells, mast cells, and progenitor cells in various tissues. CD117 plays a pivotal role in cell survival, proliferation, and differentiation. When its ligand, stem cell factor (SCF), binds to CD117, it triggers dimerization and activation of the receptor, leading to the activation of downstream signaling pathways such as Ras / MAPK and PI3K / Akt. These pathways regulate crucial cellular functions such as hematopoiesis, melanogenesis, gametogenesis, tissue mast cell development, and stem cell function.

[0163] The generation and characterization of recombinant binding proteins comprising ankyrin repeat domains with binding specificity for CD117 are described in patent application with number EP24150551 .0, which is incorporated by reference in its entirety.

[0164] In one embodiment, said ankyrin repeat domain with binding specificity for CD117 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 to any one of SEQ ID NOs: 10, 11 , 26 to 29.

[0165] In one embodiment, said ankyrin repeat domain with binding specificity for CD117 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 to SEQ ID NO: 10. In one embodiment, said ankyrin repeat domain with binding specificity for CD117 comprises or consists of the amino acid sequence of SEQ ID NO:

[0166] 10.

[0167] In one embodiment, said ankyrin repeat domain with binding specificity for CD117 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 to SEQ ID NO: 11 . In one embodiment, said ankyrin repeat domain with binding specificity for CD117 comprises or consists of the amino acid sequence of SEQ ID NO:

[0168] 11.

[0169] In one embodiment, said ankyrin repeat domain with binding specificity for CD117 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 to SEQ ID NO: 26. In one embodiment, said ankyrin repeat domain with binding specificity for CD117 comprises or consists of the amino acid sequence of SEQ ID NO: 26.

[0170] In one embodiment, said ankyrin repeat domain with binding specificity for CD117 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 to SEQ ID NO: 27. In one embodiment, said ankyrin repeat domain with binding specificity for CD117 comprises or consists of the amino acid sequence of SEQ ID NO:

[0171] 27.

[0172] In one embodiment, said ankyrin repeat domain with binding specificity for CD117 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 to SEQ ID NO: 28. In one embodiment, said ankyrin repeat domain with binding specificity for CD117 comprises or consists of the amino acid sequence of SEQ ID NO:

[0173] 28.

[0174] In one embodiment, said ankyrin repeat domain with binding specificity for CD117 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 to SEQ ID NO: 29. In one embodiment, said ankyrin repeat domain with binding specificity for CD117 comprises or consists of the amino acid sequence of SEQ ID NO:

[0175] 29.

[0176] In one embodiment, said ankyrin repeat domain with binding specificity for CD117 is capable of competing for binding with stem cell factor (SCF). In one embodiment, said ankyrin repeat domain with binding specificity for CD117 competes for binding with stem cell factor (SCF). In one embodiment, said ankyrin repeat domain with binding specificity for CD117 does not compete for binding with stem cell factor (SCF).

[0177] A suitable assay to assess whether said ankyrin repeat domain with binding specificity for CD117 is capable of competing with SCF is described in Example 6. In one embodiment, said ankyrin repeat domain with binding specificity for CD117 inhibits ligand binding with an IC50 of at most about 5 nM or less, at most about 4 nM or less, at most about 3 nM or less, at most about 2 nM or less, at most about 1 nM or less, at most about 0.9 nM or less, at most about 0.8 nM or less, at most about 0.7 nM or less, at most about 0.6 nM or less, at most about 0.5 nM or less.

[0178] In one embodiment, said ankyrin repeat domain with binding specificity for CD117 binds human soluble CD117 in PBS with a dissociation constant (KD) of or below about 10-7M, or of or below about 10-8M, or of or below about 10-9M, or of or below about 1 O-10M, or of or below about 10-11M. Suitably, the dissociation constant is determined by surface plasmon resonance (SPR), suitably in PBS, e.g. as described in Example 3.

[0179] Fusion ankyrin repeat domain specific for CD117 and CD47-binding ankyrin repeat domain

[0180] Designed ankyrin repeat domains with mutually exclusive binding specificity for two targets have been described (see WO2023110983). Such dual-specific repeat domains (also referred to as “2-in-1 domains”) can be created by combining repeat modules of two parental repeat domains. The binding specificity to the respective targets of the parental repeat domains therefore confers the binding specificity for each of the two targets. Such dual-specific repeat domains with a first binding specificity for CD117 and a second binding specificity for CD47-binding DARPins have been described in EP24150569.2.

[0181] In one embodiment, the multispecific binding protein described herein comprises an ankyrin repeat domain with a first binding region with binding specificity for a first target and a second binding region with binding specificity for a second target, wherein binding of said ankyrin repeat domain to the first and second targets is mutually exclusive. In one embodiment, said ankyrin repeat domain comprises a sensor part and a masking part, wherein said sensor part corresponds to one binding region and said masking part corresponds to the other binding region of the ankyrin repeat domain. In such constructs, said ankyrin repeat domain has a first binding specificity for a sensor target and a second binding specificity for a masking target.

[0182] In one embodiment, said first target is an ankyrin repeat domain with binding specificity for CD47, e.g., as described herein, and said second target is CD117. In one embodiment, said sensor target is CD117, and said masking target is an ankyrin repeat domain with binding specificity for CD47, e.g., as described herein.

[0183] In one embodiment, said ankyrin repeat domain comprises a first binding region with binding specificity for a CD47-binding ankyrin repeat domain and a second binding region with binding specificity for CD117, wherein binding of said ankyrin repeat domain comprising first and second binding regions to the first and second targets is mutually exclusive. Said ankyrin repeat domain binds the first target, i.e., the CD47-binding ankyrin repeat domain (“masking target”) and blocks its activity in the absence of CD117 (“sensor target”). Upon binding to CD117, steric hindrance induces the release of the CD47-binding ankyrin repeat domain, enabling binding of this ankyrin repeat domain to its target, thereby blocking the “don’t-eat-me signal”. In some embodiments, the affinity of said ankyrin repeat domain for the sensor target is higher than the affinity of said ankyrin repeat domain for the masking target. In any of the embodiments and aspects herein, the first binding region with binding specificity for a CD47-binding ankyrin repeat domain may be located N-terminally of the second binding region with binding specificity for CD117, or the first binding region with binding specificity for a CD47-binding ankyrin repeat domain may be located C-terminally of the second binding region with binding specificity for CD117 within said ankyrin repeat domain.

[0184] In one embodiment, said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, 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 to any one of SEQ

[0188] ID NOs: 12 to 16. In one embodiment, said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprises or consists of an amino acid sequence of any one of SEQ ID NO: 12 to 16.

[0189] In one embodiment, said ankyrin repeat domain 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

[0190] 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about

[0191] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0192] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 12.

[0193] In one embodiment, said ankyrin repeat domain 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

[0194] 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about

[0195] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0196] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 13.

[0197] In one embodiment, said ankyrin repeat domain 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

[0198] 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about

[0199] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0200] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 14.

[0201] In one embodiment, said ankyrin repeat domain 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

[0202] 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about

[0203] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0204] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 15.

[0205] In one embodiment, said ankyrin repeat domain comprises an amino acid sequence at least about

[0206] 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about

[0207] 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about

[0208] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 16.

[0209] In one embodiment, said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said ankyrin repeat domain to the first and second targets is mutually exclusive, is capable of competing for binding with stem cell factor (SCF). In one embodiment, said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said ankyrin repeat domain to the first and second targets is mutually exclusive, competes for binding with stem cell factor (SCF). In one embodiment, said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said ankyrin repeat domain to the first and second targets is mutually exclusive, does not compete for binding with stem cell factor (SCF). A suitable assay to assess whether said ankyrin repeat domain with binding specificity for CD117 is capable of competing with SCF is described in Example 6. In one embodiment, said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said ankyrin repeat domain to the first and second targets is mutually exclusive, inhibits ligand binding with an IC50 of at most about 5 nM or less, at most about 4 nM or less, at most about 3 nM or less, at most about 2 nM or less, at most about 1 nM or less, at most about 0.9 nM or less, at most about 0.8 nM or less, at most about 0.7 nM or less, at most about 0.6 nM or less, at most about 0.5 nM or less.

[0210] In one embodiment, said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said ankyrin repeat domain to the first and second targets is mutually exclusive, is capable of conditionally blocking the interaction of CD47 with signal-regulatory protein alpha (SIRPa).

[0211] In one embodiment, said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said ankyrin repeat domain to the first and second targets is mutually exclusive, binds to CD117 with a KD of about 10-5M or less, about 10-6M or less, about 10-7M or less, about 10-8M or less, about 10-9M or less, about 10-10M or less, about 10-11M or less, about 10-12M or less, about 10-13M or less, about 10-14M or less, from about 10-5M to about 10-15M, from about 10-6M to about 10-15M, from about 10-7M to about 10-15M, from about 10-8M to about 10-15M, from about 10-9M to about 10-15M, from about 10-10M to about 10-15M, from about 10-11M to about 10-15M, from about 10-12M to about 10-15M, from about 10-5M to about 10-14M, from about 10-6M to about 10-14M, from about 10-7M to about 10-14M, from about 10-8M to about 10-14M, from about 10-9M to about 10-14M, from about 10-10M to about 10-14M, from about 10-11M to about 10-14M, from about 10-12M to about 10-14M, from about 10-5M to about 10-13M, from about 10-6M to about 10-13M, from about 10-7M to about 10-13M, from about 10-8M to about 10-13M, from about 10-9M to about 10-13M, from about 1 O-10M to about 10-13M, from about 10-11M to about 10-13M, or from about 10-12M to about 10-13M. In further embodiments, said ankyrin repeat domain binds to CD117 with a KD value of, or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM. In one embodiment, said ankyrin repeat domain binds to CD117 with a KD value of less than or equal to about 1 nM. In another embodiment, said ankyrin repeat domain binds to CD117 with a KD value of less than or equal to about 100 pM. In another embodiment, said ankyrin repeat domain binds to CD117 with a KD value of less than or equal to about 10 pM. In yet another embodiment, said ankyrin repeat domain binds to CD117 with a KD value of less than or equal to about 1 pM.

[0212] In some embodiments, said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said ankyrin repeat domain to the first and second targets is mutually exclusive, binds to a CD47-binding ankyrin repeat domain with a dissociation constant (KD) below 10-5M. In certain embodiments, said ankyrin repeat domain binds to a CD47-binding ankyrin repeat domain with a KD of about 10-5M or less, about 10-6M or less, about 10-7M or less, about 10-8M or less, about 10-9M or less, about 10-10M or less, about 10-11M or less, about 10-12M or less, about 10-13M or less, about 10-14M or less, from about 10-5M to about 10-15M, from about 10-6M to about 10-15M, from about 10-7M to about 10-15M, from about 10-8M to about 10_15M, from about 10-9M to about 10-15M, from about 10-10M to about 10-15M, from about 10-11M to about 10-15M, from about 10-12M to about 10-15M, from about 10-5M to about 10-14M, from about 10-6M to about 10-14M, from about 10-7M to about 10-14M, from about 10-8M to about 10-14M, from about 10-9M to about 10-14M, from about 10-10M to about 10-14M, from about 10-11M to about 10-14M, from about 10-12M to about 10-14M, from about 10-5M to about 10-13M, from about 10-6M to about 10-13M, from about 10-7M to about 10-13M, from about 10-8M to about 10-13M, from about 10-9M to about 10_13M, from about 10-10M to about 10-13M, from about 10-11M to about 10-13M, or from about 10-12M to about 10-13M. In further embodiments, said ankyrin repeat domain binds to a CD47-specific binding agent with a KD value of, or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM. In one embodiment, said ankyrin repeat domain binds to a CD47-specific binding agent with a KD value of less than or equal to about 1 nM. In another embodiment, said ankyrin repeat domain binds to a CD47-specific binding agent with a KD value of less than or equal to about 100 pM. In another embodiment, said ankyrin repeat domain binds to a CD47-specific binding agent with a KD value of less than or equal to about 10 pM. In yet another embodiment, said ankyrin repeat domain binds to a CD47-specific binding agent with a KD value of less than or equal to about 1 pM. In some embodiments, the binding affinity of said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said ankyrin repeat domain to the first and second targets is mutually exclusive, to each of CD117 (first target) and CD47- specific binding agent (second target) is described in terms of two dissociation constant (KD) values, wherein KD1 represents the dissociation constant for CD117 and KD2 represents the dissociation constant for CD47-specific binding agent. In some embodiments, said ankyrin repeat domain binds to each of said first and second target with KD1 and KD2 being independently about 10-5M or less, about 10-6M or less, about 10-7M or less, about 10-8M or less, about 10-9M or less, about 1 O-10M or less, about 10-11M or less, about 10-12M or less, about 10-13M or less, about 10-14M or less, from about 10-5M to about 10-15M, from about 10-6M to about 10-15M, from about 10-7M to about 10-15M, from about 10-8M to about 10-15M, from about 10-9M to about 10-15M, from about 1 O-10M to about 10-15M, from about 10-11M to about 10-15M, from about 10-12M to about 10-15M, from about 10-5M to about 10-14M, from about 10-6M to about 10-14M, from about 10-7M to about 10-14M, from about 10-8M to about 10-14M, from about 10-9M to about 10-14M, from about 1 O-10M to about 10-14M, from about 10_11M to about 10-14M, from about 10-12M to about 10-14M, from about 10-5M to about 10-13M, from about 10-6M to about 10-13M, from about 10-7M to about 10-13M, from about 10-8M to about 10-13M, from about 10-9M to about 10-13M, from about 1 O-10M to about 10-13M, from about 10-11M to about 10-13M, or from about 10-12M to about 10-13M. In some embodiments, KD1 and KD2 are independently equal to or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM. In one embodiment, KD1 and KD2 are independently less than or equal to about 1 nM. In another embodiment, KD1 and KD2 are independently less than or equal to about 100 pM. In another exemplary embodiment, KD1 and KD2 are independently less than or equal to about 10 pM. In yet another embodiment, said first designed repeat domain binds to each of the first and second target with KD1 and KD2 being independently less than or equal to about 1 pM.

[0213] In one embodiment, said ankyrin repeat domain binds to CD117 with a first binding affinity and to the CD47-binding ankyrin repeat domain with a second binding affinity, wherein the ratio of said first binding affinity and said second binding affinity is between about 1 :1 and about 1 :105. In further embodiments, said ratio may be equal to about 1 :1 , about 1 :10, about 1 :102, about 1 :103, about 1 :104or about 1 :105.

[0214] In one embodiment, said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, wherein said first binding region binds to ankyrin repeat domain with binding specificity for CD47 with a dissociation constant (KD) of or below about 10'5M and / or to CD117 with a dissociation constant (KD) of or below about 10'5M. Half-Life Extending Moieties

[0215] A “half-life extending moiety” extends the serum half-life in vivo of the recombinant binding proteins described herein, compared to the same protein without the half-life extending moiety. Examples of half-life extending moieties include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin domain, maltose binding protein (MBP), a human serum albumin (HSA) binding domain, or polyethylene glycol (PEG).

[0216] In one embodiment, the multispecific binding proteins provided herein further comprise one or more half-life extending moieties. Preferably, said half-life extending moiety binds to human serum albumin. In some embodiments, the half-life extending moiety comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises an Fc domain. In some embodiments, the Fc domain is derived from any one of the known heavy chain isotypes: IgG (y), IgM (p), IgD (6), IgE (s), or IgA (a). In some embodiments, the Fc domain is derived from any one of the known heavy chain isotypes or subtypes: IgGi (y1), lgG2 (y2), lgG3 (y3), lgG4 (y4), IgAi (a1), lgA2 (a2). In some embodiments, the Fc domain is the Fc domain of human IgGi.

[0217] In some embodiments, the Fc domain comprises an uninterrupted native sequence (i.e., wild type sequence) of an Fc domain. In some embodiments, the immunoglobulin Fc domain comprises a variant Fc domain resulting in altered biological activity. For example, at least one point mutation or deletion may be introduced into the Fc domain so as to reduce or eliminate the effector activity (e.g., International Patent Publication No. WO 2005 / 063815), and / or to increase the homogeneity during the production of the recombinant binding protein. In some embodiments, the Fc domain is the Fc domain of human IgGi and comprises one or more of the following effector-null substitutions: L234A, L235A, and G237A (Eu numbering). In some embodiments, the Fc domain does not comprise the lysine located at the C-terminal position of human lgG1 (i.e., K447 by Eu numbering). The absence of the lysine may increase homogeneity during the production of the recombinant binding protein. In some embodiments, the Fc domain comprises the lysine located at the C-terminal position (K447, Eu numbering).

[0218] In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain binding human serum albumin comprising 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 to any one of SEQ ID NOs: 17 to 19. In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain binding human serum albumin comprising 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 to SEQ ID NO: 17. In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain binding human serum albumin comprising 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

[0219] 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about

[0220] 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about

[0221] 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 18. In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain binding human serum albumin comprising an amino acid sequence at least about 80%, at least about 81 %, at least about

[0222] 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about

[0223] 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about

[0224] 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about

[0225] 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 19.

[0226] Multispecific binding proteins

[0227] In another aspect, provided is a multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for CD47, (2) a second ankyrin repeat domain with binding specificity for the CD47-binding ankyrin repeat domain, (3) a third ankyrin repeat domain with binding specificity for CD117, wherein binding of said second ankyrin repeat domain and said third ankyrin repeat domain is mutually exclusive, and (4) a fourth ankyrin repeat domain with binding specificity for CD16a. In a more specific embodiment, the multispecific binding protein may comprise, instead of the second ankyrin repeat domain with binding specificity for the CD47-binding ankyrin repeat domain and the third ankyrin repeat domain with binding specificity for CD117, a single ankyrin repeat domain with a first binding specificity for the CD47-binding ankyrin repeat domain and a second binding specificity for CD117, wherein binding to CD47-binding ankyrin repeat domain and CD117 is mutually exclusive. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin.

[0228] Accordingly, disclosed herein are multispecific binding proteins comprising (1) a first ankyrin repeat domain with binding specificity for CD47, (2) a second ankyrin repeat domain with a first binding specificity for the CD47-binding ankyrin repeat domain and a second binding specificity for CD117, wherein binding to CD47-binding ankyrin repeat domain and CD117 is mutually exclusive, (3) a third ankyrin repeat domain with binding specificity for CD16a, and optionally (4) a fourth ankyrin repeat domain with binding specificity for CD117. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin.

[0229] In one aspect, provided is a multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for CD47, (2) a second ankyrin repeat domain with a first binding specificity for the CD47-binding ankyrin repeat domain and a second binding specificity for CD117, wherein binding to CD47-binding ankyrin repeat domain and CD117 is mutually exclusive, (3) a third ankyrin repeat domain with binding specificity for CD16a, and (4) a fourth ankyrin repeat domain with binding specificity for CD117. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin. In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to

[0230] SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 12 to 16, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 9, and optionally (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as 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

[0231] 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about

[0232] 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about

[0233] 99% or 100% identical to SEQ ID NO: 10 or 11 . Suitably, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin (HSA) comprising 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%, about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 17 to 19.

[0234] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of any one of SEQ ID NOs: 12 to 16, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and optionally (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 10 or 11. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of any one of SEQ ID NOs: 17 to 19.

[0235] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to

[0236] SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID

[0237] NO: 12, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as 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

[0238] 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about

[0239] 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about

[0240] 98%, at least about 99% or 100% identical to SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as 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

[0241] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0242] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 10. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 17.

[0243] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of SEQ ID NO: 12, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 10. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of SEQ ID NO: 17.

[0244] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to

[0245] SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID

[0246] NO: 13, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as 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

[0247] 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about

[0248] 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about

[0249] 98%, at least about 99% or 100% identical to SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as 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

[0250] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0251] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 10. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 17.

[0252] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of SEQ ID NO: 13, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 10. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of SEQ ID NO: 17.

[0253] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to

[0254] SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID

[0255] NO: 13, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as 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

[0256] 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about

[0257] 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about

[0258] 98%, at least about 99% or 100% identical to SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as 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

[0259] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0260] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 11 . Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 17. In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of SEQ ID NO: 13, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 11 . Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of SEQ ID NO: 17.

[0261] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to

[0262] SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID

[0263] NO: 14, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as 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

[0264] 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about

[0265] 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about

[0266] 98%, at least about 99% or 100% identical to SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as 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

[0267] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0268] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 11 . Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 17.

[0269] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of SEQ ID NO: 14, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 11 . Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of SEQ ID NO: 17.

[0270] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to

[0271] SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID

[0272] NO: 15, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as 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

[0273] 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about

[0274] 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about

[0275] 98%, at least about 99% or 100% identical to SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as 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

[0276] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0277] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 10. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 17.

[0278] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of SEQ ID NO: 15, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 10. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of SEQ ID NO: 17.

[0279] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to

[0280] SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID

[0281] NO: 16, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as 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

[0282] 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about

[0283] 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about

[0284] 98%, at least about 99% or 100% identical to SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as 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

[0285] 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about

[0286] 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 10. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 17.

[0287] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of SEQ ID NO: 16, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 10. Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of SEQ ID NO: 17.

[0288] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to

[0289] SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID

[0290] NO: 16, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as 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

[0291] 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about

[0292] 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about

[0293] 98%, at least about 99% or 100% identical to SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 11 . Optionally, said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 17.

[0294] In one aspect, provided is a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of SEQ ID NO: 16, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 11 . Optionally, said multispecific binding protein further comprises a fifth ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of SEQ ID NO: 17.

[0295] In one embodiment, the multispecific binding protein comprises said first, second, and third binding agents, and wherein said multispecific recombinant protein is capable of binding the respective targets of said first, second, and third binding agents simultaneously.

[0296] In one embodiment, the multispecific binding protein comprises said first, second, third, and fourth binding agents, and wherein said multispecific recombinant protein is capable of binding the respective targets of said first, second, third and fourth binding agents simultaneously.

[0297] In one embodiment, the multispecific binding protein comprises said first, second, third, fourth, and fifth binding agents, and wherein said multispecific recombinant protein is capable of binding the respective targets of said first, second, third, fourth, and fifth binding agents simultaneously.

[0298] In one embodiment, the multispecific binding protein comprises a first ankyrin repeat domain, a second ankyrin repeat domain, a third ankyrin repeat domain, a fourth ankyrin repeat domain, and / or a fifth ankyrin repeat domain, wherein said first ankyrin repeat domain specifically binds to CD47 with a dissociation constant (KD) of or below about 107M, said second ankyrin repeat domain specifically binds to CD117 with a dissociation constant (KD) of or below about 107M, said third ankyrin repeat domain specifically binds to CD117 with a dissociation constant (KD) of or below about 107M, said fourth ankyrin repeat domain specifically binds to CD16a with a dissociation constant (KD) of or below about 107M, and said fifth ankyrin repeat domain specifically binds to human serum albumin with a dissociation constant (KD) of or below about 107M. Suitably, said dissociation constants (KD) are measured in PBS, e.g., as further described in Examples 1 to 3.

[0299] In one embodiment, said multispecific binding protein is capable of conditionally blocking the interaction of CD47 with signal-regulatory protein alpha (SIRPa). A suitable assay to assess whether said ankyrin repeat domain with binding specificity for CD47 is capable of conditionally blocking the interaction of CD47 with signal-regulatory protein alpha (SIRPa) is described in Example 6. In one embodiment, said multispecific binding protein conditionally inhibits ligand binding with an IC50 of at most about 500 nM or less, at most about 400 nM or less, at most about 300 nM or less, at most about 200 nM or less, at most about 100 nM or less, at most about 90 nM or less, at most about 80 nM or less, at most about 70 nM or less, at most about 60 nM or less, at most about 50 nM or less. In one embodiment, said multispecific binding protein conditionally blocks or reduces the interaction of CD47 with signal-regulatory protein alpha (SIRPa). In one embodiment, said multispecific binding protein conditionally reduces the interaction of CD47 with signal-regulatory protein alpha (SIRPa) by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% compared to a binding protein, suitably an ankyrin repeat domain, which does not have binding specificity for CD47.

[0300] In one embodiment, said multispecific binding protein is capable of competing for binding to CD117 with stem cell factor (SCF). In one embodiment, said multispecific binding protein competes for binding to CD117 with stem cell factor (SCF). In one embodiment, said multispecific binding protein does not compete for binding to CD117 with stem cell factor (SCF). A suitable assay to assess whether said multispecific binding protein is capable of competing with SCF is described in Example 6. In one embodiment, said multispecific binding protein inhibits ligand binding with an IC50 of at most about 5 nM or less, at most about 4 nM or less, at most about 3 nM or less, at most about 2 nM or less, at most about 1 nM or less, at most about 0.9 nM or less, at most about 0.8 nM or less, at most about 0.7 nM or less, at most about 0.6 nM or less, at most about 0.5 nM or less.

[0301] In one embodiment, said multispecific binding protein is capable of increasing antibody-dependent cellular cytotoxicity (ADCC). In one embodiment, said multispecific binding protein increases antibody-dependent cellular cytotoxicity (ADCC). In one embodiment, said multispecific binding protein increases antibody-dependent cellular cytotoxicity (ADCC) by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% compared to a binding protein, suitably an ankyrin repeat domain, which does not have binding specificity for CD16a and / or which does not have binding specificity for CD47.

[0302] In one embodiment, said multispecific binding protein is capable of increasing antibody-dependent cellular phagocytosis (ADCP). In one embodiment, said multispecific binding protein increases antibody-dependent cellular phagocytosis (ADCP). In one embodiment, said multispecific binding protein increases antibody-dependent cellular phagocytosis (ADCP) by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% compared to a binding protein, suitably an ankyrin repeat domain, which does not have binding specificity for CD16a and / or which does not have binding specificity for CD47.

[0303] Suitably, said ankyrin repeat domain binding human serum albumin is located C-terminally within said multispecific binding protein. Suitably, said ankyrin repeat domain binding human serum albumin is located N-terminally within said multispecific binding protein. In some embodiments, the multispecific binding protein provided herein comprises two or more serum albumin binding ankyrin repeat domains. In some embodiments, two serum albumin binding ankyrin repeat domains are located at the N-terminus of the multispecific binding protein provided herein. In some embodiments, two serum albumin binding ankyrin repeat domains are located at the C-terminus of the multispecific binding protein provided herein. In some embodiments, a first serum albumin binding ankyrin repeat domain is located at the N-terminus of the multispecific binding protein provided herein, and a second serum albumin binding ankyrin repeat domain is located at the C-terminus of the multispecific binding protein provided herein.

[0304] In one embodiment, said multispecific binding protein comprises a first, a second, and a third binding agent, wherein said first, second and third binding agents can be arranged in any order from the N- terminus to the C-terminus. For example, the first binding agent can be at the N-terminal position or the first binding agent can be at the C-terminal position, or the first binding agent can be positioned between the second and the third binding agent. In another example, the second binding agent can be at the N-terminal position or the second binding agent can be at the C-terminal position, or the second binding agent can be positioned between the first and the third binding agent. In another example, the third binding agent can be at the N-terminal position or the third binding agent can be at the C-terminal position, or the third binding agent can be positioned between the first and the second binding agent.

[0305] In one embodiment, said multispecific binding protein comprises first, second, third and fourth binding agents, wherein said first, second, third and fourth binding agents can be arranged in any order from the N-terminus to the C-terminus. For example, the first binding agent can be at the N-terminal position or the first binding agent can be at the C-terminal position, or the first binding agent can be positioned between any of the other binding agents. In another example, the second binding agent can be at the N-terminal position or the second binding agent can be at the C-terminal position, or the second binding agent can be positioned between any of the other binding agents. In another example, the third binding agent can be at the N-terminal position or the third binding agent can be at the C- terminal position, or the third binding agent can be positioned between any of the other binding agents. In another example, the fourth binding agent can be at the N-terminal position or the fourth binding agent can be at the C-terminal position, or the fourth binding agent can be positioned between any of the other binding agents.

[0306] In one embodiment, said binding agents are covalently linked with a peptide linker. Said peptide linker may be a proline-threonine-rich peptide linker. Suitably, said peptide linker comprises between 1 and 50 amino acids. Suitably, said peptide linker comprises between 1 and 30 amino acids.

[0307] In one embodiment, any of said ankyrin repeat domains additionally comprises a G, a S or a GS at the N-terminus.

[0308] In one aspect, provided is a multispecific binding protein comprising 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: 1 to 7.

[0309] In one aspect, provided is a multispecific binding protein comprising 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.

[0310] In one aspect, provided is a multispecific binding protein comprising 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.

[0311] In one aspect, provided is a multispecific binding protein comprising 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.

[0312] In one aspect, provided is a multispecific binding protein comprising 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.

[0313] In one aspect, provided is a multispecific binding protein comprising 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.

[0314] In one aspect, provided is a multispecific binding protein comprising 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. In one aspect, provided is a multispecific binding protein comprising 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: 7.

[0315] Nucleic acids and vectors

[0316] In another aspect, the provided herein are nucleic acid molecules encoding the amino acid sequence of a multispecific binding protein provided herein. In one embodiment, the nucleic acid molecule is a DNA molecule. Nucleotide sequences encoding the multispecific binding proteins provided herein comprise, for example, any one of SEQ ID NOs: 38 to 44. In one embodiment, provided is a nucleic acid molecule encoding a multispecific binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 1 to 7. In one embodiment, provided is a nucleic acid molecule encoding the multispecific binding protein comprising the amino acid sequence of SEQ ID NO: 1. In one embodiment, provided is a nucleic acid molecule the multispecific binding protein comprising the amino acid sequence of SEQ ID NO: 2. In one embodiment, provided is a nucleic acid molecule encoding the multispecific binding protein comprising the amino acid sequence of SEQ ID NO: 3. In one embodiment, provided is a nucleic acid molecule encoding the multispecific binding protein comprising the amino acid sequence of SEQ ID NO: 4. In one embodiment, provided is a nucleic acid molecule encoding the multispecific binding protein comprising the amino acid sequence of SEQ ID NO: 5. In one embodiment, provided is a nucleic acid molecule encoding a recombinant binding protein comprising the amino acid sequence of SEQ ID NO: 6. In one embodiment, provided is a nucleic acid molecule encoding the multispecific binding protein comprising the amino acid sequence of SEQ ID NO: 7.

[0317] Also provided are nucleic acid molecules that derive from any one of SEQ ID NOs: 38 to 44 having been optimized for protein expression in a suitable host cell, such as a prokaryotic host cell, e.g., E.coli. In one embodiment, the nucleic acid molecule is codon optimized (e.g., codon optimized for expression in a host cell, suitably E.coli). The nucleic acid may be present in a vector such as a phage display vector, or in a recombinant plasmid vector. Accordingly, also provided is a cloning or expression vector comprising one or more nucleic acid sequences comprising any one of SEQ ID NOs: 38 to 44. In brief, expression vectors may be constructed comprising a nucleic acid molecule which is capable, in an appropriate host, of expressing the polypeptide encoded by the nucleic acid molecule. In one embodiment, said expression vector is suitable for the recombinant production of the isolated multispecific protein disclosed herein.

[0318] For expression of the multispecific binding protein disclosed herein, standard techniques can be applied to transfect a host cell with the expression vector, suitably comprising any one nucleic acid sequences of SEQ ID NOs: 38 to 44. The various forms of the term "transfection" are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE- dextran transfection and the like.

[0319] In one embodiment, a cloning or expression vector as described herein comprises the nucleic acid sequences comprising any one of SEQ ID NOs: 38 to 44, operatively linked to suitable promoter sequences. In one embodiment, provided is a cloning or expression vector comprising a nucleic acid molecule encoding a multispecific binding protein comprising an amino acid sequence selected from any one of SEQ ID NOs: 1 to 7, operatively linked to suitable promoter sequences.

[0320] Also provided is a vector comprising the nucleic acid encoding any of the multispecific binding proteins or ankyrin repeat domains described herein, wherein said vector is a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector.

[0321] When recombinant expression vectors encoding the multispecific binding proteins disclosed herein are introduced into suitably host cells, e.g. prokaryotic host cells, the multispecific binding proteins are produced by culturing the host cells for a period of time sufficient to allow for expression of the multispecific binding protein in the host cells or secretion of the multispecific binding protein into the culture medium in which the host cells are grown. Multispecific binding proteins can be recovered from the host cells or from the culture medium using standard protein purification methods. In one embodiment, the method comprises culturing a suitable host cell, e.g., a prokaryotic host cell and collecting the multispecific binding protein disclosed herein from the culture medium. In one embodiment, the method comprises culturing a suitable host cell, e.g., a prokaryotic host cell and collecting the multispecific binding protein disclosed herein from the host cells.

[0322] In one embodiment, provided is a host cell transfected with an expression vector comprising the nucleic acid sequences of any one of SEQ ID NOs: 38 to 44, suitable for the expression of any one of the multispecific binding proteins comprising amino acid sequences of SEQ ID NOs: 1 to 7, respectively, operatively linked to suitable promoter sequences. In one embodiment, provided is a host cell transfected with an expression vector comprising the nucleic acid sequences encoding a multispecific binding protein comprising an amino acid sequence of any one of SEQ ID NOs: 1 to 7, suitable for the expression of any one of said multispecific binding proteins. The host cells may then be further cultured under suitable conditions for the expression and production of a multispecific binding protein comprising the amino acid sequence with SEQ ID NOs: 1 to 7, respectively. Accordingly, also provided is a method for producing a multispecific protein as described herein. In one aspect, provided is a method for producing a multispecific binding protein, for example a multispecific protein comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 7, the method comprising the steps of (i) expressing said recombinant binding protein in a suitable host cell (e.g., bacteria), and (ii) purifying said recombinant binding protein (e.g., using chromatography). Said method may comprise additional steps. An example of such a method of producing a multispecific binding protein is described in Example 6.

[0323] Compositions, Uses and Methods of Treatment

[0324] Also provided are pharmaceutical compositions comprising a multispecific binding protein, a nucleic acid molecule, a vector or a cell as described herein, and a pharmaceutically acceptable carrier and / or diluent. Accordingly, in one aspect, provided is a pharmaceutical composition comprising a multispecific binding protein as described herein or a nucleic acid molecule encoding a multispecific binding protein as described herein or a vector comprising said nucleic acid molecule or a cell comprising said nucleic acid molecule or said vector, and a pharmaceutically acceptable carrier and / or diluent.

[0325] Suitable carriers, diluents, excipients or stabilizers known to one of skill in the art include, for example, saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, and / or any substances that enhance isotonicity and / or chemical stability, buffers, and preservatives. A pharmaceutically acceptable excipient typically has no significant adverse effect on the patient receiving the composition. A pharmaceutical composition may also be a combination formulation, comprising an additional active agent, such as an anti-cancer agent, or an additional bioactive compound. The compositions to be used for in vivo administration must be aseptic or sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0326] Accordingly, the pharmaceutical composition may comprise at least one multispecific binding protein as described herein and a detergent, a buffer, and a sugar. In one embodiment, such a composition comprises multispecific binding proteins as described herein and PBS.

[0327] Another object of the present disclosure is a stem cell transplantation pre-treatment method with fewer and / or less pronounced side effects compared to existing pre-treatment methods. This is attained by administering the multispecific binding proteins described herein to a subject eligible for stem cell transplantation prior to said transplantation as a pre-treatment or conditioning step, resulting in the elimination or reduction of target cells, such as malignant tumor cells and / or cancer stem cells, and / or hematopoietic stem cells. The multispecific binding protein targets, inter alia, markers specific for HSCs and / or markers expressed on cancer cells that are shared with HSCs, i.e., markers present on both cancer cells and HSCs, including, but not limited to, CD117, resulting in decreasing, destroying or killing undesired cells in a patient prior to stem cell transplantation. The multispecific binding protein directed to specific HSC target antigens can be used to deplete the subject's HSCs as a conditioning regimen prior to HSCT. When administered to a subject suffering from cancer, the multispecific binding protein can both directly attack cancer cells and HSCs expressing the marker antigens. This approach can be used to reduce or eliminate the cells expressing these marker antigens, including tumor cells as well as cancer stem cells (CSCs), and also eliminate HSCs. Once the HSCs and / or CSCs have been reduced or eliminated, the subject can undergo autologous or allogeneic stem cell transplantation. Pretreatment of a patient using the multispecific binding proteins described herein will give rise to fewer side effects such drug-induced toxicities and risk of infections compared to other existing conditioning regimens, also allowing HSCT in older and / or more fragile patients.

[0328] Thus, the present disclosure is directed to a multispecific binding protein described herein, a pharmaceutical composition comprising such a recombinant binding protein, a nucleic acid encoding such a multispecific binding protein, a vector comprising a nucleic acid encoding such a multispecific binding protein, or a cell comprising a nucleic acid encoding such a multispecific binding protein or the vector comprising a nucleic acid encoding such a multispecific binding protein for use as a medicament. Said medicament may be used in a conditioning regimen. In one embodiment, said medicament is used in a conditioning regimen.

[0329] The present disclosure is also directed to the use of a multispecific binding protein described herein, a pharmaceutical composition comprising such a recombinant binding protein, a nucleic acid encoding such a multispecific binding protein, a vector comprising a nucleic acid encoding such a multispecific binding protein, or a cell comprising a nucleic acid encoding such a multispecific binding protein or the vector comprising a nucleic acid encoding such a multispecific binding protein in the manufacture of a medicament. Said medicament may be used in a conditioning regimen. In one embodiment, said medicament is used in a conditioning regimen.

[0330] The present disclosure is also directed to a method of treatment comprising administering to a subject in need thereof a therapeutically effective amount of the multispecific binding protein described herein, a pharmaceutical composition comprising such a recombinant binding protein, a nucleic acid encoding such a multispecific binding protein, a vector comprising a nucleic acid encoding such a multispecific binding protein, or a cell comprising a nucleic acid encoding such a multispecific binding protein or the vector comprising a nucleic acid encoding such a multispecific binding. Said method of treatment may be a conditioning regimen. In one embodiment, said medicament is used in a conditioning regimen.

[0331] A conditioning regimen may be required in a subject with a malignant, with a pre-malignant or with a non-malignant disorder. For example, the multispecific binding proteins described herein can be used to treat any non-malignant condition / disorder wherein stem cell transplantation could be beneficial, such as hematologic diseases or hematological malignancies. Said multispecific binding proteins can be used as a conditioning regimen in a subject prior to receiving stem cell transplantation, and / or prior to re-transplantation of stem cells. In one embodiment, said conditioning is used prior to transplantation of genetically modified hematopoietic stem cells, e.g. by viral gene transfer or through genome editing. Hematologic diseases that may benefit from such conditioning may be sickle cell disease, or beta thalassemia or other hemoglobinopathies. In one embodiment, autologous, syngeneic or allogeneic hematopoietic stem cells are transplanted that have been genetically modified. If the reason for the transplantation is a hematological malignancy, the multispecific binding proteins described herein can also be used for the treatment of said hematological malignancy, e.g., by reducing relapse after stem cell transplantation due to more efficient killing of malignant cells before the transplantation, while also having an additional beneficial effect of reduced impact on the recipient's immune cells / stem cells. Treating subjects in need of treatment with these multispecific binding proteins will result in the reduction or elimination of those cells expressing these targets, including CSCs and / or HSCs. Therefore, the subject may be in need of either autologous or allogeneic hematopoietic stem cell transplantation. Accordingly, said methods or uses may be employed for selectively depleting or ablating a hematopoietic stem cell (HSC) or progenitor cell population in a target tissue of the subject. Said methods and uses may also be used for engrafting stem cells in a subject by (a) selectively depleting or ablating the endogenous hematopoietic stem cell or progenitor cell population in a target tissue of the subject; and (b) administering a stem cell population to the target tissue of the subject, wherein the administered stem cell population engrafts in the target tissue of the subject. The treatment can therefore be used as a pre-treatment before stem cell transplantation, and / or after (e.g., priorto a re-transplantation) if relapse of the underlying malignancy occurs. Accordingly, in one embodiment said subject is suffering from a hematologic disease, hematological malignancy, or solid tumor. Common types of hematological diseases that can be treated with the described methods and uses, include but are not limited to leukemias, lymphomas and myelodysplasia syndromes. In one embodiment, said hematological malignancy is leukemia. In one embodiment, the malignancy is acute leukemia, e.g., acute myeloid leukemia or acute lymphocytic leukemia. In one embodiment, the malignancy is myelodysplastic syndrome (MDS). In a further embodiment, the disease / malignancy is any disorder or condition requiring or benefitting from stem cell transplantation. A further object can be the treatment of relapse after HSCT of the underlying malignancy. In one embodiment, said subject is suffering from a non-malignant disorder. In one embodiment, said non-malignant disorder is an autoimmune condition.

[0332] In an embodiment the hematopoietic stem cell transplantation is autologous, allogeneic, syngeneic, or xenogeneic. In one embodiment, the stem cell transplantation is a first stem cell transplantation. In one embodiment the stem cell transplantation is a re-transplantation, e.g. after relapse.

[0333] When the subject in need of treatment suffers from a malignancy, the multispecific binding proteins specifically bind to at least one activating molecule on immune cells, e.g. effector cells, and to at least one marker shared in cancer cells and HSCs. Suitably, the marker expressed on the cancer cells is also present on hematopoietic stem cells but not on other hematopoietic cells, said marker including, but not limited to, CD117. In one embodiment, said medicament is used in the treatment of cancer. Said cancer may be a CD117-positive cancer, e.g. gastrointestinal stromal tumor (GIST).

[0334] A typical route of administration of the multispecific binding protein described herein, the nucleic acid encoding such a multispecific binding protein, or the vector or cell comprising a nucleic acid encoding such a multispecific binding protein, or the pharmaceutical composition comprising such a multispecific binding protein, nucleic acid, vector, or cell is parenteral administration, e.g., intravenous, or subcutaneous. In parental administration, the pharmaceutical composition is formulated in a unit dosage injectable form such as a solution, suspension, or emulsion, in association with the pharmaceutically acceptable excipients as described above. The dosage and mode of administration depends on the individual to be treated and the disease.

[0335] 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 over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure is dictated by and are directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound. The multispecific binding protein described herein, the nucleic acid encoding such a multispecific binding protein, or the vector or cell comprising a nucleic acid encoding such a multispecific binding protein, or the pharmaceutical composition comprising such a multispecific binding protein, nucleic acid, vector, or cell may also be administered in combination with one or more other therapies. The term “use in combination with”, as used herein, refers to a co-administration, which is carried out under a given regimen. This includes synchronous administration of the different compounds as well as time-shifted administration of the different compounds (e.g., compound A is given once and compound B is given several times thereafter, or vice versa, or both compounds are given synchronously and one of the two is also given at later stages).

[0336] In one aspect, also provided is a kit comprising the multispecific binding protein described herein, the nucleic acid encoding such a multispecific binding protein, or the vector or cell comprising a nucleic acid encoding such a multispecific binding protein, or the pharmaceutical composition comprising such a multispecific binding protein, nucleic acid, vector, or cell. In one aspect, provided is a kit comprising the multispecific protein described herein, for example a multispecific binding protein comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 7, and / or a nucleic acid encoding the multispecific protein described herein, for example a multispecific binding protein comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 7, and / or a pharmaceutical composition comprising the multispecific protein described herein, for example a multispecific binding protein comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 7.

[0337] SEQUENCE TABLE

[0338]

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346] EXAMPLES

[0347] Starting materials and reagents disclosed below are known to those skilled in the art, are commercially available and / or can be prepared using known techniques.

[0348] Materials

[0349] Chemicals were purchased from Sigma-Aldrich (USA). Oligonucleotides were from Microsynth (Switzerland). Unless stated otherwise, DNA polymerases, restriction enzymes and buffers were from New England Biolabs (USA) or Fermentas / ThermoFisher Scientific (USA). Inducible E. coli expression strains were used for cloning and protein production, e.g. E. coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA). If appropriate, proteins were produced with an N-terminal His-tag (such as SEQ ID NO: 20) for ease of purification.

[0350] Molecular Biology

[0351] Unless stated otherwise, methods are performed according to known protocols (see, e.g., Sambrook J., Fritsch E.F. and Maniatis T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, New York).

[0352] Designed ankyrin repeat protein libraries

[0353] Methods to generate designed ankyrin repeat protein libraries have been described, e.g. in U.S. Patent No. 7,417,130; Binz et al., J. Mol. Biol. 332, 489-503, 2003; Binz et al. 2004, loc. cit. By such methods designed ankyrin repeat protein libraries having randomized ankyrin repeat modules and / or randomized capping modules can be constructed. For example, such libraries can be assembled based on a fixed N-terminal capping module or a randomized N-terminal capping module, and a fixed C-terminal capping module or a randomized C-terminal capping module. Preferably, such libraries are assembled to not have any of the amino acids C, G, M, N (in front of a G residue) and P at randomized positions of repeat or capping modules.

[0354] Furthermore, randomized modules in such libraries may comprise additional polypeptide loop insertions with randomized amino acid positions. Examples of such polypeptide loop insertions are complementarity determining region (CDR) loop libraries of antibodies or de novo generated peptide libraries. For example, such a loop insertion could be designed using the structure of the N-terminal ankyrin repeat domain of human ribonuclease L (Tanaka, N., Nakanishi, M, Kusakabe, Y, Goto, Y., Kitade, Y, Nakamura, K.T., EMBO J. 23(30), 3929-3938, 2004) as guidance. In analogy to this ankyrin repeat domain, where ten amino acids are inserted in the beta-turn present close to the boarder of two ankyrin repeats, ankyrin repeat proteins libraries may contain randomized loops (with fixed and randomized positions) of variable length (e.g. 1 to 20 amino acids) inserted in one or more beta-turns of an ankyrin repeat domain.

[0355] Any such N-terminal capping module of an ankyrin repeat protein library suitably comprises the RILLAA, RILLKA or RELLKA motif and any such C-terminal capping module of an ankyrin repeat protein library suitably comprises the KLN, KLA or KAA motif.

[0356] The design of such an ankyrin repeat protein library may be guided by known structures of an ankyrin repeat domain interacting with a target. Examples of such structures, identified by their Protein Data Bank (PDB) unique accession or identification codes (PDB-IDs), are 1 WDY, 3V31 , 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1 N11 , 1 S70 and 2ZGD.

[0357] Examples of designed ankyrin repeat protein libraries, such as N2C and N3C designed ankyrin repeat protein libraries, have been described (U.S. Patent No. 7,417,130; Binz et al. 2003, loc. cit.; Binz et al. 2004, loc. cit.). The digit in N2C and N3C describes the number of randomized repeat modules present between the N-terminal and C-terminal capping modules.

[0358] The nomenclature used to define the positions inside the repeat units and modules is based on Binz et al. 2004, loc. cit. with the modification that borders of the ankyrin repeat modules and ankyrin repeat units are shifted by one amino acid position. For example, position 1 of an ankyrin repeat module of Binz et al. 2004 (loc. cit.) corresponds to position 2 of an ankyrin repeat module of the current disclosure and consequently position 33 of an ankyrin repeat module of Binz et al. 2004, loc. cit. corresponds to position 1 of a following ankyrin repeat module of the current disclosure.

[0359] Example 1 : Selection of binding proteins comprising an ankyrin repeat domain with binding specificity for CD47

[0360] Selection of CD47-specific ankyrin repeat proteins

[0361] The selection of CD47-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Pluckthun, loc. cit.) using part of the extracellular domain of CD47 (UniProt Ref. No: Q08722, residues 19 to 141) as target protein, libraries of ankyrin repeat proteins as described above, and established protocols (see, e.g., Zahnd, C., Amstutz, P. and Pluckthun, A., Nat. Methods 4, 69-79, 2007), and is further described in WO2024 / 251695.

[0362] Pools from ribosome display were expressed in E. coli cells in 96-well plates. DARPin proteins were expressed in E. coli cells with a Flag-tag and a His-tag. Crude extracts thereof were prepared to test binding of the Flag-tagged DARPin proteins to human CD47 recombinant protein in an HTRF assay.

[0363] Several ankyrin repeat proteins binding to human CD47 were tested for SIRPIa competition using the PathHunter Jurkat SIRPa Signaling Bioassay Kit purchased from Eurofins DiscoverX products LLC. Ligand engagement through co-culture of Jurkat CD47 presenting cells and Jurkat SIPRI a signaling cells results in phosphorylation and intracellular pathway activation leading to a chemiluminescence signal. This signal can be inhibited in the presence of an anti-CD47 DARPin or anti-SIRPa antibody disrupting ligand-receptor interaction. An IC50 value of 53.5 nM was obtained for the CD47-binding ankyrin repeat domain with SEQ ID NO: 8.

[0364] Determination of dissociation constants (KD) of ankyrin repeat proteins with binding specificity for human CD47 by Surface Plasmon Resonance (SPR) analysis

[0365] The binding affinity of a purified ankyrin repeat protein to biotinylated recombinant human CD47-Fc target was analyzed using a ProteOn XPR 36 instrument (NAHLC200M, Xantec ProteOn Sensor Chip) and the measurement was performed according to standard procedures. The CD47-binding ankyrin repeat domain with SEQ ID NO: 8 (“aCD47”) was subcloned into a derivative of the pQE30 (Qiagen) expression vector, containing an N-terminal His-tag, and expressed and purified as described above.

[0366] Biotinylated human CD47-Fc target was diluted in PBST (PBS, pH 7.4 containing 0.005% Tween 20®) and coated on an NAHLC200M chip (BioRad) to a level of around 350 and 800 resonance units (RU). The interaction of ankyrin repeat protein and human CD47 was then measured by injecting 200 pl running buffer (PBS, pH 7.4 containing 0.005% Tween 20®) containing serial dilutions of ankyrin repeat proteins covering a concentration range between 16.7 nM, 5.6 nM, 1.85 nM and 0.62 nM for multi-trace SPR measurements, followed by a running buffer flow for at least 20 minutes at a constant flow rate of 100 pl / min (off-rate measurement). The regeneration was performed using 30 pl of 10 mM Glycine pH 2.5. The signals (i.e. resonance unit (RU) values) of an empty lane (no target coated) and a reference injection (i.e. injection of running buffer only) were subtracted from the RU traces obtained after injection of ankyrin repeat protein (double-referencing). Binding parameters (KD, on-rate, off-rate) against CD47 were as shown in Table 1. Dissociation constants (KD) were calculated from the estimated on- and off-rates using standard procedures.

[0367] Table 1 : Binding parameters for ankyrin repeat proteins with binding specificity for human CD47 by Surface Plasmon Resonance (SPR)

[0368] Example 2: Selection of binding proteins comprising an ankyrin repeat domain with binding specificity for CD16a

[0369] Selection of CD16a-specific ankyrin repeat proteins

[0370] The selection of CD16a-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Pluckthun, loc. cit.) using the biotinylated extracellular domains of human CD16a (UniProt Ref. No: P08637) as target protein, libraries of ankyrin repeat proteins as described above, and established protocols (See, e.g., Zahnd, C., Amstutz, P. and Pluckthun, A., Nat. Methods 4, 69-79, 2007), and is further described in WO2024 / 251628.

[0371] Selected ankyrin repeat proteins specifically binding to CD16a were identified by Homogeneous Time Resolved Fluorescence (HTRF) assay using crude extracts of ankyrin repeat protein-expressing Escherichia coli cells using standard protocols.

[0372] Variants with increased affinity to and / or reduced off-rate from target protein were generated using affinity maturation. Thereby, one initially identified binding protein was selected for affinity maturation. The affinity maturation procedure entailed saturation mutagenesis of each randomized position and one non-randomized position (i.e. position 5 in 2ndinternal repeat) of the ankyrin repeat domain used as a starting point. Sequences generated by the affinity maturation procedure were screened for lower off-rates by competition HTRF, resulting in the ankyrin repeat domain with SEQ ID NO: 9 (“aCD16a”). Determination of dissociation constants (KD) of recombinant ankyrin repeat proteins with binding specificity for CD16a by Surface Plasmon Resonance (SPR) analysis

[0373] The binding affinity of the CD16a ankyrin repeat domain with SEQ ID NO: 9 (“aCD16a“) on recombinant CD16a target was analysed by SPR. Briefly, SPR measurements were performed using a ProteOn XPR36 instrument (BioRad). The running buffer was PBS pH 7.4 containing 0.005% Tween 20® (PBST). Biotinylated CD16a target was immobilized on a NLC chip (BioRad) to a level of about 3200 RU. The interactions of purified aCD16a with the target were measured by injecting the construct at 100 nM (and 1 :3 dilutions) with an association of 120 s and dissociation of 300 s using a constant flow of 100 pl / min. The target was regenerated between the individual measurements using 16mM H3PO4. The signals were double referenced against the running buffer (PBST) treated control lanes. The KD was calculated with a 1 :1 Langmuir model fitting from the estimated on- and off-rates using standard procedures. Results are shown in Table 2.

[0374] Table 2: Binding parameters for ankyrin repeat proteins with binding specificity for human CD16a by Surface Plasmon Resonance (SPR).

[0375] Example 3: Selection of binding proteins comprising an ankyrin repeat domain with binding specificity for CD117

[0376] Selection of CD117-specific ankyrin repeat proteins

[0377] The selection of CD117-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Pluckthun, loc. cit.) using the extracellular domain of CD117 (UniProt Ref. No: P10721-2, residues 26 to 516) as target protein, libraries of ankyrin repeat proteins as described above, and established protocols (see, e.g., Zahnd, C., Amstutz, P. and Pluckthun, A., Nat. Methods 4, 69-79, 2007), and is further described in EP24150551 .0.

[0378] Pools from the ribosome display were expressed in Escherichia coli cells in 96 well plates with a Flagtag and a His-tag. Crude extracts thereof were prepared to test binding of the Flag-tagged designed ankyrin repeat proteins to human CD117 target protein in an HTRF (Homogeneous Time Resolved Fluorescence) assay.

[0379] In a further step, selected designed ankyrin repeat proteins with binding specificity for CD117 were sequence engineered in order to improve serum half-life and biophysical properties.

[0380] Determination of dissociation constants (KD) of ankyrin repeat proteins with binding specificity for human CD117 by Surface Plasmon Resonance (SPR) analysis

[0381] The binding affinities of purified ankyrin repeat proteins to biotinylated recombinant human CD117-His target were analyzed using a ProteOn XPR 36 instrument (NAHLC200M, Xantec ProteOn Sensor Chip) and the measurement was performed according to standard procedures. Ankyrin repeat proteins were subcloned into derivatives of the pQE30 (Qiagen) expression vector, containing an N- terminal His-tag, and expressed and purified as described above. Briefly, biotinylated human CD117- His target was diluted in PBST (PBS, pH 7.4 containing 0.005% Tween 20®) and coated on an NAHLC200M chip (BioRad) to a level of around 650-700 resonance units (RU). The interaction of ankyrin repeat protein and human CD117 was then measured by injecting 200 pl running buffer (PBS, pH 7.4 containing 0.005% Tween 20®) containing serial dilutions (1 :3) of ankyrin repeat proteins, starting at optimized concentrations for each DARPin (3-20 nM), followed by a running buffer flow for at least 20 minutes at a constant flow rate of 100 pl / min (off-rate measurement). The regeneration was performed using 30 pl of 10 mM glycine, pH 2.5. The signals (i.e. resonance unit (RU) values) of an empty lane (no target coated) and a reference injection (i.e. injection of running buffer only) were subtracted from the RU traces obtained after injection of ankyrin repeat protein (double-referencing). Binding parameters (KD, on-rate, off-rate) against CD117 were determined for the constructs as shown in Table 3.

[0382] Table 3: Binding parameters for ankyrin repeat proteins with binding specificity for human CD117 by Surface Plasmon Resonance (SPR).

[0383] Assessment of competitive cell binding of selected CD117 specific ankyrin repeat proteins

[0384] In order to identify CD117 specific ankyrin repeat proteins which compete with SCF in binding to CD117 target, a cell binding competition assay was performed. Briefly, Kasumi-3 cells (CD117 expressing cell line; ATCC CRL2725) were seeded on a 96 well plate. After adding Fc block for 10 minutes at 4°C, the cells were washed and 1 nM of biot-hSCF was added to the cells for 30 minutes at 4°C, followed by centrifugation (350g, 4 minutes) and washed twice by adding 100 pl of FACS buffer. Then a titrated amount of DARPins or of a control benchmark monoclonal anti-CD117 antibody which does not compete with SCF (clone 104D2, Invitrogen, #MA1 -10072) were added to the cells (from 100 nM or 50 nM, 4-fold dilution) for 30 minutes at 4°C. The cells were then washed twice as described above and incubated with streptavidin-AF647 in order to detect bound SCF to the CD117 receptor on cells. After 20 minutes of incubation at 4°C, cells were washed twice and incubated with cell fix buffer for 20 min at 4°C. Cells were then washed, resuspended in 200ul of PBS / 2mM EDTA and measured in the Attune Nxt Flow Cytometer. In the presence of SCF competing DARPins a decrease of the signal was obtained in a dose dependent manner. aCD117-1 and aCD117-3 were identified as competing with SCF (“epitope A” CD117 specific DARPins), while aCD117-2 does not compete with SCF (“epitope B” CD117 specific DARPins). IC50 values of the DARPin proteins competing with SCF are shown in Table 4Fehler! Verweisquelle konnte nicht gefunden werden..

[0385] Table 4: IC50 values corresponding to the cell binding competition against SCF.

[0386] Cell binding by selected CD117-binding designed ankyrin repeat proteins

[0387] Designed ankyrin repeat proteins were further tested for CD117-specific cell binding. CD117 binding was confirmed by a cell binding assay using Kasumi-1 cancer cell line expressing CD117. In brief, the CD117 expressing cells were washed Ix with 500 pl PBS, spun at 350g for 5 minutes and the supernatant was discarded. Then, a titration of CD117 designed ankyrin repeat proteins diluted in FACS buffer was added to the pellet of cells, resuspended and incubated for 30 minutes at 4°C. The cells were washed twice with 200 pl cold PBS, spun at 350g for 5 minutes and the supernatant was discarded. 50 pl of anti-DARPin antibody AF-647 with live / dead aqua (1 :3000), was added to the cells and incubated for 30 mins at 4°C, in the dark. Then the cells were washed twice in 200 pl ice cold PBS and spun for 4 min at 400g. 50 pl of cell fix was added and the cells were incubated for 60 min at 4°C, then they were washed with PBS / 2mM EDTA, spun for 5 min at 500g and resuspended in 200 pl of PBS / 2mM EDTA. Flow cytometry analysis was performed using a Attune Nxt Flow Cytometer. All tested proteins show specific binding to the CD117 target on cells as shown in Table 5.

[0388] Table 5: EC50 values for CD117-binding ankyrin repeat proteins.

[0389] Example 4: Functional assessment of selected CD16a-CD117-bispecific binding

[0390] DARPin dependent cellular cytotoxicity

[0391] Selected CD117-specific ankyrin repeat proteins were formatted as bispecific immune cell engagers with an anti-CD16a specific ankyrin repeat domain (see Table 6). The cytotoxic activity of these engagers was tested in a DARPin-dependent cellular cytotoxicity (DDCC) assay using Jurkat-NFAT- CD16a reporter cells. Upon binding of DARPin to CD16a, these cells express the Lucia luciferase reporter gene. The extent of induced DDCC can therefore be quantified by a luminescent readout. Kasumi-1 cells, which express the CD117 receptor, were used as target cells in this assay. Briefly, Kasumi-1 target cells were plated in 96 well U-bottom plates one day prior to the assay at a density of 10,000 cells per well in DMEM medium supplemented with 10% FBS and were incubated overnight at 37°C. DARPin proteins serially diluted in assay medium were added to the Kasumi-1 cells. Finally, Jurkat-Lucia NFAT-CD16a Reporter cells were added at a concentration of 50,000 cells per well, so as to achieve an E:T ratio of 5:1. The assay incubation duration was 24h after which the assay plates were centrifuged at 300g for 5min. 50 pL of the supernatant from each plate was added onto Luminescence plates (Corning 3610) and mixed together with an equal volume (50 pL) of QuantiLuc substrate which was prepared as per the manufacturer’s guidelines. The resulting signal was measured as luminescence using a Tecan plate reader (Tecan Infinite M1000 Pro Reader).

[0392] Luminescence values are shown with a standard non-linear regression three parameter logistic model fit. As shown in Table 6, all tested constructs show specific and dose dependent cytotoxicity. The maximum levels of cytotoxicity observed correlated with the level of binding observed for the different CD117-CD16a specific DARPins tested.

[0393] Table 6: EC50 values and maximal signal (RLU-relative light units) obtained in the DDCC reporter assay for CD16a-CD117 bispecific constructs. The bispecific constructs further comprised an N- terminal His tag (SEQ ID NO: 20) for ease of purification.

[0394] NK cell degranulation

[0395] The ability of the bispecific ankyrin repeat proteins, aCD16a-CD117-1 , aCD16a-CD117-2, and aCD16a-CD117-3, to mediate activation of primary NK cells was further assessed by NK cell degranulation observed in the presence of target expressing cells. Peripheral Blood Mononuclear cells (PBMCs) were isolated from buffy coats from healthy donors using the human NK cell isolation kit (Miltenyi Biotec) according to manufacturer’s instructions. After isolation, NK cells were kept overnight in medium containing human interleukin-2 (hlL-2) at 37°C. Next, Kasumi-1 target cells, NK cells, titrations of the tested proteins and the anti-CD107a BV421 antibody (Biolegend) were added to a 96 well plate and incubated for 2 hours at 37°C. Cells were then centrifuged at 350g for 4 min at 4°C and washed with PBS, followed by cell resuspension with human Fc block (BD Biosciences) and incubation for 15 min at 4°C. After centrifugation, an antibody mix (CD56 BV605 BD and CD16 FITC Biolegend) was added to the cells in FACS buffer which were incubated for 30 min at 4°C. Cells were centrifuged, washed and stained with the Live Dead Zombie NIR (Biolegend) for 20 min at 4°C, then were washed and fixed / permeabilized with the FoxP3 / Transcription Factor staining buffer set (Invitrogen) following the instructions provided by the manufacturer. Cells were finally analysed by flow cytometry in the Attune NxT device.

[0396] Live NK cells (CD56+) were gated and levels of degranulation were obtained by assessing the degranulation marker CD107a. Levels of CD16 were also assessed since engagement of CD16 can lead to CD16 downregulation. Figure 2 shows that all tested DARPin proteins mediated efficient NK cell degranulation through CD16a engagement.

[0397] Macrophage induced cellular phagocytosis The activity of the bispecific ankyrin repeat proteins, aCD117-aCD16a-1 , aCD117-aCD16a-2, and aCD117-aCD16a-3, was also tested in a DARPin-dependent cellular phagocytosis (DDCP) assay by flow cytometry. Macrophages derived from monocytes isolated from cryopreserved Peripheral Blood Mononuclear cells (PBMCs) from healthy donors were used as effector cells. Kasumi-1 cells, which express CD117, were used as target cells. Briefly, monocytes were isolated from cryopreserved PBMCs using a CD14 ultrapure isolation kit (Miltenyi Biotec, 130-118-906), according to manufacturer’s instructions. After isolation, monocytes were differentiated into MO-like macrophages by in vitro culture with M-CSF (Miltenyi Biotec, 130-093-866) in Cellgenix GMP DC medium for six days (final well concentration of 0.05 ug / ml). Macrophages were then harvested, washed and seeded overnight in an assay plate. The next day, Kasumi-1 target cells were harvested, labelled with a proliferation dye (Cell Trace TM Yellow cells, ThermoFisher) according to the manufacturer’s instructions and pre-incubated with the tested proteins. Kasumi-1 cells were then added on top of the pre-seeded macrophages at an effector to target ratio of 5:1 for 4 hours at 37°C. After co-culture, plates were centrifuged and cells were harvested using dissociation buffer (Cell dissociation buffer, Gibco, 13151014). Cells were washed and stained for viability (Zombie Aqua viability dye, Biolegend, 30 min at 4°C). After a washing step, cells were incubated with Fc blocking reagent (Miltenyi Biotec, 130-059-901) for 5 min at 4°C. Cells were then centrifuged, washed twice and stained with an antibody mix (containing anti-CD11 b APC Miltenyi Biotec 130-110-554) for 30 min at 4°C. Cells were centrifuged and washed twice and 30 pl of fix buffer was added for 20 min at RT. Finally, cells were washed twice, resuspended in 150ul of PBS + 2nM EDTA and acquired on the Attune NxT flow cytometer. The percentage of DDCP corresponds to the percentage of CD11 b+CellTrace+ cells. As can be seen in Figure 3, all tested proteins mediated potent cellular phagocytosis by macrophages in a dose dependent manner.

[0398] Example 5: Selection of binding proteins comprising a 2-i n-1 ankyrin repeat domain with binding specificity for CD117 and CD47-binding ankyrin repeat domain

[0399] Selection of ankyrin repeat proteins with binding specificity for CD47-binding DARPins

[0400] Using ribosome display (Hanes, J. and Pluckthun, A., PNAS 94, 4937-42, 1997), ankyrin repeat proteins with binding specificity for CD47-binding DARPins were selected from DARPin libraries essentially as described by Binz et al. 2004 (loc. cit.). A CD47-binding DARPin comprising the amino acid sequence with SEQ ID NO: 8 described in Example 1 was used as target material for the ribosome display selection. Generation of this ankyrin repeat protein with binding specificity for CD47- binding DARPins as well as generation of 2-in-1 binding domains is further described in EP24150569.2.

[0401] The binding of the selected clones towards the targets was assessed by crude extract Homogeneous Time Resolved Fluorescence (HTRF). Briefly, pools from the ribosome display were expressed in E. coli cells with an N-terminal Flag-tag (SEQ ID NO: 22). Crude extracts thereof were prepared to test binding of the Flag-tagged DARPin proteins to biotinylated CD47-binding DARPins. Clones were shown to bind specifically to CD47-binding DARPins. Generation of2-in-1 binding domains

[0402] Selected ankyrin repeat proteins with binding specificity for CD47-binding DARPins were then fused with the CD117-binding ankyrin repeat domains described in Example 3. The fusion proteins were further engineered, including replacement of the “RILLA” motif was replaced with “RELLK” in the N- terminal capping module of CD117-binding ankyrin repeat domains, mutation of position 15 in the N- terminal capping module, transformation of a terminal capping module into an internal repeat module, or vice versa as well as engineering of potential target interaction residues of some repeat modules to create a range of binding affinities for the CD47-binding ankyrin repeat domain.

[0403] For subsequent characterization, 2-in-1 domains were cloned following standard procedures and expressed with a N-terminal Flag-tag of SEQ ID NO: 22.

[0404] Selected 2-in-1 domains were then assessed for their binding behavior to CD117 or CD47-binding DARPin compared to the binding behavior of their respective parental DARPins. A titration of the 2-in- 1 domains to evaluate their binding response to targets CD117 or CD47-binding DARPin was performed in an ELISA. Biotinylated human CD117 target material was purchased from AcroBiosystems (Catalogue num. CD7-H82E6) which also comprises a His and Avi-tag. Each of CD47-binding DARPin of SEQ ID NOs: 25 and 26 were tagged with a N-terminal His-tag of SEQ ID NO: 20 and with a C-terminal Avi-tag of SEQ ID NO: 23, and biotinylated. Targets were coated at 20nM on neutravidin coated Nunc Maxisorp 96-well plates (Thermo 442404). Serial dilutions (1 :3) were performed. Signal was detected using an anti-Flag-tag-HRP antibody (SIGMA, A8592) and a Tecan sunrise reader (OD 450 nm, ref 620 nm). BC50 (half-maximum binding concentration) values are shown in Table 7.

[0405] Table 7: BC50 (half-maximum binding concentration) values of selected 2-in-1 binding domains with binding specificity to CD117 and CD47-binding ankyrin repeat domain.

[0406] Mutual binding exclusivity assessment of the 2-in-1 domains by competitive ELISA

[0407] In this experiment, the mutual binding exclusivity property of representative 2-in-1 domains were further assessed in a competitive binding ELISA. A schematic view of the experimental setup is shown in Figure 4A. The tested domains were 2-in-1 DARPin #01 with SEQ ID NO: 12, 2-in-1 DARPin #02 with SEQ ID NO: 13, and 2-in-1 DARPin #04 with SEQ ID NO: 15.

[0408] In a first step, an optimal concentration of each tested 2-in-1 domain was determined by ELISA on the CD117 target (Fc-tagged human CD117, AcroBiosystems CD7-H5255), which was coated on 96 well plates (F96 MaxiSorp NUNC Immuno plate). 2-in-1 domains were titrated and the BC90 concentration (i.e. 90% maximum binding concentration) was selected at a fixed concentration of each 2-in-1 domain for the subsequent challenge with the competitor. The competitor was the CD47-binding DARPin of SEQ ID NO: 8, further comprising an N-terminal His-tag of SEQ ID NO: 20 and a C- terminal Avi-tag of SEQ ID NO: 23.

[0409] In a second step, a titration of the competitor (5000, 1250, 312.50, 78.13, 19.53, 4.88, 1 .22 and 0.31 nM) was added to the 2-in-1 domain (used at the corresponding fixed BC90 cone.) and incubated for 1 hour prior to detection. The signal was detected via the flag-tag (HRP Anti-DDDDK tag, Abeam, ab2493) on each 2-in-1 domain, which allowed to visualize the extent of competition of the competitor with the interaction of the 2-in-1 domain and the coated CD117 target. A reduction of the signal in function of the increasing competitor concentration therefore indicates a mutually exclusive binding character of the 2-in-1 domain.

[0410] Figure 4B shows the resulting measures with fitted curves, where the plots on the left show the 2-in-1 titration (points) and the evolution of the signal at the fixed BC90 concentration (triangles) where the titrated competitor is applied. The plots on the right in Figure 4B show the corresponding evolution of the signal in function of the competitor concentration. Accordingly, mutual binding exclusivity to CD117 and CD47-binding DARPin was observed for all tested 2-in- 1 domains. Curves represent a fitted four-parameters sigmoid model.

[0411] Example 6: Generation and Characterization of multispecific constructs

[0412] Multispecific binding proteins in various formats were generated (see Table 8) alongside appropriate controls (see Table 9). The order of the different domains as indicated in the tables reflects the actual sequence of the domains from N-terminus to C-terminus in the molecular structure of the proteins. The individual domains were linked with a linker with SEQ ID NO: 25, e.g., in the format of CD47- binding domain (“aCD47”) - linker - 2-in- 1 domain (“2-in- 1 ”) - linker - CD117-binding domain (“aCD117”) - linker - CD16a-binding domain ("aCD16a”) - linker - HSA binding domain (“aHSA”). All proteins additionally comprised a His-tag (SEQ ID NO: 20) at the N-terminus for ease of purification. Constructs were expressed and purified using their His-tag according to standard protocols. Briefly, E. coli BL21 cells were transformed with the ankyrin repeat proteins, plated on LB-agar (containing 1% glucose and 50 pg / ml ampicillin) and then incubated overnight at 37°C. For each construct, a single colony was picked into 50 ml of TB medium (containing 1% glucose and 50 pg / ml of ampicillin) and incubated overnight at 37°C, shaking at 220 rpm. The stationary overnight cultures were used to inoculate TB medium (containing 50 pg / ml ampicillin) and incubated at 37°C, shaking at 220 rpm. At an absorbance of 1 .0 to 1 .5 at 600 nm, the cultures were induced with 0.5 mM IPTG and incubated further for 4 to 5 hrs. The cultures were centrifuged, and the resulting pellets were re-suspended in 25 ml of TBS500 (50 mM Tris-HCI, 500 mM NaCI, pH 8) and lysed (sonication). Following the lysis, the samples were mixed with 50 KU DNase / ml and incubated for 15 minutes prior to a heat-treatment step for 30 minutes at 62.5°C, centrifuged and the supernatant was collected and filtrated. Tergitol (1% (v / v) final concentration) and imidazole (20 mM final concentration) were added to the homogenate. Proteins were purified over a Ni-nitrilotriacetic (Ni-NTA) acid column followed by a size exclusion chromatography on an AKTAxpress™ system according to standard protocols.

[0413] These multispecific proteins all comprised a CD47-binding ankyrin repeat domain, a 2-in-1 ankyrin repeat domain comprising (i) a binding region binding the CD47-binding ankyrin repeat domain (“mask”) and (ii) a CD117-binding region, a CD16a-binding ankyrin repeat domain, as well as an HSA-binding ankyrin repeat domain. Some variants also comprised a further CD117-binding ankyrin repeat domain. All proteins additionally comprised a His-tag (SEQ ID NO: 20) at the N-terminus for ease of purification.

[0414] Table 8: Multispecific proteins in various domain formats. The individual domains were linked with a linker with SEQ ID NO: 25 in the format of CD47-binding domain (“aCD47”) - linker - 2-in-1 domain (“2-in- 1 ”) - linker - CD117-binding domain (“aCD117”) - linker - CD16a-binding domain ("aCD16a”) - linker - HSA binding domain (“aHSA”).

[0415] Table 9: Control proteins. Ni2C: non-binding randomised control; HSA: human serum albumin; anti- aCD47: ankyrin repeat domain binding ankyrin repeat domain with binding specificity for CD47 (“aCD47”); a[target]: ankyrin repeat domains with binding specificity for [target]. Individual domains were linked with peptide linker with SEQ ID NO: 25.

[0416] Cell binding assay

[0417] Cell binding assays were carried out on the AML cell line Kasumi-1 (DSMZ ACC 220) (CD1177CD47+) as well as engineered cell lines CHO-k1 hCD117 (CD117+) (lentiviral transduction of CHO-k1 cell line to express human CD117), CHO-k1 hCD47 (CD47+) (lentiviral transduction of CHO- k1 cell line to express human CD47) to address the question whether the constructs bind to CD117, and selectively release the CD47-binding agent in the presence of CD117.

[0418] Per well of a 96-well U-bottom plate, 50,000 cells were seeded, spun down at 350g for 4 minutes at 4 °C and the supernatant decanted. Multispecific binding proteins and controls were prepared in FACS buffer (PBS + 2 % FBS + 10 pM HSA) and added to the cell pellets. Cells and binding proteins were incubated for 30 min at 4°C, and washed by adding 150 pl FACS buffer and centrifuging at 350g for 4 min. Supernatant was decanted, and cells washed with 200 pl FACS buffer, centrifuged at 350g for 4 min and the supernatant decanted. Finally, cells were stained with 50 pl per well of live-dead green (1 :3000) and anti-penta his-AF647 (1 :200) diluted in FACS buffer. Cells were incubated for 30 min at 4°C and washed twice as described above. Finally, cells were resuspended in 50 pL / well of 1 :10 diluted cell fix buffer in water, incubated at 4°C for 20 min, followed by the addition of 150 pl PBS / 2mM EDTA. Cells were centrifuged at 400g for 4 min, supernatant aspirated, and the cells resuspended in 200 pL PBS / 2mM EDTA, and acquired on an Attune NxT flow cytometer.

[0419] As shown in Figure 5A-H and Table 10, multispecific binding proteins show significantly improved binding to Kasumi-1 cells, compared to CHO-k1 hCD47 (CD47+), indicating that the presence of CD117 is necessary and sufficient to selectively release (“unmask”) the CD47-binding ankyrin repeat domain from the mask of the 2-in-1 domain. All multispecific binding proteins bind to CHO-k1 hCD117 (CD1177CD47 ) cells. Shown are the measured median fluorescent intensity (MFI AF647). Curves represent a fitted four-parameters sigmoid model.

[0420]

[0421] Table 10: EC50 values for multispecific binding proteins and controls obtained in cell binding assay on CD1177CD47-, CD1177CD47+, and CD1177CD47+cells. N.d. not determinable.

[0422] Cell binding competition assay

[0423] In order to address the question whether the selective release of the CD47-binding agent in the presence of CD117 occurs, the multispecific binding proteins were tested on CD1177CD47+cells (Kasumi-1) versus CD47+cells (CHO-k1 hCD47), in the presence of biotinylated CD47-binding ankyrin repeat domain. A schematic view of the experimental setup is provided in Figure 6A. If the CD47-binding ankyrin repeat domain is released from the conditional mask in the presence of CD117, the CD47-binding agent competes with biotinylated CD47-binding ankyrin repeat domain for binding to CD47 on target cells in a dose dependent manner. If the CD47-binding agent is not released from the conditional mask in the presence of CD117, the CD47-binding agent cannot bind to CD47 on the target cells, and therefore does not compete with biotinylated CD47-binding ankyrin repeat domain. The lack of competition on CHO-k1 hCD47 cells confirms CD117 mediated release of the conditional mask.

[0424] Kasumi-1 or CHO-K1 hCD47 cells were plated at a density of 50,000 cells per 96-well plate. Cells were collected by centrifugation at 350g for 4 min at 4°C and removal of the supernatant. 50 pl of increasing concentrations of multispecific binding proteins diluted in FACS buffer (PBS + 2 % FBS + 10 pM HSA) were added to the cells and incubated for 30 min at 4°C and washed twice in FACS buffer. Biotinylated CD47-binding ankyrin repeat domain comprising the amino acid sequence with SEQ ID NO: 8 was added to the cells at a concentration of 10 nM in FACS buffer and incubated for 30 min at 4°C. Cells were washed twice in FACS buffer, resuspended in 50 pl streptavidin AF647 (Invitrogen, S32357) with live dead green (Invitrogen, L34970) (1 :3000) and incubated for 20 min at 4°C. Cells were washed and fixed with 1 % paraformaldehyde (Himedia, TCL119) for 30 min at 4°C, followed by the addition of 2 mM EDTA in PBS. Cells were centrifuged, supernatant removed and resuspended in 2 mM EDTA in PBS. Fluorescence was determined by Attune NxT Flow Cytometer. As shown in Figure 6B-I and Table 11 , the capacity of the multispecific binding proteins to compete with biotinylated CD47-binding ankyrin repeat domain for binding to CD47 target is higher in the presence of CD117. This shows that the presence of CD117 is necessary and sufficient for efficient unmasking of the CD47-binding ankyrin repeat domain comprised in the multispecific binding proteins disclosed herein. Shown are the measured median fluorescent intensity (MFI AF647). Curves represent a fitted four-parameters sigmoid model.

[0425] Table 11 : IC50 values for multispecific binding proteins and controls obtained in competitive cell binding assay on CD1177CD47+(CHO-K1 hCD47) and CD1177CD47+(Kasumi-1) cells. N.d. not determinable.

[0426] CD47 / SIRPa competition assay

[0427] In order to address the question whether in the presence of CD117 only, the CD47-binding ankyrin repeat domain is released from its binding to the anti-CD47-binding ankyrin repeat domain, it was assessed whether the CD47 / SIRPa pathway was inhibited. The multispecific binding proteins were tested for SIRPIa competition using the PathHunter Jurkat SIRPa Signaling Bioassay Kit (Eurofins DiscoverX products LLC). Ligand engagement through co-culture of Jurkat CD47 presenting cells (CD477CD117 ) or Kasumi-3 cells (CD477CD117+) and Jurkat SIRPIa signaling cells results in phosphorylation and intracellular pathway activation leading to a chemiluminescence signal. This signal can be inhibited in the presence of a binding protein that binds to CD47 or SIRPa, thereby disrupting ligand-receptor interaction. As shown in Figure 7A and B, multispecific binding proteins #1 to 7 (Figure 7A) were able to conditionally inhibit CD47 signaling in the presence of CD117. CD47 signaling is not inhibited in Jurkat-hCD47 cells, which do not express CD117. Here, the CD47 ankyrin repeat domain remains bound to its mask.

[0428] Assessment of cellular cytotoxicity mediated by multispecific binding proteins

[0429] It was also assessed whether the multispecific binding proteins induced activation of DARPin dependent cellular cytotoxicity (DDCC). The assay was performed essentially as described above in Example 4, except that Kasumi-1 target cells were plated at a density of 100,000 cells per well while Jurkat-Lucia NFAT-CD16a Reporter cells were added at a concentration of 100,000 cells per well, so as to achieve an E:T ratio of 1 :1 . In order to assess the levels of specific DDCC activity, Jurkat cells (CD477CD16+) in the absence of target cells were used.

[0430] Multispecific binding proteins #1 to 7 showed specific and dose dependent cytotoxicity in the presence of CD117 and CD47, while DDCC was significantly lower or not detectable for the multispecific binding proteins in the absence of CD117 as shown in Figure 8A and Table 12. As shown in Table 12, in the absence of a masking moiety (Control #1), potent DDCC is obtained in the presence but also absence of target cells showing unspecific activity. This unspecific activity is abolished or significantly decreased in the absence of a CD47-specific ankyrin repeat domain (Control #2) or in the presence of a CD47-mask which is released in a CD117-dependent manner.

[0431] Table 12: EC50 values obtained in the DDCC reporter assay for multispecific binding proteins and controls. N.d.: not determinable.

[0432] In a second experiment, it was assessed whether NK cells from healthy donors were able to induce specific lysis of CD34+ mobilised cells from healthy donors. NK cells were isolated from PBMC using established protocols and CD16a expression was confirmed by flow cytometry analysis. Mobilised CD34+ cells from healthy donors were commercially obtained (Allcells). Target expression of CD117 and CD47 on CD34+ cells was confirmed by flow cytometry analysis.

[0433] 10'000 target cells for E:T 5:1 were plated to a U-bottom plate. 50 pl of multispecific binding protein MSC #2 , a multispecific binding protein with a non-binding DARPin instead of the CD47-binding domain, or a non-binding DARPin were added at an 8-point titration (between 10 nM and 0.001 nM) and incubated for 30 min at 37°C / 5% CO2. Effector NK cells (CD56+, CD16+, CD47+) were added at the indicated E:T ratio and incubated for 24h at 37°C / 5% CO2. Specific lysis was calculated based on the reduction of the target CD34+ population (in percentage) after 24 h

[0434] As shown in Figure 8B, specific lysis was obtained upon 24 h coculture of PBMC-purified NK cells and CD34+ CD47+ CD117+ mobilized cells from healthy donors in the presence of MSC #2, whereas the cytotoxic mediated effect was reduced in the absence of a CD47-binding domain.

[0435] Assessment of macrophage induced cellular phagocytosis mediated by multispecific binding proteins

[0436] It was further tested whether the multispecific binding proteins were able to induce effector cells (M0 macrophages) to phagocytose target cells. To assess whether macrophage-induced cellular phagocytosis is conditional to the presence of CD117, the assay was carried out on Kasumi-1 (CD477CD117+) versus CHO-hCD47 (CD477CD117 ) target cells.

[0437] The assay was performed essentially as described above in Example 4. The multispecific binding protein was applied at four different concentrations (1 nM, 0.2 nM, 0.04 nM, and 0.008 nM) to Kasumi- 1 target cells, and at two different concentrations (1 nM, 0.2 nM) to CHO-hCD47 target cells.

[0438] Multispecific binding proteins #1 to 7 induced specific and dose dependent phagocytosis in the presence of CD117 and CD47, while DDCP was at background level in the absence of CD117 as shown in Figure 9. In the absence of a CD47-mask (see Control#4), higher levels of unspecific DDCP of CHO-hCD47 cells were obtained, while this was significantly reduced in the presence of a CD47- mask (see Control #3). Lack of CD117 conditional release of the CD47-mask results in lower DDCP levels compared to constructs where conditional unmasking occurs (multispecific binding proteins #1 to 7). This demonstrates that conditional unmasking of the CD47-binding agent comprised in the multispecific binding proteins upon binding to CD117 on target cells leads to efficient and specific DDCP through blockade of CD47, while also engaging the CD16a effector function of macrophages. In the absence of CD47 blockade, the levels of phagocytosis of the target cells by the effector cells is lower. Conditional Blockade of CD47 and target engagement in the presence of CD16a receptor engagement triggers effective phagocytosis.

[0439] In a second experiment, selected proteins tested for their ability to induce phagocytosis of Kasumi-1 cells by MO-like macrophages. Macrophages were generated as described above and re-seeded at 15'000 cells per well. The next day, Kasumi-1 cells were pHrodo-labelled (manufacturer’s instructions) and 60'000 cells added to achieve an E:T ratio of 1 :4. Titrations (5 nM, 1.2, 0.3, 0.08 and 0.02 nM) of multispecific binding protein MSC#2 or control antibodies (Fc-active (lgG1) anti-CD117 Ab based on JSP-191 , magrolimab (anti-CD47 antibody lgG4) or a combination of both as well as a non-binding control DARPin containing a CD16a binding domain (SEQ ID NO: 9) were added to the co-culture. Cells were then observed for 48h using Incucyte scan using phase and red fluorescent channel at 10x magnification and scanned every hour for the first 24 hours and every other hour thereafter. 2 images / well were captured per timepoint.

[0440] Figure 9C demonstrates higher macrophage-mediated phagocytosis activity of MSC#2 compared to an Fc-active anti-CD117 antibody, magrolimab (anti-CD47 antibody) or a combination of both.

[0441] Furthermore, the specific activity of MSC #2 was demonstrated by its ability to induce phagocytosis of pHrodo-labelled CD117+CD47+ (Kasumi-1 cell line), but not of CD117-CD47+ (Raji cell line) by M0- like macrophages. The experimental setup was as described above. MSC #2 was tested at different concentrations (100 nM, 33 nM, 11 nM). As shown in Figure 9D-E, levels of phagocytosis of CD117- target cells were comparable to untreated cells (D), whereas MSC #2 stimulated significant levels of phagocytosis of Kasumi-1 target cells by MO-like macrophages (E).

[0442] Blockade of c-Kit signaling

[0443] In order to address the question whether the CD117-binding ankyrin repeat domains comprised in the multispecific binding proteins blocks CD117, multispecific binding proteins #1 to 7 and appropriate controls were tested using the PathHunter express c-Kit functional assay (Eurofins DiscoverX). This reporter assay is designed to measure the activation of the receptor tyrosine kinase (RTK) pathway specifically associated with CD117 receptor. When CD117 on U2OS reporter cells is activated by its ligand SCF, it undergoes dimerization, triggering the recruitment of SH2-EA. This recruitment results in the assembly of a functional enzyme complex, which facilitates the hydrolyzation of a substrate, and the enzymatic activity leads to the generation of a chemiluminescent signal. This signal can be inhibited in the presence of a binding protein that binds to CD117, thereby disrupting ligand-receptor interaction.

[0444] The assay was performed according to manufacturer’s instructions. Briefly, U2OS reporter cells were seeded and cultured for 48 hours at 37°C / 5% CO2. Titrations of multispecific binding proteins and control constructs were prepared, and added to the cells, which were incubated for 1 hr. Human SCF was added and incubated for 3 hrs at room temperature. Detection agent was added, cells incubated for 1 hr at room temperature, followed by detection of the luminescent signal on a Tecan plate reader. Also included in the assay were monoclonal antibodies binding to CD117, AMG191 (WO2007127317), which competes with SCF for binding to CD117, and 104D2 (ThermoFisher Scientific, #MA1 -10072), which does not compete with SCF for binding to CD117. As shown in Table 13, the multispecific binding proteins MSC #1 to 7 block signaling by SCF. Further, no agonist activity was observed by the multispecific binding proteins.

[0445]

[0446] Table 13: IC50 values obtained in the c-Kit reporter assay for multispecific binding proteins and an antibody control. N.d. not determinable.

[0447] Example 7: Pharmacokinetic analysis of recombinant proteins in female BALB / c mice

[0448] In order to determine whether selected multispecific ankyrin repeat proteins have an appropriate serum half-life in vivo, the pharmacokinetic profiles of MSC#2 (SEQ ID NO: 2), MSC#5 (SEQ ID NO: 5), MSC#6 (SEQ ID NO: 6) and MSC#7 (SEQ ID NO: 7), each comprising a C-terminal human serum albumin binding ankyrin repeat domain (SEQ ID NO: 17) for half-life extension and further an N- terminal His-tag, were analysed in mice.

[0449] In vivo administration and sample collection

[0450] Each fusion protein was administered as a single intravenous bolus injection in phosphate-buffered saline (PBS) solution into the tail vein of 6 mice. The target dose level was 1 mg / kg with an application volume of 5 mL / kg.

[0451] Four serum samples were collected from each mouse. Blood samples for pharmacokinetic investigations were collected from the saphenous vein at 5 min, 6 h, 24 h, 48 h, 72 h, 96 h and 168 h post compound administration. Blood was kept at room temperature to allow clotting followed by centrifugation and collection of serum.

[0452] Bioanalytics by ELISA to measure ankyrin repeat proteins in serum samples

[0453] 100 pL per well of 0.5 ug / mL rabbit anti-DARPin 1.1.1 antibody in PBS was coated onto a NUNC MaxiSorp ELISA plate overnight at 4°C. After washing each well three times with 300 pL PBST (PBS supplemented with 0.1% Tween20), the wells were blocked with 300 pL PBST supplemented with 0.25% Casein (PBST-C) for 1 h at room temperature (RT) on a Heidolph Titramax 1000 shaker (450 rpm). Plates were washed as described above. 100 pL of diluted serum samples (1 :20 - 1 :312500 in 1 :5 dilution steps) or ankyrin repeat protein standard curve samples (0 and 50 - 0.0008 nmol / L in 1 :3 dilution steps) were applied for 2 h, at RT, shaking at 450 rpm. Plates were washed as described above.

[0454] 100 pL of 0.125 pg / mL human anti-DARPin 1.4.8 (H1) antibody in PBST-C was added and the plates were incubated at RT (22°C) with orbital shaking (450 rpm) for 1 h. Plates were washed as described above.

[0455] Wells were then incubated with 100 pL HRP conjugated anti-human IgG antibody and incubated for 1 h, at RT, shaking at 450 rpm. Plates were washed as described above. The ELISA was developed using 100 pL / well TMB substrate solution for 10 minutes and stopped by the addition of 100 pL 1 mol / L H2SO4. The difference between the absorbance at 450 nm and the absorbance at 620 nm was calculated. Samples were measured in duplicate on two different plates. Figure 10 shows the serum concentration of MSC #2 (A), MSC #5 (B), MSC #6 (C) and MSC #7 (D) respectively, as a function of time after the single intravenous administration into mice. The traces indicate roughly monoexponential elimination of the compounds.

[0456] Pharmacokinetic analysis

[0457] Pharmacokinetic data analysis was performed using Version 8.3 of the WinNonlin program as part of Phoenix 64, Pharsight, North Carolina. Calculation of the pharmacokinetic parameters based on the mean concentration-time data of the animals dosed via intravenous bolus injection was performed with non-compartmental analysis (NCA model 200-202, IV bolus, linear trapezoidal linear interpolation). The following pharmacokinetic parameters were calculated: AUCinf, AUCIast, AUC_%extrapol, Cmax, Tmax, Cl_pred, Vss_pred, t1 / 2

[0458] Maximum serum concentrations (Cmax) and the times of their occurrence (Tmax) were obtained directly from the serum concentration-time profiles. The area under the serum concentration-time curve (AUCinf) was determined by the linear trapezoidal formula up to the last sampling point (Tlast) and extrapolation to infinity assuming mono-exponential decrease of the terminal phase. The extrapolation up to infinity was performed using Clast I Az, where Az denotes the terminal rate constant estimated by log linear regression and Clast denotes the concentration estimated at Tlast by means of the terminal log-linear regression. Total serum clearance (Cl_pred) and the apparent terminal half-life were calculated as follows: Cl_pred = i.v. dose I AUCinf and t1 / 2 = In2 I Az. The steady-state volume of distribution Vss was determined by: Vss = i.v. dose • AUMCinf / (AUCinf)2. AUMCinf denotes the total area under the first moment of drug concentration-time curve extrapolated to infinity using the same extrapolation procedure as described for calculation of AUCinf. To calculate PK parameters based on concentrations given in nmol / L dose values given as mg / kg were converted to nmol / kg by using the molecular weight of the ankyrin repeat proteins. Table 14 shows the summary of pharmacokinetic characteristics of the four tested ankyrin repeat proteins MSC #2, MSC #5, MSC #6 and MSC #7 following single intravenous administration of 1 mg / kg.

[0459] Table 14: Pharmacokinetic parameters for four exemplary multi specific ankyrin repeat proteins. Each ankyrin repeat protein comprises a C-terminal human serum albumin binding ankyrin repeat domain (SEQ ID NO: 17).

[0460] Example 8: In Vivo efficacy evaluation of exemplary multi-specific binding proteins in CD34+ humanized mice.

[0461] Two different designed ankyrin repeat proteins with binding specificity for human serum albumin, CD47, CD117 and CD16a - control #1 protein (SEQ ID NO:30, without a masking moiety) and multispecific binding protein #2 (SEQ ID NO: 2, comprising a masking moiety for CD47) - were tested in a CD34+ humanized mouse model and compared to a benchmark anti-CD117 monoclonal antibody (based on the sequence of AMG191) and a benchmark anti-CD47 monoclonal antibody (based on the sequence of magrolimab).

[0462] Materials and Methods

[0463] Animals: 50 female NSG mice, age of animals at study initiation 21 to 22 weeks (provider Jackson laboratories). NSG mice were humanized by engraftment of human cord blood-derived CD34+ stem cells isolated from two donors. 25 NSG mice were engrafted per donor.

[0464] Method: 7 to 23 days before the start of the treatment the frequency of human CD45 cells (hCD45) in the blood was assessed in 100 pl blood collected from each animal and flow cytometry analysis was performed using antibodies listed in Table 15. Mice were randomized to have equal mean frequency of human CD45 cells in the blood and similar standard deviation in each treatment group.

[0465]

[0466] Table 15: Flow cytometry panel used for humanization assessment in blood.

[0467] Test and control molecules: Control #1 and multispecific binding protein #2 were produced at a concentration of 6.5 mg / ml and 10.8 mg / ml, respectively; benchmark anti-CD117 antibody at 1.43 mg / ml and benchmark anti-CD47 antibody at 1 .51 mg / ml were provided by a commercial provider.

[0468] Treatment groups: 50 mice were enrolled in the study. All animals were randomly allocated to the 5 different study groups (ten mice per group with five each for the two cord blood donors). The treatment start date is denoted as day 0 (DO).

[0469] On day 0 treatment was started with intravenous or intraperitoneal injections according to Table 16.

[0470] Table 16: Allocation of Treatment Groups and Treatment Scheme. Every group comprised ten mice (5 per cord blood donor). D, day.

[0471] On day 7 mice were euthanized, blood collected in EDTA-coated tubes and bone marrow samples were processed within 2 hours for flow cytometry analyses. Bone marrow cell suspension was counted and 106cells were stained for analysis. 100 pl of blood samples were stained for analysis. The antibody cocktail for the surface staining was prepared using antibodies listed in Table 17. After surface staining, cell suspension was fixed and acquired on flow cytometry device Cytek Aurora (4Laser, 16V-14B-10YG-8R).

[0472] Table 17: Flow cytometry panel used for bone marrow and blood analysis on the day of termination.

[0473] Results

[0474] Treatment with multispecific binding protein #2 targets human CD117+ HSCs engrafted in CD34+ humanized NSG mice, while preventing elimination of peripheral hCD45+ cells observed with unconditional anti-CD47 blockade.

[0475] In blood, multispecific binding protein #2 did not have a negative impact on the total hCD45% compartment, unlike the combined treatment with anti-CD117 antibody and anti-CD47 antibody, where hCD45+ cells were depleted after treatment (Figure 11 A). CD117+ cells, with a stem-like signature (CD117+CD34+ within CD45RA-HLADR+CD45dim), were reduced in frequency in the bone marrow of mice treated with multispecific binding protein #2 (depicted dose: 1 mg / kg). This reduction follows the same trend observed in the group treated with the anti-CD117 antibody (Figure 11B).

[0476] CD117+ cells within a CD34- compartment in the bone marrow, were also reduced in frequency upon treatment with multispecific binding protein #2, with significantly greater reduction in comparison than with the anti-CD117 antibody-treated group (Figure 11C).

Claims

CLAIMS1 . A multispecific binding protein comprising (1) a first binding agent with binding specificity for an immunoregulatory protein, (2) a second binding agent with a first binding region with binding specificity for a first target, wherein said first target is the first binding agent, and a second binding region with binding specificity for a second target, wherein said second target is a stem cell-associated antigen, and wherein binding of said second binding agent to the first and second targets is mutually exclusive.

2. The multispecific binding protein of claim 1 , wherein the first binding region of said second binding agent reversibly binds said first binding agent, and wherein said first binding region is released upon binding of the second binding region to said stem cell-associated antigen.

3. The multispecific binding protein of any one of claims 1 or 2, comprising a further binding agent with binding specificity for an immune cell-associated antigen.

4. The multispecific binding protein of any one of the preceding claims, comprising a further binding agent with binding specificity for said stem cell-associated antigen.

5. The multispecific binding protein of any one of the preceding claims, wherein said immunoregulatory protein is CD47.

6. The multispecific binding protein of any one of the preceding claims, wherein said immune cell-associated antigen is CD16a.

7. The multispecific binding protein of any one of the preceding claims, wherein said stem cell- associated antigen is CD117.

8. The multispecific binding protein of any one of the preceding claims, wherein said binding agents are ankyrin repeat domains.

9. The multispecific binding protein of any one of the preceding claims, wherein a. said first binding agent comprises an ankyrin repeat domain with binding specificity for CD47, comprising an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 8; b. said second binding agent comprises an ankyrin repeat domain comprising an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 12 to 16; c. said further binding agent with binding specificity for an immune cell-associated antigen comprises an ankyrin repeat domain with binding specificity for CD16a, comprising an amino acid sequence at least about 80% identical to the amino acid sequence of SEQ ID NO: 9; and / or d. said further binding agent with binding specificity for a stem cell-associated antigen comprises an ankyrin repeat domain with binding specificity for CD117 comprising anamino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NO: 10 or 11.

10. The multispecific binding protein of any one of the preceding claims, wherein said multispecific binding protein further comprises a half-life extending moiety.11 . The multispecific binding protein of claim 10, wherein the half-life extending moiety comprises a designed ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 17 to 19.

12. The multispecific binding protein of any one of the preceding claims, comprising an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 1 to 7.

13. A nucleic acid encoding the recombinant binding protein of any one of the preceding claims.

14. A vector comprising the nucleic acid of claim 13.

15. A cell comprising the nucleic acid of claim 13 or the vector of claim 14.

16. A method of producing a multispecific binding protein, the method comprising culturing the cell of claim 15 and collecting the multispecific binding protein from the cell and / or the culture medium.

17. A pharmaceutical composition comprising the multispecific binding protein of any one of claims 1 to 12, the nucleic acid of claim 13, the vector of claim 14, or the cell of claim 15, and a pharmaceutically acceptable carrier and / or diluent.

18. A method of treatment in a subject in need thereof, the method comprising administering to the subject an effective amount of the multispecific binding protein of any one of claims 1 to 12, the nucleic acid of claim 13, the vector of claim 14, the cell of claim 15, or the pharmaceutical composition of claim 17.

19. A method of selectively depleting or ablating a hematopoietic stem cell (HSC) or progenitor cell population in a subject in need thereof, the method comprising administering to the subject an effective amount of the multispecific binding protein of any one of claims 1 to 12, the nucleic acid of claim 13, the vector of claim 14, the cell of claim 15, or the pharmaceutical composition of claim 17.

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