Designed repeat domains with dual binding specificity for CD117 and CD47-binding agent

Abstract

The present invention relates to recombinant binding proteins comprising a first designed ankyrin repeat domain which has a first binding specificity for a first target and a second binding specificity for a second target, wherein the binding of said first domain to said first and second targets is mutually exclusive, and wherein the first target is CD117 and the second target is a CD47-specific binding agent. The invention also provides further recombinant proteins comprising said first dual-specific repeat domain and additional therapeutic agents such as binding agents. Further disclosed are nucleic acids encoding such recombinant proteins, pharmaceutical compositions comprising such recombinant proteins or nucleic acids and the use of such recombinant proteins, nucleic acids or pharmaceutical compositions in methods for treating diseases, such as cancer in a mammal, including a human. Designed ankyrin repeat domains with binding specificity for a CD47-specific ankyrin repeat domain are also disclosed.

Description

[0001] DESIGNED REPEAT DOMAINS WITH DUAL BINDING SPECIFICITY FOR CD117 AND CD47-BINDING AGENT

[0002] FIELD OF THE DISCLOSURE

[0003] The present invention relates to recombinant binding proteins comprising a first designed ankyrin repeat domain which has a first binding specificity for a first target and a second binding specificity for a second target, wherein the binding of said first domain to said first and second targets is mutually exclusive, and wherein the first target is CD117 and the second target is a CD47-specific binding agent. The invention also provides further recombinant proteins comprising said first dual-specific repeat domain and additional therapeutic agents such as binding agents. Further disclosed are nucleic acids encoding such recombinant proteins, pharmaceutical compositions comprising such recombinant proteins or nucleic acids and the use of such recombinant proteins, nucleic acids or pharmaceutical compositions in methods for treating diseases, such as cancer in a mammal, including a human. Designed ankyrin repeat domains with binding specificity for a CD47-specific ankyrin repeat domain are also disclosed.

[0004] BACKGROUND

[0005] Selective cell depletion shows a great potential for treating a number of medical conditions, and is used, for example, for conditioning of patients for stem cell transplantation, treatment of autoimmune diseases, e.g. by B cell depletion therapy, and treatment of certain cancers (Lee et al. (2020) Nature Reviews Drug Discovery, volume 20, pp. 179-199).

[0006] Conditioning is a process by which a patient is prepared (i.e., “conditioned”) for receiving a transplant containing stem cells, such as, e.g. hematopoietic stem cells (HSCs). Conditioning procedures thereby promote the successful engraftment of a stem cell transplant and they are performed prior to engraftment in order to create conducive conditions for the patient to receive the transplant.

[0007] Currently, there are a number of non-specific (i.e., non-targeting) conditioning methods used in hematopoietic stem cell transplantation (HSCT)-related indications and hemoglobinopathies, including, but not limited to, the use of irradiation (e.g., total body irradiation (TBI)) and DNA alkylating / modifying agents, both of which are highly toxic, not only to hematopoietic cells, but also to non-hematopoietic cells and many of the patient's organs. These harsh conditioning regimens typically result in the destruction of the recipient patient's immune system and niche cells and can in many cases lead to life-threatening complications (Russel et al; Mol Ther Methods Clin Dev; 2021 ; 21 : 710-727; Radtke; Mol Ther Methods Clin Dev; 2023; 28: 385-386).

[0008] Accordingly, the development of more targeted conditioning regimens that selectively deplete an endogenous hematopoietic stem cell population, while avoiding the undesirable toxicity of the aforementioned non-specific conditioning methods, is needed. Depletion of stem cells, such as HSCs, may be facilitated by targeting certain molecules expressed on HSCs, including, for example, CD117. CD117 (also referred to as c-KIT or Stem Cell Factor Receptor (SCRF), UniProt: P10721) is a 145 kDa single transmembrane, receptor tyrosine kinase that binds the ligand Stem Cell Factor (SCF, UniProt: P21583). SCF induces homodimerization of cKIT, which activates its tyrosine kinase activity and signalling through both the PI3-AKT and MAPK pathways (Kindblom et al., Am J. Path. 1998 152(5):1259). CD117 is highly expressed on HSCs and this expression pattern makes CD117 a potential target for conditioning procedures useful for a broad range of diseases. DARPins having binding specificity to CD117 have been described in EP24150551 .0.

[0009] In the context of conditioning, targeting CD117 alone was shown to be insufficient to mediate efficient HSC depletion, and the advantage of conditioning with a combination of CD117 and CD47 specific mAbs showed improved depletion of endogenous HSCs from the bone marrow in pre-clinical studies, potentially enabling HSC transplantation comparable to high dose radiation (Marjon et al., Blood 134, 4428 (2019)). CD47 is a ubiquitously expressed innate immune checkpoint receptor that serves as a universal “don’t eat me” signal of phagocytosis, this receptor is often up-regulated by haematological and solid cancers to evade immune surveillance. CD47 inhibits phagocytosis by interacting with the transmembrane protein SIRPa on the surface of phagocytes, such as macrophages, neutrophils and dendritic cells, forming a CD47-SIRPa signalling complex which triggers this "don't eat me" signal. However, CD47 is also broadly expressed on normal cells, such as hematopoietic cells, red blood cells (RBCs) and platelets, thus posing safety and efficacy challenges, as targeting CD47, for example with a neutralizing antibody, could affect healthy cells, potentially leading to toxic effects (Ye at al; Acta Pharm Sin B. 2023 Apr;13(4):1467-1487). Additionally, broad expression of CD47 could also lead to rapid elimination of CD47 binding agents, leading to poor pharmacokinetics and decreased efficacy (Dizman et al; Cancers (Basel). 2021 Dec; 13(24): 6229). Many of the anti-CD47 antibodies that have been reported are known to cause agglutination of RBCs upon binding to CD47 on the cell surface, which significantly hampers the therapeutic utility of such antibodies (Velliquette et al; Transfusion; 2019 Feb;59(2):730-737; Sun et al; Leuk Res Rep; 2021 Sep 14;16).

[0010] Hence current CD47 blockade strategies applying mAbs come with limitations (Bouwstra, R., Meerten, T. & Bremer, E. Clin. Transl. Med. 12, e943 (2022)) and despite the availability of several conditioning approaches (Arai et al; Mol Ther; 2018; 26(5): 1181-1197), there remains a need for safe and efficient methods and compositions targeting stem cells, which can be used for conditioning. Such methods and compositions may also be useful for treating diseases, where selective cell depletion would be therapeutic.

[0011] SUMMARY

[0012] In previous work, designed ankyrin repeat domains having mutually exclusive binding specificity for two targets have been disclosed (WO2023110983). Such dual-specific repeat domains (also referred to herein as “2-in-1 domains” or “2-in-1 DARPins”) can be created by combining repeat modules of two parental repeat domains. The binding specificity for each of the first and second targets is therefore inherited from the binding specificity to the respective targets of the parental repeat domains.

[0013] In the present invention, such dual-specific repeat domains having a first binding specificity for CD117 and a second binding specificity for a CD47-binding agent, such as a CD47-binding DARPin, have been created. These dual-specific domains have particularly useful properties when applied e.g. in multi-domain immune cell engager DARPin constructs, enabling a target dependent switch for binding eitherto CD117 (e.g. where CD117 target serves as a HSC localiser) or to a CD47-binding agent, such as a CD47-binding DARPin (e.g. where a CD47-binding DARPin target in the unbound (or unmasked) state can interfere with binding of SIRPa on target (stem) cells, herewith inhibiting their “don’t eat me” signal). Effectively, such a switch mechanism provides for a CD117-dependent activation or deactivation of CD47 engagement by respectively unmasking or masking the CD47-binding agent, which together with an immune cell engager effector component, such as a CD16a binding agent, can provide a targeted and therefore safer HSC depletion strategy. Such as switch mechanism may also be used in other formats and for other therapeutic approaches.

[0014] Therefore, the present invention provides a recombinant binding protein comprising a first designed ankyrin repeat domain which has a first binding specificity for a first target and a second binding specificity for a second target, wherein the binding of said first domain to said first and second targets is mutually exclusive, and wherein the first target is CD117 and the second target is a CD47-specific binding agent. The invention also provides further recombinant proteins comprising said first dual-specific repeat domain and additional therapeutic agents such as binding agents. Further disclosed are nucleic acids encoding such recombinant proteins, pharmaceutical compositions comprising such recombinant proteins or nucleic acids and the use of such recombinant proteins, nucleic acids or pharmaceutical compositions in methods for treating diseases, such as cancer in a mammal, including a human. Designed ankyrin repeat domains with binding specificity for a CD47-specific ankyrin repeat domain are also disclosed herein.

[0015] 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).

[0016] E1 . A recombinant binding protein comprising a first designed ankyrin repeat domain having a first binding specificity for a first target and a second binding specificity for a second target, wherein binding of said first designed repeat domain to said first and second targets is mutually exclusive and wherein the first target is CD117 and the second target is a CD47-specific binding agent.

[0017] E2. The recombinant binding protein according to E1 , wherein the first designed repeat domain binds to said first target with a dissociation constant (KD) below 10-5M and / or wherein the first designed repeat domain binds to said second target with a dissociation constant (KD) below 10-5M.

[0018] E3. The recombinant binding protein according to any one of E1 to E2, wherein the first designed repeat domain comprises an N-terminal capping module, a C-terminal capping module, and at least one internal repeat module.

[0019] E4. The recombinant binding protein according to any one of E1 to E3, wherein the first designed repeat domain comprises between three and six internal repeat modules.

[0020] E5. The recombinant binding protein according to any one of E1 to E4, wherein the first designed repeat domain comprises a sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 38 to 67. E6. The recombinant binding protein according to any one of E1 to E5, wherein the protein further comprises a therapeutic agent.

[0021] E7. The recombinant binding protein according to E6, wherein the therapeutic agent is a binding agent having specificity for a tumor-associated antigen, a cell type-associated antigen and / or an immune cell surface-expressed antigen.

[0022] E8. The recombinant binding protein according to E7, wherein the tumor-associated antigen and / or the cell type-associated antigen is CD117.

[0023] E9. The recombinant binding protein according to any one of E7 to E8, wherein the immune cell surface- expressed antigen is CD16a.

[0024] E10. The recombinant binding protein according to any one of E1 to E9, wherein the protein further comprises a CD47-specific binding agent.

[0025] E11. The recombinant binding protein according to E10, wherein the CD47-specific binding agent is a second designed ankyrin repeat domain.

[0026] E12. The recombinant binding protein according to E11 , wherein the second designed ankyrin repeat domain is the second target of the first designed ankyrin repeat domain.

[0027] E13. The recombinant binding protein according to any one of E11 to E12, wherein the second designed ankyrin repeat domain comprises a sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 5, 6, 68, 69, 70, 71 , 72 or 73.

[0028] E14. An isolated nucleic acid encoding the binding protein according to any one of E1 to E13.

[0029] E15. A cloning or expression vector comprising the nucleic acid of E14, wherein said vector is suitable for the recombinant production of a recombinant binding protein as defined in any one of E1 to E13.

[0030] E16. The vector of E15, wherein said vector is a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector.

[0031] E17. A host cell comprising the nucleic acid of E14 or the vector of any one of E15 to E16.

[0032] E18. A pharmaceutical composition comprising (i) the recombinant binding protein according to any one of E1 to E13 and / or (ii) the nucleic acid according to E14, and optionally a pharmaceutically acceptable carrier or diluent.

[0033] E19. A method of conditionally binding a CD47-specific binding agent to CD47 expressed on the surface of cells, the method comprising the step of administering to a subject the recombinant binding protein according to any one of E10 to E13.

[0034] E20. The method of E19, wherein said binding a CD47-specific binding agent to CD47 expressed on the surface of cells is conditional on the presence of CD117 on the surface of said cells.

[0035] E21 . A method of treating a medical condition, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of the binding protein according to any one of E1 to E13, the nucleic acid according to E14, or the pharmaceutical composition according to E18. E22. A process for the production of a recombinant binding protein according to any one of E1 to E13, comprising culturing a host cell according to E17, purifying and recovering said recombinant binding protein.

[0036] BRIEF DESCRIPTION OF THE FIGURES

[0037] Figure 1 : Size exclusion chromatography (SEC) profiles of masking DARPins (i.e. DARPins with binding specificity for a CD47-binding DARPin, also referred to as “Masks”), which underwent a one-week stress test incubation at 60°C (left plots), compared to the reference sample stored at -70°C (right plots). Masks of the main selection campaign (MaskCH to Mask13 of SEQ ID NOs: 10 to 22, respectively) are shown in Fig. 1A and Mask14 to Mask26 (of SEQ ID NOs: 23 to 35, respectively) which are further engineered variants are shown in Fig. 1 B. X-axis in min, Y-axis in mAU.

[0038] Figure 2: Response curves from multi-trace SPR analysis of masking DARPins (MaskOI to Mask06 and Mask08 to Maskl 3) binding to CD47-binding DARPins of SEQ ID NO: 5 (in column L2) or of SEQ ID NO: 6 (in column L3). Serial dilutions (100 nM starting cone, and 1 :3 dilutions) of purified masking DARPins were applied to a sensor chip with immobilized target DARPins for on-rate and off-rate measurements. The obtained SPR trace analyses were used to determine the binding affinity of the masking DARPins to CD47- binding DARPin. X-axis: time [sec], Y-axis: response [RU, Resonance Units]

[0039] Figure 3: SEC profiles of 30 selected dual-specific DARPins according to the invention (also referred to as 2-in-1 DARPins or 2-in-1 domains) which have a first binding specificity for a CD47-binding DARPin and a second binding specificity for CD117. Details of the 2-in-1 domains are shown in Table 6. X-axis in min, Y- axis in mAU.

[0040] Figure 4: ELISA titration curves of 2-in-1 DARPinOI to 2-in-1 DARPin30 against CD117 (Fig. 4A), against CD47-binding DARPin of SEQ ID NO: 5 (Fig. 4B) or against CD47-binding DARPin of SEQ ID NO: 6 (Fig. 4C). Mono-specific DARPins with binding specificity for the coated target, and which correspond to the parental DARPins used in the respective 2-in- 1 domains were tested in each assay for comparison. Details about the parental DARPins used in each 2-in-1 domain are shown in Table 6. Curves represent a fitted four-parameters sigmoid model.

[0041] Figure 5: Results of a cell binding assay in which selected 2- in - 1 domains were tested for binding to CD117- expressing Kasumi-1 cells in comparison to mono-specific parental CD117-binding DARPins. Median fluorescent intensities (MFI) measured by flow cytometry are shown.

[0042] Figure 6: Results of a competitive binding ELISA showing the mutual binding exclusivity property of representative 2-in-1 domains of the invention. A schematic view of the experimental setup is shown in Fig. 6A. 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: 5. 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 domains and using an anti-flag-tag antibody. For all 2-in-1 domains shown (Fig. 6B), 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.

[0043] Figure 7: Cell binding data is shown where two-domain (2D) DARPin constructs comprising a first 2-in-1 domain and a second CD47-binding repeat domain were tested for binding on hCD47-expressing CHO-k1 cells. This setup allows to assess the masking effect (or blocking window) of the 2-in-1 domain on the CD47-binding DARPin, when compared to a mono-specific CD47-binding DARPin. The observed blocking windows correlate with the BC50 values of the 2-in-1 domains forthe CD47-binding DARPin (2-in-1 DARPin 14 comprised in 2D construct 01 has a BC50 of 3800 nM, 2-in-1 DARPin 22 comprised in 2D construct 02 has a BC50 of 55 nM and 2-in-1 DARPin 12 comprised in 2D construct 03 has a BC50 of 0.05 nM).

[0044] DETAILED DESCRIPTION OF THE INVENTION

[0045] Designed ankyrin repeat domains are structural units of designed ankyrin repeat proteins. Designed repeat protein libraries, including 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 target-specific designed repeat domains that bind to their target with high affinity. Such targetspecific designed repeat domains in turn can be used as valuable components of recombinant binding proteins for the treatment of diseases.

[0046] Repeat domains of the invention

[0047] In one aspect, the invention relates to a recombinant binding protein comprising a first ankyrin repeat domain having binding specificity for a CD47-specific binding agent. This first ankyrin repeat domain can be subsequently used for the creation of 2-in-1 domains of the invention.

[0048] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% amino acid sequence identity with any one of SEQ ID NOs: 10 to 35.

[0049] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 10. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 10.

[0050] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 11. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 11 . In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 12. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 12.

[0051] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 13. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 13.

[0052] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 14. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 14.

[0053] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 15. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 15.

[0054] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 16. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 16.

[0055] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 17. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 17.

[0056] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 18. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 18.

[0057] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 19. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 19.

[0058] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 20. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 20.

[0059] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 21. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 21 .

[0060] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 22. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 22.

[0061] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 23. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 23.

[0062] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 24. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 24.

[0063] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 25. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 25.

[0064] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 26. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 26.

[0065] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 27. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 27.

[0066] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 28. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 28.

[0067] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 29. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0068] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 30. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 30. In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 31. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 31 .

[0069] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 32. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 32.

[0070] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 33. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 33.

[0071] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 34. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 34.

[0072] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 35. In one embodiment, said first ankyrin repeat domain comprises or consists of the amino acid sequence of SEQ ID NO: 35.

[0073] In one embodiment, the potential interaction residues in said first ankyrin repeat domain are identical to the corresponding positions in any one of the ankyrin repeat domains of SEQ ID NOs: 10 to 35.

[0074] In one embodiment, said first ankyrin repeat domain comprises a sequence selected from SEQ ID NO: 36 or 37.

[0075] In further embodiments, said first ankyrin repeat domain comprises a sequence selected from the group consisting of (1) SEQ ID NO: 36 and (2) sequences in which up to 5, up to 4, up to 3, up to 2 or up to 1 framework residues other than position 95 in SEQ ID NO: 36 are substituted by another amino acid. In further embodiments, said first ankyrin repeat domain comprises a sequence selected from the group consisting of (1) SEQ ID NO: 37 and (2) sequences in which up to 5, up to 4, up to 3, up to 2 or up to 1 framework residues other than positions 15, 80, 83 and 124 in SEQ ID NO: 37 are substituted by another amino acid.

[0076] In one embodiment, said first repeat domain having binding specificity for a CD47-specific binding agent binds to said agent with a dissociation constant (KD) of or below about 10-7M, of or below about 10-8M or of or below about 10-9M. Thus, in one embodiment, said first repeat domain binds to said agent with a KD of or below about 10-7M. In another embodiment, said first repeat domain binds to said agent with a KD of or below about 10-8M. In another embodiment, said first repeat domain binds to said agent with a KD of or below about 10-9M. Preferably, said binding is measured in PBS.

[0077] The term CD47-specific binding agent referred to in the above embodiments encompasses any molecule capable of specifically binding CD47, and 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 CD47, and can comprise natural proteins and proteins modified or genetically engineered, e.g., to include non-natural residues and / or to lack natural residues.

[0078] In a particular embodiment, said CD47-specific binding agent is an ankyrin repeat domain. In some embodiments, the recombinant binding protein of the invention comprises a first ankyrin repeat domain having binding specificity for a CD47-specific ankyrin repeat domain. In some embodiment, said CD47- specific ankyrin repeat domain comprises an amino acid sequence with at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with any one of SEQ ID NOs: 5, 6, 68, 69, 70, 71 , 72 or 73.

[0079] In another aspect, the invention provides a recombinant binding protein comprising a first designed ankyrin repeat domain having a first binding specificity for a first target and a second binding specificity for a second target, wherein binding of said first designed ankyrin repeat domain to said first and second targets is mutually exclusive and wherein the first target is CD117 and the second target is a CD47-specific binding agent.

[0080] In one embodiment, said first designed repeat domain binds to CD117 with a dissociation constant (KD) below 10-5M. In some embodiments, said first designed repeat domain 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 1 O-1CIM 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 first designed 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 exemplary embodiment, said first designed repeat domain binds to CD117 with a KD value of less than or equal to about 1 nM. In another exemplary embodiment, said first designed repeat domain binds to CD117 with a KD value of less than or equal to about 100 pM. In another exemplary embodiment, said first designed repeat domain binds to CD117 with a KD value of less than or equal to about 10 pM. In yet another exemplary embodiment, said first designed repeat domain binds to CD117 with a KD value of less than or equal to about 1 pM.

[0081] In some embodiments, said first designed repeat domain binds to the CD47-specific binding agent with a dissociation constant (KD) below 10-5M. In certain embodiments, said first designed repeat domain binds to the CD47-specific binding agent 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 10’1° M 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 1 O’8M to about 1 O’14M, from about 1 O’9M to about 1 O’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 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 first designed repeat domain binds to the 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 exemplary embodiment, said first designed repeat domain binds to the CD47-specific binding agent with a KD value of less than or equal to about 1 nM. In another exemplary embodiment, said first designed repeat domain binds to the CD47-specific binding agent with a KD value of less than or equal to about 100 pM. In another exemplary embodiment, said first designed repeat domain binds to the CD47- specific binding agent with a KD value of less than or equal to about 10 pM. In yet another exemplary embodiment, said first designed repeat domain binds to the CD47-specific binding agent with a KD value of less than or equal to about 1 pM.

[0082] In some embodiments, the binding affinity of said first designed repeat domain 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 exemplary embodiments, said first designed repeat domain binds to each of said first and second targets 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 exemplary 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

[0083] 2 fM, or about 1 fM. In one exemplary embodiment, KD1 and KD2 are independently less than or equal to about 1 nM. In another exemplary 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 exemplary 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.

[0084] In one embodiment, said first designed repeat domain binds to CD117 with a first binding affinity and to the CD47-specific binding agent with a second binding affinity, wherein the ratio of said first binding affinity to said second binding affinity is between 103:1 and 1 :10s. In further embodiments, said ratio is equal to about 103:1 , 102:1 , 10:1 , 1 :1 , 1 :10, 1 :102, 1 :103, 1 :104, 1 :105or 1 :106.

[0085] In one embodiment, said first and second targets are located on different molecules.

[0086] In some embodiments, said first designed repeat domain comprises at least 1 repeat module, at least 2 repeat modules, at least 3 repeat modules, at least 4 repeat modules, at least 5 repeat modules or at least 6 repeat modules. In some embodiments, said first designed repeat domain comprises between 1 and 6 repeat modules, between 2 and 6 repeat modules, between 3 and 6 repeat modules, between 4 and 6 repeat modules or between 5 and 6 repeat modules. In some embodiments, said first designed repeat domain comprises an N-terminal capping module and / or a C-terminal capping module. In some embodiments, said first designed repeat domain comprises an N-terminal capping module and / or a C- terminal capping module and at least 1 internal repeat module, at least 2 internal repeat modules, at least

[0087] 3 internal repeat modules or at least 4 internal repeat modules. In some embodiments, said first designed repeat domain comprises an N-terminal capping module and / or a C-terminal capping module and between 1 and 6 internal repeat modules, between 1 and 6 internal repeat modules or between 1 and 5 internal repeat modules.

[0088] In some embodiments, said first designed repeat domain comprises an N-terminal capping module, a C- terminal capping module and one, two, three, four, five or six internal repeat modules.

[0089] In one preferred embodiment, said first designed repeat domain is an ankyrin repeat domain.

[0090] In some embodiments, said first designed repeat domains according to the invention comprise a paratope. In some embodiments, such paratope is essentially separated in two regions, where each of said region comprises repeat modules contributing to the first or second binding specificity. In such embodiments, the paratope regions do not overlap. In other embodiments, some repeat modules may contribute to both binding specificities due to one or more potential target interaction residues which interact with both targets, and therefore both regions of the paratope may overlap to a certain extent.

[0091] In a particular embodiment, said first designed repeat domain comprises an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 38 to 67 and (2) sequences with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with any one of SEQ ID NOs: 38 to 67. Thus, in a more particular embodiment, said designed repeat domain comprises an amino acid sequence selected from the group consisting of (1) any one of SEQ ID NOs: 38 to 67 and (2) sequences with at least 80% amino acid sequence identity with any one of SEQ ID NOs: 38 to 67. Such recombinant binding proteins are characterized in Example 3.

[0092] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 38. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 38.

[0093] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 39. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 39.

[0094] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 40. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 40. In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 41 . Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 41 .

[0095] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 42. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 42.

[0096] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 43. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 43.

[0097] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 44. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 44.

[0098] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 45. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 45.

[0099] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 46. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 46.

[0100] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 47. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 47.

[0101] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 48. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 48.

[0102] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 49. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 49.

[0103] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 50. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 50.

[0104] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 51 . Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 51 .

[0105] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 52. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 52.

[0106] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 53. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 53.

[0107] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 54. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 54.

[0108] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 55. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 55.

[0109] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 56. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 56.

[0110] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 57. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 57.

[0111] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 58. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 58.

[0112] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 59. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 59. In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 60. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 60.

[0113] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 61 . Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 61 .

[0114] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 62. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 62.

[0115] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 63. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 63.

[0116] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 64. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 64.

[0117] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 65. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 65.

[0118] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 65. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 66.

[0119] In one embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with SEQ ID NO: 67. Thus, in one exemplary embodiment, said first ankyrin repeat domain comprises an amino acid sequence with at least 95% amino acid sequence identity with SEQ ID NO: 67.

[0120] In any of the embodiments relating to sequence identity or substitutions disclosed herein, residues selected for substitutions can be located at randomized or non-randomized positions of the repeat domain. Accordingly, in some embodiments, the substituted residues are selected among residues located at nonrandomized positions of said repeat domain. In other embodiments, the substituted residues are selected among residues located at randomized positions of said repeat domain. In other embodiments, residues selected for substitutions can be framework residues or target interaction residues. Accordingly, in some embodiments, the substituted residues are selected among the framework residues comprised in said repeat domain. In other embodiments, the substituted residues are selected among the potential target interaction residues comprised in said repeat domain. In other embodiments, the substituted residues are selected among all residues comprised in said repeat domain. In some embodiments, the randomized positions of the repeat domain correspond to the positions of potential target interaction residues. In further embodiments, the non-randomized positions correspond to the positions of framework residues. Preferred positions of framework and potential target interaction residues are shown in Table A.

[0121] Table A

[0122] A substitution as recited herein is preferably a conservative substitution according to Table B.

[0123] Table B

[0124] Furthermore, the sequence of any repeat domain of the present invention may optionally comprise at its N- terminus, a G, an S, or a GS and the sequence of any repeat domain of the present invention may optionally end with KLN (C-terminus).

[0125] Recombinant proteins The designed repeat domains or recombinant binding proteins of the invention can be genetically fused to further components, such as, e.g., a therapeutic agent, and such fusions are also referred to as “recombinant protein” or “constructs” interchangeably.

[0126] Accordingly, in one aspect, the invention provides a recombinant protein comprising the designed ankyrin repeat domains or the recombinant binding protein disclosed herein. In one embodiment, said recombinant protein further comprises a CD47-specific binding agent. The term CD47-specific binding agent encompasses any molecule capable of specifically binding CD47, and 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 CD47, and can comprise natural proteins and proteins modified or genetically engineered, e.g., to include non-natural residues and / or to lack natural residues.

[0127] Accordingly, in one embodiment, said recombinant protein comprises a first designed ankyrin repeat domain having a first binding specificity for a first target and a second binding specificity for a second target, wherein binding of said first designed ankyrin repeat domain to said first and second targets is mutually exclusive and wherein the first target is CD117 and the second target is a CD47-specific binding agent, and wherein said recombinant protein further comprises a CD47-specific binding agent. In one embodiment, said CD47-specific binding agent is a second designed ankyrin repeat domain. In one embodiment, said CD47-specific binding agent is a second designed ankyrin repeat domain; in such cases, the recombinant protein of the invention is a two-domain ankyrin repeat protein. Suitable binding agents with binding specificity for CD47 are disclosed in WO2024 / 251695.

[0128] In one embodiment, said second designed ankyrin repeat domain comprises an amino acid sequence having at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with any one of SEQ ID NOs: 5, 6, 68, 69, 70, 71 , 72 or 73.

[0129] In one embodiment, said second designed ankyrin repeat domain is the second target of the first designed ankyrin repeat domain. In some embodiments, said first and second repeat domains are linked by a peptide linker, such as e.g. a linker of SEQ ID NO: 77. In one embodiment, said recombinant protein comprises an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity with any one of SEQ ID NOs: 74 to 76. Such proteins are characterized in Example 3.5.

[0130] Accordingly, in one embodiment, the recombinant protein of the invention comprises a first designed ankyrin repeat domain having a first binding specificity for a first target and a second binding specificity for a second target, wherein binding of said first designed ankyrin repeat domain to said first and second targets is mutually exclusive and wherein the first target is CD117 and the second target is a CD47-specific designed ankyrin repeat domain and a second designed ankyrin repeat domain having binding specificity for CD47, wherein said second repeat domain is the second target of said first repeat domain.

[0131] In one embodiment, said recombinant protein of the invention is capable of competing for binding with stem cell factor (SCF). In one embodiment, said recombinant protein competes for binding with stem cell factor (SCF). In one embodiment, said recombinant protein does not compete for binding with stem cell factor (SCF). In one embodiment, said recombinant 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. In one embodiment, said recombinant protein inhibits SCF signalling.

[0132] In one embodiment, said recombinant protein further comprises a therapeutic agent. Such recombinant proteins of the invention can be generated in different formats. In one embodiment, said therapeutic agent is fused on the N-terminal side of said recombinant protein. In one embodiment, said therapeutic agent is fused on the C-terminal side of said recombinant protein. In some embodiments, said therapeutic agent is directly linked to said recombinant protein. In some embodiments, a peptide linker is used to link said recombinant protein to said therapeutic agent. In some embodiments, said therapeutic agent is separated from said recombinant protein by further molecules such as or one or more ankyrin repeat domains.

[0133] In one embodiment, said therapeutic agent is a binding agent. Such binding agents encompass any molecule capable of specifically binding a target, and include, for example, antibodies, antibody fragments, aptamers, peptides, alternative scaffolds, antibody mimics, repeat proteins, e.g., designed ankyrin repeat proteins and receptor proteins, and can comprise natural proteins and proteins modified or genetically engineered, e.g., to include non-natural residues and / or to lack natural residues.

[0134] In some embodiment, said binding agent has specificity for a tumor-associated antigen, a cell type- associated antigen and / or an immune cell surface-expressed antigen. An example of tumor-associated antigen and / or cell type-associated antigen is CD117. The amino acid sequence of human CD117 is shown in UniProt (www.uniprot.org) Ref. No. P10721. Accordingly, in one embodiment, said binding agent has specificity for CD117. Suitable binding agents with binding specificity for CD117 are disclosed in EP24150551 .0. An example of immune cell surface-expressed antigen is CD16a (Fc gamma lll-A receptor). The amino acid sequence of human CD16a (hCD16a) is shown in UniProt Ref. No. P08637. Accordingly, in one embodiment, said binding agent has specificity for CD16a. Suitable binding agents with binding specificity for CD16a are disclosed in WO2024 / 251628.

[0135] In further embodiments, said binding agent comprised in the recombinant protein of the invention comprises or consists of a designed ankyrin repeat domain. Accordingly, in one embodiment, the recombinant protein of the invention comprises a first designed ankyrin repeat domain having a first binding specificity for a first target and a second binding specificity for a second target, wherein binding of said first designed ankyrin repeat domain to said first and second targets is mutually exclusive and wherein the first target is CD117 and the second target is a CD47-specific designed ankyrin repeat domain, a second designed ankyrin repeat domain having binding specificity for CD47, wherein said second repeat domain is the second target of said first repeat domain, a third designed ankyrin repeat domain having binding specificity for CD117 and a fourth designed ankyrin repeat domain having binding specificity for CD16a.

[0136] In one embodiment, the recombinant protein of the invention including a second designed ankyrin repeat domain having binding specificity for CD47 is capable of blocking the interaction of CD47 with signal- regulatory protein alpha (SIRPa). In one embodiment, said recombinant protein 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.

[0137] In one embodiment, the recombinant proteins provided herein further comprise one or more half-life extending moieties. Preferably, said half-life extending moiety binds to human serum albumin. In one embodiment, the half-life extending moiety comprises an ankyrin repeat domain binding human serum albumin.

[0138] In one embodiment, the components comprised in the recombinant proteins provided herein are covalently linked with a peptide linker. Said peptide linker may be a proline-threonine-rich peptide linker of e.g. SEQ ID NO: 77. Suitably, said peptide linker comprises between 1 and 50 amino acids. Suitably, said peptide linker comprises between 1 and 30 amino acids. Other linkers known in the art may also be used to link repeat domains (see, e.g., WO2021116469).

[0139] Nucleic acids and vectors

[0140] In another aspect, the invention relates to an isolated nucleic acid encoding the amino acid sequence of the designed ankyrin repeat domain or the recombinant protein of the invention.

[0141] Further disclosed are vectors comprising any nucleic acid of the invention. Accordingly, in another aspect, a recombinant expression vector comprising a nucleic acid according to the invention is disclosed, wherein the vector optionally comprises an expression control sequence, allowing expression in prokaryotic or eukaryotic host cells of the encoded polypeptide, operably linked to said nucleic acid. The nucleic acid sequence can be inserted in the recombinant vector by methods well known to a person skilled in the art such as, for example, those that are described in MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al, 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y., 2001.

[0142] Nucleic acids are well known to the skilled person in the art. Nucleic acids were used to produce designed ankyrin repeat domains or recombinant binding proteins of the invention in E. coli, e.g. as described in U.S. Patent No. 7,417,130.

[0143] In one embodiment, the invention relates to a method for producing a recombinant binding protein of the present invention. In one embodiment, the invention relates to a method for producing a recombinant binding protein, for example a recombinant binding protein comprising the amino acid sequence of any one of SEQ ID NOs: 5 to 6, 10 to 35, 78 to 67, 74 to 76, 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. Exemplary methods of producing a recombinant binding protein of the present invention are described the Examples.

[0144] Compositions

[0145] The invention further relates to pharmaceutical compositions comprising a designed ankyrin repeat domain, a recombinant protein and / or a nucleic acid described herein and a pharmaceutically acceptable carrier or diluent. The invention also relates to uses and methods of treatment using said pharmaceutical compositions disclosed herein. The methods and uses encompassed by the present invention are described in more detail below.

[0146] The pharmaceutical compositions described herein may be prepared using methods known in the art.

[0147] The pharmaceutical compositions optionally comprise a pharmaceutically acceptable carrier or excipient or diluent. Standard pharmaceutical carriers include a phosphate buffered saline solution, water, emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents.

[0148] The pharmaceutical compositions may comprise any other pharmaceutically acceptable ingredients, including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, colouring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavour enhancers, flavouring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin penetrants, solubilizing agents, solvents, stabilizing agents, suppository bases, surface active agents, surfactants, suspending agents, sweetening agents, therapeutic agents, thickening agents, tonicity agents, toxicity agents, viscosityincreasing agents, water-absorbing agents, water-miscible cosolvents, water softeners, or wetting agents. See, e.g., the Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0149] In one embodiment, the invention provides a pharmaceutical composition comprising (i) a recombinant protein according to the invention or (ii) a nucleic acid according to the invention, and at least one pharmaceutically acceptable carrier or diluent.

[0150] Therapeutic uses and methods according to the invention

[0151] In another aspect, the invention relates to a method of conditionally binding a CD47-specific binding agent to CD47 expressed on the surface of cells, the method comprising the step of administering to a subject in need thereof the recombinant protein disclosed herein. In one embodiment, said binding a CD47-specific binding agent to CD47 expressed on the surface of cells is conditional on the presence of CD117 on the surface of said cells. Accordingly, in some embodiments, the recombinant protein of the invention comprising a first designed ankyrin repeat domain having a first binding specificity for a first target and a second binding specificity for a second target, wherein binding of said first designed ankyrin repeat domain to said first and second targets is mutually exclusive and wherein the first target is CD117 and the second target is a CD47-specific binding agent, acts as a CD117 dependent on / off switch for masking or unmasking the CD47-specific binding agent. In some embodiment, said recombinant protein further comprises a second designed ankyrin repeat domain having binding specificity for CD47, wherein said second repeat domain is the second target of said first repeat domain. In another aspect, the invention provides a method of treating a medical condition, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of the designed ankyrin repeat domain of the invention, the recombinant protein of the invention, the nucleic acid of the invention or the pharmaceutical composition of the invention.

[0152] Further provided is the designed ankyrin repeat domain, the recombinant protein, the nucleic acid, or the pharmaceutical composition of the invention for use in a method of treating a medical condition.

[0153] In one embodiment, the invention relates to the use of the designed ankyrin repeat domain, the recombinant protein, the nucleic acid, or the pharmaceutical composition according to the present invention for the treatment of a disease. Forthat purpose, the designed ankyrin repeat domain, the recombinant protein, the nucleic acid, or the pharmaceutical composition according to the present invention is administered to a subject in need thereof, in a therapeutically effective amount.

[0154] In one embodiment, the invention relates to a method of treatment of a medical condition, the method comprising the step of administering, to a subject in need of such a treatment, a therapeutically effective amount of the designed ankyrin repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention.

[0155] In one embodiment, the invention relates to the use of the designed ankyrin repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the present invention for the treatment of a medical condition.

[0156] In one embodiment, the invention relates to the designed ankyrin repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention for use in the treatment of a medical condition.

[0157] In one embodiment, the invention relates to the use of the designed ankyrin repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention, as a medicament for the treatment of a medical condition.

[0158] In one embodiment, the invention relates to a process for the manufacturing of a medicament for the treatment of a medical condition, wherein the designed ankyrin repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention is an active ingredient of the medicament.

[0159] In one embodiment, the invention relates to the use of the designed ankyrin repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention, for manufacturing of a medicament.

[0160] In one embodiment, the invention relates to the use of the designed ankyrin repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention, for manufacturing of a medicament for the treatment of a medical condition.

[0161] In the context of the invention, the terms “medical condition”, “disease” and “disorder” are used interchangeably and include but are not limited to cancer. In one embodiment, said medical condition is a cancer. In one embodiment, said cancer is a CD117-positive cancer, e.g. gastrointestinal stromal tumor (GIST). In one embodiment, the method according to the invention is a conditioning regimen. 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 designed ankyrin repeat domain or recombinant protein 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 designed ankyrin repeat domain or recombinant protein 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. If the reason for the transplantation is a hematological malignancy, the designed ankyrin repeat domain or recombinant protein 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 the designed ankyrin repeat domain or recombinant protein will result in the reduction or elimination of those cells expressing these targets, including cancer stem cells (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 used for selectively depleting or ablating an endogenous HSC or progenitor cell population in a 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., prior to 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.

[0162] In some embodiments, said subject is a mammal. In preferred embodiments, the subject is a human.

[0163] In some embodiments, a single administration of said designed ankyrin repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention may be sufficient. In other embodiments, repeated administration may be necessary. Various factors will impact on the number and frequency of administrations, such as the age and general health of the subject, as well as the nature and typical dosage regime of the therapeutic agent.

[0164] Definitions Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art.

[0165] The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms unless otherwise noted. If aspects of the invention are described as "comprising" a feature, embodiments also are contemplated "consisting of or "consisting essentially of the feature. 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 of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about" as that term would be interpreted by the person skilled in the relevant art. The term “about” as used herein is equivalent to ± 10% of a given numerical value, unless otherwise stated.

[0166] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0167] The term "nucleic acid" refers to a polynucleotide molecule, which may be a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule, either single stranded or double stranded, and includes modified and artificial forms of DNA or RNA. A nucleic acid may either be present in isolated form or be comprised in recombinant nucleic acid molecules or vectors.

[0168] In the context of the present invention 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". Such protein domains are well known to the practitioner skilled in the art.

[0169] 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 well 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.

[0170] In the context of the present invention, the term "polypeptide" relates to a molecule consisting of a chain of multiple, i.e. two or more, 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. Polypeptides are well-known to the person skilled in the art.

[0171] The term “target” refers to an individual molecule such as a nucleic acid, a polypeptide or protein, a carbohydrate, or any other naturally or non-naturally occurring molecule, including any part of such individual molecule, or complexes of two or more of such molecules. The target may be a whole cell or a tissue sample, or it may be any non-natural molecule or moiety. Preferably, the target is a naturally occurring or nonnatural polypeptide or a polypeptide containing chemical modifications, for example modified by natural or non-natural phosphorylation, acetylation, or methylation.

[0172] Patent application W02002 / 020565 and Forrer et al., 2003 (Forrer, P., Stumpp, M.T., Binz, H.K., Pluckthun, A., 2003. FEBS Letters 539, 2-6), contain a general description of repeat protein features and repeat domain features, techniques and applications. The term "repeat 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.

[0173] 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. Optionally, a repeat domain also comprises an N-terminal and / or a C-terminal capping module. For clarity, a capping module can be a repeat module, and as such can contribute to the two or more consecutive repeat modules of a repeat domain. Such repeat domains, repeat modules, and capping modules, sequence motives, as well as structural homology and sequence homology are well known to the practitioner in the art from examples of ankyrin repeat domains (Binz et al., J. Mol. Biol. 332, 489-503, 2003; Binz et al., 2004, loc. cit. ; W02002 / 020565; WO2012 / 069655), leucine-rich repeat domains (W02002 / 020565), tetratricopeptide repeat domains (Main, E.R., Xiong, Y., Cocco, M.J., D'Andrea, L., Regan, L., Structure 11 (5), 497-508, 2003), and armadillo repeat domains (W02009 / 040338). It is further well known to the practitioner in the art that such repeat domains are different from proteins comprising repeated amino acid sequences, where every repeated amino acid sequence is able to form an individual domain (for example FN3 domains of Fibronectin).

[0174] The term "ankyrin repeat domain" refers to a repeat domain comprising two or more consecutive ankyrin repeat modules as structural units, wherein said ankyrin repeat modules have structural and sequence homology. 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 binding domains of the instant invention are designed repeat domains. Preferably, a designed repeat domain of the invention is a designed ankyrin repeat domain.

[0175] The term "repeat modules" refers to the repeated amino acid sequence and structural units of the designed repeat domains, which are originally derived from the repeat units of naturally occurring repeat proteins. Each repeat module comprised in a repeat domain is derived from one or more repeat units of a family or subfamily of naturally occurring repeat proteins, preferably the family of ankyrin repeat proteins. Furthermore, each repeat module comprised in a repeat domain may comprise a “repeat sequence motif’ deduced from homologous repeat modules obtained from repeat domains selected on a target and having the same target specificity. A repeat module as used in the present invention encompasses internal repeat modules and capping modules such as N-terminal and C-terminal capping modules. An “internal repeat module” refers to a repeat module that is flanked by two repeat modules. In other words, an internal repeat module is N-terminally flanked by one repeat module and C-terminally flanked by another repeat module. In the context of the present disclosure, an N-terminal capping module corresponds to positions 1 to 30 in any repeat domain sequence of SEQ ID NOs: 1 to 6 and 10 to 73. For N2C repeat domains, internal repeat modules correspond to positions 31 to 63 (IR1) and 64 to 96 (IR2) and the C-terminal capping module corresponds to positions 97 to 124. For N3C repeat domains, internal repeat modules correspond to positions 31 to 63 (IR1), 64 to 96 (IR2) and 97 to 129 (IR3) and the C-terminal capping module corresponds to positions 130 to 157. More generally, the boundaries of each repeat module in function of the total number of internal repeat modules x comprised in a “NxC” repeat domain are preferably determined using the following formula:

[0176] N-terminal capping module: positions 1 to 30;

[0177] The nthinternal repeat module: positions anto bn, where an= 31 + 33*(n-1) and bn= 30 + 33*n, where n goes from integer values 1 to x; and

[0178] C-terminal capping module: positions c to d, where c = 31 + 33*x and d = 58 + 33*x.

[0179] Accordingly, repeat modules comprised in the repeat domains disclosed herein can be defined using this formula. One such exemplary N-terminal capping module is provided in SEQ ID NO: 79, one such exemplary internal repeat module is provided in SEQ ID NO: 80, and one such exemplary C-terminal capping module is provided in SEQ ID NO: 81. A person skilled in the art would understand how to adapt the formula in case an ankyrin repeat domain comprises one or more amino acids typically not present and / or lacks one or more amino acids typically present.

[0180] Accordingly, the term "ankyrin repeat module" refers to a repeat module, which is originally derived from the repeat units of naturally occurring ankyrin repeat proteins. Ankyrin repeat proteins are well known to the person skilled in the art. Designed ankyrin repeat proteins have been described previously; see, e.g., International Patent Publication Nos. WQ2002 / 020565, WQ2010 / 060748, WQ2011 / 135067, WQ2012 / 069654, WQ2012 / 069655, WQ2014 / 001442, WO2014 / 191574, WQ2014 / 083208, WQ2016 / 156596, and WQ2018 / 054971 , all of which are incorporated by reference in their entireties. Typically, an ankyrin repeat module comprises about 31 to 33 amino acid residues that form two alpha helices, separated by loops.

[0181] Repeat modules may comprise positions with amino acid residues which have not been randomized in a library for the purpose of selecting target-specific repeat domains ("non-randomized positions" or “fixed positions” used interchangeably herein) and positions with amino acid residues which have been randomized in the library for the purpose of selecting target-specific repeat domains ("randomized positions"). Non-randomized positions comprise framework residues and may also comprise potential target interaction residues. The randomized positions comprise potential target interaction residues. “Have been randomized” means that two or more amino acids were allowed at an amino acid position of a repeat module, for example, wherein any of the usual twenty naturally occurring amino acids were allowed, or wherein most of the twenty naturally occurring amino acids were allowed, such as amino acids other than cysteine, or amino acids other than glycine, cysteine and proline.

[0182] The term “paratope” or “binding surface” refers to the region of a repeat domain which provides binding specificity for one or more target(s). A paratope as used herein comprises potential target interaction residues and framework residues. The positions of paratope residues are defined per repeat module as follows: in internal repeat modules, positions corresponding to positions 3, 4, 6, 11 , 14 and 15 with reference to SEQ ID NO: 80 comprising potential target interaction residues, and a position corresponding to position 10 comprising a framework residue; in N-terminal capping modules, positions corresponding to positions 4, 5, 8, 11 and 12 with reference to SEQ ID NO: 79 comprising potential target interaction residues, and a position corresponding to position 7 comprising a framework residue; in C-terminal capping modules, positions corresponding to positions 3, 4, 6, 14 and 15 with reference to SEQ ID NO: 81 comprising potential target interaction residues, and positions corresponding to positions 10 and 11 comprising framework residues.

[0183] The term "repeat sequence motif refers to an amino acid sequence, which is deduced from one or more repeat modules. Preferably, said repeat modules are from repeat domains having binding specificity for the same target. Such repeat sequence motifs comprise framework residue positions and target interaction residue positions. Said framework residue positions correspond to the positions of framework residues of the repeat modules. Likewise, said target interaction residue positions correspond to the positions of target interaction residues of the repeat modules. Repeat sequence motifs comprise non-randomized positions and randomized positions.

[0184] The term “repeat unit” refers to amino acid sequences comprising sequence motifs of one or more naturally occurring proteins, wherein said "repeat units" are found in multiple copies, and exhibit a defined folding topology common to all said motifs determining the fold of the protein. Examples of such repeat units include leucine-rich repeat units, ankyrin repeat units, armadillo repeat units, tetratricopeptide repeat units, HEAT repeat units, and leucine-rich variant repeat units.

[0185] The term "target interaction residues" refers to amino acid residues of a repeat module, which contribute to the direct interaction with a target. Such contribution of a residue can be tested, e.g., in a binding assay, for example in a mutagenesis study performed to identify residues required, sufficient, and / or necessary for a repeat domain to bind a target with its original binding affinity or quantity (i.e. its binding affinity or quantity in the absence of any mutations). In the context of the present invention, if a loss of at least 10% binding interaction between a repeat domain and a target results from a residue mutation within said domain, as compared to the binding interaction between the non-mutated repeat domain and the target, said residue is considered to contribute to the interaction with said target. Target interaction residues can also be determined by structural analyses of a repeat domain bound to a target.

[0186] The terms "framework residues" refers to amino acid residues of a repeat module, which contribute to the folding topology, i.e. which contribute to the fold of said repeat module or which contribute to the interaction with a neighboring module. Such contribution may be the interaction with other residues in the repeat module, 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. The term “framework residues” does not include the amino acid residues located at the positions within the designed ankyrin repeat domain that correspond to the positions of potential target interaction residues listed in Table A.

[0187] 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 well known to practitioners in structural biology and / or bioinformatics.

[0188] The term “binding specificity”, “has binding specificity for a target”, “specifically binding to a target”, “binding to a target with high specificity”, “specific for a target” or “target specificity” and the like means that a binding protein or binding domain binds to a target with a lower dissociation constant (i.e. it binds with higher affinity) than it binds to an unrelated protein such as the E. coli maltose binding protein (MBP). Preferably, the dissociation constant (“KD”) for the target is at least 102; more preferably, at least 103; more preferably, at least 104; or more preferably, at least 105times lower than the corresponding dissociation constant for MBP. Methods to determine dissociation constants of protein-protein interactions, such as surface plasmon resonance (SPR) based technologies (e.g. SPR equilibrium analysis) or isothermal titration calorimetry (ITC) are well 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.

[0189] 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 koir / kon. non

[0190] [B] + [T] [BT] koff 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.

[0191] A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present invention. For example, as exemplified herein, the binding affinity of a particular binding moiety to a drug molecule target can be expressed as 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 (kotr)”, to the association rate, or “on-rate (kon)”. Thus, KD equals kotr / kon and is expressed as a molar concentration (M), and the smaller the KD, the stronger the affinity of binding.

[0192] 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; 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., analysing 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. Preferably, the KD values are determined in PBS and by SPR. The term “PBS” means a phosphate buffered water solution containing 137 mM NaCI, 10 mM phosphate and 2.7 mM KCI and having a pH of 7.4.

[0193] The term "dual binding specificity" or “dual-specific” refers to the property that designed repeat domains of the invention have binding specificity for two targets, wherein the two targets are two epitopes where each one of said two epitopes is preferably located on a different target molecule. The epitopes are preferably different from each other.

[0194] The term “mutual exclusivity” or “mutually exclusive” as used in mutually exclusive binding refers to the property of designed repeat domains of the invention to be able 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 to the repeat domain influences binding of said second target to the repeat domain (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 invention, a binding interaction between a designed repeat domain of the invention and its first and second targets is considered to be mutually exclusive if, when bound to one of said first and second targets, said repeat domain 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 to the repeat domain is compared to the binding of the second target to the repeat domain. 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 competitive binding assay is shown in Example 3.4. The term "therapeutic agent" as used herein refers to a drug, molecule, nucleic acid, protein, composition or other substance that provides a therapeutic effect. The terms "therapeutic agent" and "drug" are used interchangeably.

[0195] 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.

[0196] 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.

[0197] Solid tumors typically also comprise tumor stroma.

[0198] The invention is not restricted to the particular embodiments described in the Examples.

[0199] SEQUENCES

[0200]

[0201]

[0202]

[0203]

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211] EXAMPLES

[0212] Materials

[0213] 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 / Thermo Fisher Scientific (USA). Inducible E. coll expression strains were used for cloning and protein production, e.g. E. coll XL1-blue (Stratagene, USA) or BL21 (Novagen, USA).

[0214] Molecular Biology

[0215] 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).

[0216] Designed ankyrin repeat protein libraries

[0217] Methods to generate designed ankyrin repeat protein libraries have been described, e.g. in U.S. Patent No. 7,417,130; Binz et al. 2003, loc. cit.; 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 could accordingly be assembled based on a fixed N-terminal capping module or a randomized N-terminal capping module, one or more randomized repeat modules, and a fixed C-terminal capping module or a randomized C-terminal capping module (see, e.g., the N- terminal capping modules and C-terminal capping modules provided in WO 2021 / 116462). 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.

[0218] Furthermore, such 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 border of two ankyrin repeats, ankyrin repeat protein 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.

[0219] An N-terminal capping module of an ankyrin repeat protein library preferably possesses the RILLAA, RILLKA or RELLKA motif and any such C-terminal capping module of an ankyrin repeat protein library preferably possesses the KLN, KLA or KAA motif.

[0220] 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 1WDY, 3V31 , 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1 N11 , 1 S70 and 2ZGD.

[0221] 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.

[0222] 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.

[0223] Example 1 : Parental DARPins for creating 2-in-1 ankyrin repeat domains

[0224] Summary

[0225] In previous work, designed ankyrin repeat domains having mutually exclusive binding specificity for two targets have been disclosed (WO2023110983). Such dual-specific repeat domains (also referred to as 2- in-1 domains or 2-in-1 DARPins) can be created by combining repeat modules of two parental repeat domains. The binding specificity for each of the first and second targets is therefore inherited from the binding specificity to the respective targets of the parental repeat domains. In the present invention, such dual-specific repeat domains with a first binding specificity for CD117 (or c-Kit) and a second binding specificity for CD47-binding DARPins have been created. These dual-specific domains have particularly useful properties when applied in multi-DARPin domain constructs, enabling a target dependent conditional switch for binding to either CD117 (e.g. CD117 target serves as HSC localiser) or CD47-binding DARPins (e.g. a CD47-binding DARPin target in the unmasked state can interfere with binding of SIRPa on a target (stem) cell). Accordingly, such 2-in-1 domains can be beneficial e.g. in the context of hematopoietic stem cell transplantation (HSCT).

[0226] 1.1 Masking DARPins

[0227] The first parental DARPins required as source DARPin forthe creation of the 2-in-1 domains ofthe invention are DARPins with a binding specificity to CD47-binding DARPins. Their primary role is to bind CD47-binding DARPins and herewith mask their capability to interact with the CD47 protein. Such DARPins with binding specificity to CD47-binding DARPins are therefore also referred to as masking DARPins (or Masks). A selection and screening campaign to produce masking DARPins has been performed as further detailed below.

[0228] 1. 1. 1 Biotinylated CD47-binding DARPins target preparation for Masks selection

[0229] Two different CD47-binding DARPins (i.e. of SEQ ID NO: 5 and 6) were used as target material for the ribosome display selection. These CD47-binding DARPins are disclosed in detail in WO2024 / 251695. The DARPin sequences were cloned with an N-terminal 6xHis-tag of SEQ ID NO: 7 and a C-terminal Avi-tag of SEQ ID NO: 8 and purified by AKTA Xpress over IMAC-SEC, then in vitro biotinylated and further purified by an additional SEC run. From 500ml expression of each of the proteins, 67-202mg protein were obtained after IMAC-SEC. 12mg of each DARPin were biotinylated a yield of 6.3-6.8 mg of each biotinylated protein were obtained after the final SEC. The biotinylation efficiency was estimated to be 60-90% and all other QC criteria were passed. Endotoxin values were below 0.4EU / mg, purity by SEC was 98-100%, the MW was confirmed by MALS and no impurities were detected by SDS-PAGE.

[0230] 1. 1.2 Selection of binding proteins comprising an ankyrin repeat domain with binding specificity for CD47- binding DARPins

[0231] Using ribosome display (Hanes, J. and Pluckthun, A., PNAS 94, 4937-42, 1997), many ankyrin repeat proteins with binding specificity for CD47-binding DARPins were selected from DARPin libraries similar as described by Binz et al. 2004 (loc. cit.). The binding of the selected clones towards the above described CD47-binding DARPins target was assessed by crude extract Homogeneous Time Resolved Fluorescence (HTRF), indicating that hundreds of masking DARPins were successfully selected. For example, the ankyrin repeat domains of SEQ ID NOs: 10-35 constitute amino acid sequences of selected masking ankyrin repeat domains with binding specificity for CD47-binding DARPins. More specifically, Masks of SEQ ID NOs: 10 to 17 were selected against biotinylated CD47-binding DARPin of SEQ ID NO: 5 and Masks of SEQ ID NOs: 18 to 22 were selected against biotinylated CD47-binding DARPin of SEQ ID NO: 6.

[0232] The resulting clones were obtained from two selection arms where four or six rounds of standard ribosome selections respectively were employed, using decreasing target concentration and increasing washing stringency to increase selection pressure from round 1 to round 4 or 6 (Binz et al. 2004, loc. cit.).

[0233] 1. 1.3 Selected clones bind specifically to CD47-binding DARPins as shown by crude extract HTRF

[0234] The pools from the ribosome display were expressed in E. coll cells in 96 well plates. Eight plates of DARPin proteins from ribosome display round 4 and 4 plates of DARPin proteins from round 6 were expressed with a N-terminal Flag-tag (of SEQ ID NO: 9) from the pools selected on each CD47-binding DARPin. Crude extracts thereof were prepared to test binding of the Flag-tagged DARPin proteins to biotinylated CD47- binding DARPins in an HTRF assay. Briefly, E. coliXLA blue 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 an individual well of a 96-deep-well plate with 1.2 mL TB medium (containing 1 % glucose and 50 pg / ml ampicillin) and incubated overnight at 37°C, shaking at 230 rpm. Fresh TB medium (containing 50 pg / ml ampicillin; 0.99 mL per well of a 96-deep-well plate) was inoculated with overnight culture (1 :10) and incubated at 37°C at 230 rpm. After 2 h the culture was induced by addition of IPTG (0.5 mM final concentration) and incubated for further 5-6 h at 37°C 230 rpm. Harvest was done by centrifugation (6 min 5000 x g) followed by cell disruption with 10 pl B-PER™ II Bacterial Protein Extraction Reagent (Cat. No. 78260; Thermo Fisher Scientific, Waltham, Massachusetts, United States; supplemented with DNAse I (200 Units / ml) and lysozyme (0.4 mg / ml)) according to the manufacturer’s protocol and addition of 150 pl PBS, pH7.4. The crude extracts were used at 1 :2000 dilution (final) in the assay. Binding was performed against 10 nM (final concentration) of biotinylated CD47 binding DARPins with 1 :300 (final concentration) of streptavidin-Tb FRET donor conjugate (Cisbio) and 1 :300 (final concentration) of MAb Anti Flag M2-d2 FRET acceptor conjugate (Cisbio) in a well of a 384-well plate and incubated for 60 minutes at room temperature (RT). The HTRF was read-out on a Tecan M1000 using a 340 nm excitation wavelength and a 665 ±10 nm emission filter. All dilutions were prepared in PBS-TB (PBS, pH 7.4 containing 0.2% BSA and 0.1 % Tween 20®).

[0235] Amino acid sequences of selected masking ankyrin repeat domains that specifically bind to CD47-binding

[0236] DARPins of SEQ ID NO: 5 and / or SEQ ID NO: 6 are provided in SEQ ID NOs: 10 to 22, i.e. Mask01 (SEQ

[0237] ID NO: 10), Mask02 (SEQ ID NO: 11), Mask03 (SEQ ID NO: 12), Mask04 (SEQ ID NO: 13), Mask05 (SEQ

[0238] ID NO: 14), Mask06 (SEQ ID NO: 15), Mask07 (SEQ ID NO: 16), Mask08 (SEQ ID NO: 17), Mask09 (SEQ

[0239] ID NO: 18), Mask10 (SEQ ID NO: 19), Mask11 (SEQ ID NO: 20), Mask12 (SEQ ID NO: 21) and Mask13 (SEQ ID NO: 22).

[0240] 1. 1.4 Purification of masking ankyrin repeat proteins

[0241] For further analysis, the selected clones showing specific CD47-binding DARPin binding in the crude cell extract HTRF, as described above, were expressed in E. coli cells, with a His-tag (of SEQ ID NO: 7). 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 25 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 500 ml 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 4-5 h. 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 and resins known to the person skilled in the art.

[0242] Highly soluble ankyrin repeat proteins were purified from E. coli culture (up to 200 mg ankyrin repeat protein per litre of culture) with a purity >95% as estimated from 4-12% SDS PAGE. These mono-domain DARPins were characterized biophysically by size exclusion chromatography, ProteOn surface plasmon resonance (SPR) target affinity assessment, and SDS-PAGE.

[0243] 1. 1.5 Engineered masking variants

[0244] Additional sequence engineering was made on four of the above-described Masks, leading to the following mask variants:

[0245] Based on Mask03 (SEQ ID NO: 12):

[0246] Mask14 (SEQ ID NO: 23) and Mask15 (SEQ ID NO: 24).

[0247] Based on Mask05 (SEQ ID NO: 14): Mask16 (SEQ ID NO: 25), Mask17 (SEQ ID NO: 26), Mask18 (SEQ ID NO: 27), Mask19 (SEQ ID NO: 28), Mask20 (SEQ ID NO: 29), Mask21 (SEQ ID NO: 30) and Mask22 (SEQ ID NO: 31).

[0248] Based on Mask06 (SEQ ID NO: 15):

[0249] Mask23 (SEQ ID NO: 32) and Mask24 (SEQ ID NO: 33).

[0250] Based on Mask11 (SEQ ID NO: 20):

[0251] Mask25 (SEQ ID NO: 34) and Mask26 (SEQ ID NO: 35).

[0252] 1. 1.6 SEC and stress test analysis

[0253] AKTA purified masking DARPins in PBS, pH7.4 were aliquoted into sterile glass vials (Schmidlin: LPP 11 09 0620) and stressed by incubation at 60°C for 1 week. The applied stress conditions allow a prediction of the biophysical properties after 2 years storage at 4°C. For each Mask, an aliquot was stored at -70°C as a reference. The reference and the heat-stressed samples were exposed to one freeze-thaw cycle before analysis. The samples were evaluated for multimerization, aggregation and fragmentation by SDS- PAGE and analytic SEC. Stressed samples were then compared to the respective reference samples. No colour change, turbidity or precipitation was detected by visual inspection. The proteins were run at 2 mg / ml in PBS, pH7.4 over a Superdex 200 Increase 5 / 150 GL column (GE healthcare). Traces were analyzed at 280 nm (referenced by 360nm). No significant generation of multimer or degradation peaks were observed for the 60°C samples in comparison to the reference samples stored at -70°C.

[0254] Corresponding chromatograms are shown in Figure 1A (Mask01 to Mask13) and Figure 1 B (Mask14 to Mask26).

[0255] 1. 1. 7 SPR analysis

[0256] Biotinylated CD47-binding DARPins of SEQ ID NO: 5 and 6 in PBST (PBS, pH 7.4 containing 0.005% Tween 20®) were immobilized as target on a neutravidin chip (NAHLC200M, Xantec ProteOn Sensor Chip) on a ProteOn XPR 36 instrument according to standard procedures. In L2, CD47-binding DARPin of SEQ ID NO: 5 was immobilized at about 200 RU; in L3, CD47-binding DARPin of SEQ ID NO: 6 was immobilized at about 275 RU.

[0257] The interaction of Mask01 to Mask06 and Mask08 to Mask13 with the targets was then measured by injecting 367 pl PBST containing serial dilutions (1 :3) of the masking DARPins (100 nM, 33 nM, 11 nM, 3.7 nM), followed by a running buffer (PBST) flow for 15 minutes at a constant flow rate of 100 pl / min (off-rate measurement). The regeneration was performed using 30 pl 100 mM H3PO4. 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 the masking DARPins (doublereferencing). Binding parameters (Kd) against the CD47-binding DARPins were determined forthe masking DARPins as shown in Table 1 .

[0258] Table 1 longer off-rate measurement required to obtain more reliable value.

[0259] Figure 2 shows the corresponding SPR curves. All masking DARPins (except Mask12 for which Kd value determination was not optimal) displayed some cross-reactivity to the CD47-binding DARPin they were not selected on because of the sequence similarity (one identical repeat) of the 2 CD47-binding DARPins. The specific binding affinity to the CD47-binding DARPin on which the Mask were selected on was at least 25- fold higher than the affinity to the other CD47-binding DARPin.

[0260] 1.2 CD117-binding DARPins

[0261] The second parental DARPins required as source DARPin for the creation of the 2-in-1 domains of the invention are DARPins with a binding specificity to CD117 (referred to as CD117-binding DARPins or CD117 parent). Such DARPins with binding specificity to CD117 have been disclosed in EP24150551.

[0262] Four CD117-binding DARPins were used as source for the creation of various 2-in-1 domains having mutually exclusive binding specificity to CD117 and CD47-binding DARPins. For three of them (CD117 parent 01 to 03), binding to CD117 is known to compete with the CD117 ligand (SCF), while the fourth one (CD117 parent 04) binds CD117 in a non-competing way with this ligand (EP24150551). An overview is shown in Table 2.

[0263] Table 2

[0264] Example 2: Profiling of the parental DARPins

[0265] The generation of 2-in-1 repeat domains (also referred to as 2-in-1 DARPins) can be separated into two steps, which are summarized below:

[0266] First step: a profiling of each parental DARPin is performed to understand if and which one of the terminal capping modules as well as the internal repeat modules are involved in the binding to the respective targets. The profiling also helps to determine if the terminal capping modules can be replaced by internal repeat modules. In this first step, variants of the parental DARPins are created (by shortening or introducing nonbinding dummy repeat modules at specific positions within the DARPin) and subsequent binding properties of the variants to the targets are assessed by ELISA. As the result of the profiling, one obtains an understanding of which parental DARPins can be fused on which side of a 2-in-1 domain, and to which extent, without major loss of binding affinity.

[0267] Second step (see Example 3): the 2-in-1 domains are designed and tested based on the profiling outcome. The aim is to obtain 2-in-1 domains maintaining the affinity to CD117 (i.e. 1sttarget of the 2-in-1 domains) and also generate a diverse range of affinities to the CD47-binding DARPin (i.e. the 2ndtarget of the 2-in-1 domains).

[0268] Example 2 presents the outcome of the profiling (first step) made for each of the parental DARPins. The profiling method applied here follows the method described in more details in Example 9, section 9.1 of WO2023110983.

[0269] All parental DARPins used for the creation of 2-in-1 domains are summarized in Table 3.

[0270] Table 3

[0271] 2.1 Mask profiling

[0272] For each parental Mask (i.e. Mask05, Mask06 and Mask11), 5 variants were created for the N-terminal investigation and 5 variants were created for the C-terminal investigation (not shown). Profiling results are shown in Table 4.

[0273] Table 4: Profiling of parental Masks DARPins. where X means the repeat module cannot be modified or extended; Y means the repeat module type can be changed (e.g. transformed from an internal repeat module to a terminal capping module); Z means the repeat module can be removed without expecting major binding affinity loss; U means not investigated; N is the N-terminal capping module; C is the C-terminal capping module; and IR1 , IR2 and IR3 are the first, second and third internal repeat modules from the N-terminal end, respectively.

[0274] 2.2 CD117-binding DARPin profiling For each parental CD117-binding DARPin (i.e. CD117 parent 01 , 02, 03 and 04), 5 variants were created for the N-terminal investigation and 5 variants were created for the C-terminal investigation (not shown). Profiling results are shown in Table 5.

[0275] Table 5: Profiling of parental CD117 DARPins. where X means the repeat module cannot be modified or extended; Y means the repeat module type can be changed (e.g. transformed from an internal repeat module to a terminal capping module); Z means the repeat module can be removed without expecting major binding affinity loss; N is the N-terminal capping module; C is the C-terminal capping module; and IR1 , IR2 and IR3 are the first, second and third internal repeat modules from the N-terminal end, respectively.

[0276] Example 3: Design, creation and characterisation of 2-in-1 domains

[0277] Based on the outcome of the parental DARPins profiling, 89 2-in- 1 domains based on the parental DARPins shown in Table 3 were designed. These designs included various combinations of said parental DARPins or their profiling variants. For CD117 parent sequences 01 , 02 and 04, additional mutations in the N-cap to replace the “RILLA” motif with “RELLK” in the N-terminal capping module (i.e. in positions 19 to 23 in SEQ ID NOs: 1 , 2 and 4) were performed in cases where said CD117 parent sequences were used as parental DARPin for the N-terminal side of a 2-in-1 domain. For some designs, another example of mutated residue in the N-terminal capping modules of the 2-in-1 domains compared to the corresponding parental N- terminal capping modules was position 15 (numbered according to any of the parental DARPins of Table 3). Where required, mutations were additionally made in 2-in- 1 domains which include a transformed repeat module (e.g. a repeat module transformed from a terminal capping module to an internal repeat module, or vice versa) to adapt these transformed modules to the conventional ankyrin repeat module structure. To create a range of binding affinities of the 2-in-1 domains to the CD47-binding DARPin target, some mutations were also made in the potential target interaction residues of the repeat modules originating from the masking parental DARPins.

[0278] Among the 89 initial clones, thirty 2-in-1 domains were selected as most relevant for further characterisation. These thirty selected 2-in-1 domains were classified per family and are shown in Table 6.

[0279] Table 6

[0280] For subsequent characterization, 2-in-1 domains were cloned following standard procedures and expressed with a N-terminal Flag-tag of SEQ ID NO: 9. The 2-in-1 domains were expressed in high throughput by IMAC purification in 96-wells plates followed by a desalting plate to remove the imidazole content, and then stored in PBS pH 7.4.

[0281] 3.1 : SEC analysis of the 2-in-1 domains

[0282] SEC analysis of the expressed 2-in- 1 domains was assessed at a concentration of 10OuM (in PBS at pH7.4) and read at 280nm (referenced by 360nm), according to standard procedures. Corresponding chromatograms are shown in Figure 3. 3.2: Binding assessment of the 2-i n-1 domains to CD117 or CD47-binding DARPin

[0283] The rationale of this experiment was to compare the dose-response binding behaviour of the 2-in- 1 domains with the binding behaviour 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: 5 and 6 were tagged with a N-terminal His-tag of SEQ ID NO: 7 and with a C-terminal Avi-tag of SEQ ID NO: 8, 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 (OD450nm, ref 620nm).

[0284] Resulting titration curves are shown in Figure 4A for target CD117, in Figure 4B for target CD47-binding DARPin of SEQ ID NO: 5 and in Figure 4C for target CD47-binding DARPin of SEQ ID NO:6. BC50 (half- maximum binding concentration) values are shown in Table 7 (ND = not determined).

[0285] Table 7

[0286] 3.3: Cell binding

[0287] A selection of 12 purified 2-in-1 domains (i.e. 2-in-1 DARPinOI , 2-in-1 DARPinO3, 2-in-1 DARPinO5, 2-in-1 DARPinO6, 2-in-1 DARPinO7, 2-in-1 DARPin14, 2-in-1 DARPin18, 2-in-1 DARPin19 and 2-in-1 DARPin22) were tested for CD117 cell binding using a cancer cell line expressing CD117 (Kasumi-1 cells, CL0400, DMSZ). In brief, the CD117 expressing cells were washed 1x with 500 pl PBS, spun at 350xg for 5 minutes and the supernatant was discarded. Then, a titration of the selected 2-in-1 domains diluted in FACS buffer was added to the pellet of cells, resuspended and incubated for 30 minutes at 4°C. Cells were washed twice with 200 pl cold PBS, spun at 350g for 5 minutes and the supernatant was discarded. 50 pl of anti- penta His AF647 antibody (1 :200, Qiagen, 1019252) with live / dead green (1 :3000, thermo Fischer L34970), 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. CD117 parent 01 to 04 were used as controls.

[0288] Curves showing the measured median fluorescent intensity (MFI) are show in Figure 5. Curves represent a fitted four-parameters sigmoid model.

[0289] 3.4: Mutual binding exclusivity assessment of the 2-in-1 domains by competitive ELISA

[0290] In this experiment, the mutual binding exclusivity property of representative 2-in-1 domains of the invention were further assessed in a competitive binding ELISA. A schematic view of the experimental setup is shown in Figure 6A. The tested domains are: 2-in-1 DARPinOI , 2-in-1 DARPinO4, 2-in-1 DARPinW, 2-in- 1 DARPinW, 2-in-1 DARPinW, 2-in-1 DARPinW, 2-in-1 DARPinW, 2-in-1 DARPinW, 2-in-1 DARPin20, 2-in-1 DARPin21 , 2-in-1 DARPin22, 2-in-1 DARPin23, 2-in-1 DARPin24, 2-in-1 DARPin25 and 2-in- 1 DARPin30.

[0291] 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, Catalogue num. 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 as fixed concentration of each 2-in- 1 domain for the subsequent exposure to the competitor. The competitor was the CD47-binding DARPin of SEQ ID NO: 5, further comprising an N-terminal His-tag of SEQ ID NO: 7 and a C-terminal Avi-tag of SEQ ID NO: 8. 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 detection of the signal was made via the flag-tag (HRP Anti-DDDDK tag, abeam, ab2493) on each 2-in-1 domain, which allows 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.

[0292] Figure 6B 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) at which the titrated competitor was applied. The plots on the right in Figure 6B show the corresponding evolution of the signal in function of the competitor concentration. Accordingly, a property of mutual binding exclusivity to CD117 and CD47-binding DARPin is observed for all tested 2-in-1 domains. Curves represent a fitted four- parameters sigmoid model.

[0293] 3.5: Blocking window assessment in 2D format

[0294] A cell binding experiment was performed on a hCD47-expressing CHO cell line (in house lentiviral transduction of CHO-k1 cell line to express human hCD47) to evaluate the blocking (or masking) window provided by the 2-in-1 domains when linked to a CD47-binding DARPin.

[0295] Representative 2-in-1 domains according to the invention were therefore tested in a two-domain (2D) DARPin format, i.e. 2-in-1 DARPins 12, 14 and 22 (of SEQ ID NOs: 49, 51 and 59, respectively) were linked to a CD47-binding DARPin of SEQ ID NO: 5, where the CD47-binding DARPin is located N-terminally of the respective 2-in-1 domains.

[0296] A blocking window can be determined by comparing the binding behaviour of the 2D constructs with the corresponding mono-domain (1 D) CD47-binding DARPin (i.e. SEQ ID NO: 5). In the 2D constructs, the 2- in-1 domain having binding specificity for CD47-binding DARPin can mask the CD47-binding DARPin and therefore hinder binding of the CD47-binding DARPin to CD47 expressed on the cells.

[0297] An overview of the tested 2D constructs is shown in Table 8.

[0298] Table 8

[0299] The 2D constructs were cloned to express an N-terminal “MGS” tag and a C-terminal His6 tag of SEQ ID NO: 78. The CD47-binding domain was linked to the 2-in-1 domain by a linker of SEQ ID NO: 77. The 1 D control binder in this experiment (“1 D CD47-binding DARPin”) was cloned to express an N-terminal flag tag of SEQ ID NO: 9 in front of SEQ ID NO: 5. Cloning and expression of these proteins followed standard procedures known by the skilled in the art. The 2D constructs were purified in high throughput by IMAC purification in 96-wells plates and the 1 D CD47-binding DARPin was purified by IMAC purification followed by size exclusion chromatography on an AKTAxpress™ system according to standard protocols and resins known to the person skilled in the art. As a negative control, 2-in-1 DARPin12 (1 D) as described in section 3.2 was also tested; as expected, no binding of the 2-in-1 domain to CD47 expressed on the cells was observed.

[0300] Curves showing the measured median fluorescent intensity (MFI) are shown in Figure 7, where the curves represent a fitted four-parameters sigmoid model. As can be seen, the binding of the 2D constructs correlates with the BC50 values of each 2-in-1 domain comprised in said 2D constructs, where the highest blocking window was observed for the 2D DARPinO3 comprising the 2-in-1 DARPin with lowest BC50 value (2-in-1 DARPin 12).

[0301] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The aspects within the specification provide an illustration of aspects of the invention and should not be construed to limit the scope of the invention. The skilled artisan readily recognizes that many other aspects are encompassed by the invention. All publications, patents, and GenBank sequences cited in this disclosure are incorporated by reference in their entirety. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material. The citation of any references herein is not an admission that such references are prior art to the present invention.

[0302] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific aspects of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1 . A recombinant binding protein comprising a first designed ankyrin repeat domain having a first binding specificity for a first target and a second binding specificity for a second target, wherein binding of said first designed repeat domain to said first and second targets is mutually exclusive and wherein the first target is CD117 and the second target is a CD47-specific binding agent.

2. The recombinant binding protein according to claim 1 , wherein the first designed repeat domain binds to said first target with a dissociation constant (KD) below 10-5M and / or wherein the first designed repeat domain binds to said second target with a dissociation constant (KD) below 10-5M.

3. The recombinant binding protein according to any one of claims 1 to 2, wherein the first designed repeat domain comprises an N-terminal capping module, a C-terminal capping module, and at least one internal repeat module.

4. The recombinant binding protein according to any one of claims 1 to 3, wherein the first designed repeat domain comprises between three and six internal repeat modules.

5. The recombinant binding protein according to any one of claims 1 to 4, wherein the first designed repeat domain comprises a sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 38 to 67.

6. The recombinant binding protein according to any one of claims 1 to 5, wherein the protein further comprises a therapeutic agent.

7. The recombinant binding protein according to claim 6, wherein the therapeutic agent is a binding agent having specificity for a tumor-associated antigen, a cell type-associated antigen and / or an immune cell surface-expressed antigen.

8. The recombinant binding protein according to claim 7, wherein (i) the tumor-associated antigen and / or the cell type-associated antigen is CD117, and / or (ii) the immune cell surface-expressed antigen is CD16a.

9. The recombinant binding protein according to any one of claims 1 to 8, wherein the protein further comprises a CD47-specific binding agent.

10. The recombinant binding protein according to claim 9, wherein the CD47-specific binding agent is a second designed ankyrin repeat domain, optionally wherein the second designed ankyrin repeat domain is the second target of the first designed ankyrin repeat domain.

11. The recombinant binding protein according to claim 10, wherein the second designed ankyrin repeat domain comprises a sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 5, 6, 68, 69, 70, 71 , 72 or 73.

12. An isolated nucleic acid encoding the binding protein according to any one of claims 1 to 11.

13. A pharmaceutical composition comprising (i) the recombinant binding protein according to any one of claims 1 to 11 and / or (ii) the nucleic acid according to claim 12, and optionally a pharmaceutically acceptable carrier or diluent.

14. A method of conditionally binding a CD47-specific binding agent to CD47 expressed on the surface of cells, the method comprising the step of administering to a subject the recombinant binding protein according to any one of claims 10 to 11.

15. The method of claim 14, wherein said binding a CD47-specific binding agent to CD47 expressed on the surface of cells is conditional on the presence of CD117 on the surface of said cells.

16. A method of treating a medical condition, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of the binding protein according to any one of claims 1 to 11 , the nucleic acid according to claim 12, or the pharmaceutical composition according to claim 13.

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