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WO2025146487A1 - Protéines de liaison multispécifiques - Google Patents

Protéines de liaison multispécifiques Download PDF

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Publication number
WO2025146487A1
WO2025146487A1 PCT/EP2025/050108 EP2025050108W WO2025146487A1 WO 2025146487 A1 WO2025146487 A1 WO 2025146487A1 EP 2025050108 W EP2025050108 W EP 2025050108W WO 2025146487 A1 WO2025146487 A1 WO 2025146487A1
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Prior art keywords
binding
ankyrin repeat
multispecific
protein
repeat domain
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Valérie Perrine CALABRO
Alexander Link
Anja Christina SCHLEGEL
Natalia VENETZ ARENAS
Simon FONTAINE
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Molecular Partners AG
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Molecular Partners AG
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics

Definitions

  • the present disclosure relates to multispecific binding proteins that specifically bind to (i) a stem cell marker, (ii) an immunoregulatory protein and (iii) an immune cell-associated antigen. More specifically, the disclosure relates to multispecific binding proteins which are useful for the pretreatment of a subject prior to receiving a stem cell transplantation.
  • a subject may be suffering from a condition in which a stem cell transplantation is considered beneficial, for example a hematologic disease or a hematological malignancy, such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML).
  • MDS myelodysplastic syndrome
  • AML acute myeloid leukemia
  • HSCs Hematopoietic stem cells
  • HSC Hematopoietic stem cell transplantation
  • HSCs active hematopoietic stem cells collected from various sources, such as bone marrow, peripheral blood, or umbilical cord blood, to restore normal blood cell production in individuals with damaged or defective bone marrow or immune systems. This procedure serves as a critical therapy for eliminating bone marrow-related diseases like leukemia or correcting congenital immunodeficiencies.
  • HSCT HSCT
  • Prevailing conditioning methodologies such as irradiation (e.g., total body irradiation) and DNA alkylating/modifying agents are non-targeted.
  • irradiation e.g., total body irradiation
  • DNA alkylating/modifying agents are non-targeted.
  • These aggressive conditioning regimens effectively decimate the recipient's immune and other cells, and also trigger detrimental repercussions across numerous organ systems resulting possibly in organ damage and secondary malignancies and frequently precipitate life-threatening complications.
  • the severe toxicity of these conditioning regimens severely limits the clinical applicability of bone marrow transplantation as they are contraindicated for large groups of patients.
  • the safety of transplantation could significantly improve, making this powerful form of cell therapy accessible to a broader range of patients.
  • Therapeutic modalities that selectively target the endogenous hematopoietic stem cell population, thereby sparing cells and tissues, such as platelets, white blood cells, and red blood cells may provide improved conditioning regimens.
  • the depletion of host HSCs and facilitating the integration of donor HSCs has been shown experimentally in animal models using monoclonal anti-CD117 antibodies, including in combination with blockade of CD47 (Chhabra et al, Sci Transl Med. 2016 Aug 10;8(351):351 ra105), signifying the potential for targeted conditioning regimens using biologic agents.
  • the cell surface marker CD117 can be used to identify specific hematopoietic progenitor cells residing in the bone marrow.
  • CD117 also recognized as c-kit or stem cell factor (SCF) receptor, is pivotal in governing diverse cellular processes, including cell survival, proliferation, and differentiation, particularly HSCs. High expression levels of CD117 are observed in HSCs, multipotent progenitors (MPPs), and common myeloid progenitors (CMPs), serving as a marker for these cell types. During hematopoiesis, CD117 is present on early progenitor cells, orchestrating their self-renewal and guiding their differentiation into distinct blood cell lineages.
  • SCF stem cell factor
  • CD47 localized on the cell surface of most cells, acts as a key regulator of phagocytosis by engaging with multiple ligands. Its widespread expression encompasses various cell types and tissues, playing a crucial role as a cellular ligand for SIRPa, predominantly found on myeloid cells, including macrophages, granulocytes, myeloid dendritic cells, and mast cells. Of particular importance is its role in inhibiting phagocytosis through the CD47-SIRPa signaling complex, serving as a “don’t-eat-me” signal for phagocytes.
  • CD47 is expressed on the surface of most healthy cells, including red blood cells, and targeting CD47 is associated with numerous side effects, including depletion of red blood cells.
  • the present invention is directed to multispecific binding proteins. More particularly, the present invention is directed to a multispecific binding protein that specifically binds to (i) a stem cell marker, (ii) an immunoregulatory protein and (iii) an immune cell-associated antigen. Such multispecific binding proteins may comprise one or more ankyrin repeat domains. Such multispecific binding proteins may be used in the targeted depletion of hematopoietic stem cells through activation of innate immune cells, particularly in the context of hematopoietic stem cell transplantation (HSCT).
  • HSCT hematopoietic stem cell transplantation
  • a multispecific binding protein comprising (1) a first binding agent with binding specificity for an immunoregulatory protein, (2) a second binding agent with binding specificity for the first binding agent, and (3) a third binding agent with binding specificity for a stem cell-associated antigen, wherein binding of said second binding agent and said third binding agent is mutually exclusive.
  • E2 The multispecific binding protein of E1 , wherein said second binding agent reversibly binds said first binding agent.
  • E3 The multispecific binding protein of E1 or E2, wherein said first binding agent is released from binding to the second binding agent upon binding of the third binding agent to said stem cell-associated antigen.
  • E5. The multispecific binding protein of E4, wherein the first binding region of said second binding agent reversibly binds said first binding agent, and wherein said first binding region is released upon binding of the second binding region to said stem cell-associated antigen.
  • E14 The multispecific binding protein of any one of E8 to E12, wherein said second binding agent and said further binding agent with binding specificity for a stem cell-associated antigen bind to a different epitope on the stem cell-associated antigen.
  • E30 The multispecific binding protein of any one of E8 to E29, wherein said further binding agent with binding specificity for a stem cell-associated antigen comprises an ankyrin repeat domain with binding specificity for CD117.
  • E31 The multispecific binding protein of any one of E8 to E30, wherein said further binding agent with binding specificity for a stem cell-associated antigen comprises an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 10, 11 , or 26 to 29.
  • E33 The multispecific binding protein of any one of E8 to E32, wherein said further binding agent with binding specificity for a stem cell-associated antigen comprises an ankyrin repeat domain with binding specificity for CD117, wherein said ankyrin repeat domain with binding specificity for CD117 competes with ligand binding.
  • E34 The multispecific binding protein of any one of E1 to E33, wherein the multispecific binding protein conditionally blocks CD47 signaling.
  • E35 The multispecific binding protein of any one of E1 to E34, wherein the multispecific binding protein blocks stem cell factor (SCF) signaling.
  • SCF stem cell factor
  • E36 The multispecific binding protein of any one of E1 to E35, wherein said binding agents can be arranged in any order.
  • E37 The multispecific binding protein of any one of E1 to E36, wherein said binding agents are covalently linked with a peptide linker.
  • E38 The multispecific binding protein of E37, wherein said peptide linker is a proline-threonine-rich peptide linker.
  • E39 The multispecific binding protein of E37 or E38, wherein the amino acid sequence of said peptide linker has a length from 1 to 50 amino acids.
  • E40 The multispecific binding protein of any one of E1 to E39, wherein any of said ankyrin repeat domains additionally comprises a G, a S or a GS at the N-terminus.
  • E41 The multispecific binding protein of any one of E1 to E40, wherein said multispecific binding protein further comprises a half-life extending moiety.
  • E42 The multispecific binding protein of E41 , wherein said half-life extending moiety is a binding agent that specifically binds to human serum albumin.
  • E43 The multispecific binding protein of E42, wherein said binding agent that specifically binds to human serum albumin comprises a designed ankyrin repeat domain with binding specificity for human serum albumin.
  • E44. The multispecific binding protein of E43, wherein said ankyrin repeat domain with binding specificity for human serum albumin comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 17 to 19.
  • E45 The multispecific binding protein of E43 or E44, wherein said ankyrin repeat domain with binding specificity for human serum albumin is located at the C-terminus or at the N-terminus of the multispecific recombinant binding protein.
  • E46 The multispecific binding protein of any one of E1 to E45, wherein said multispecific recombinant binding protein comprises an amino acid sequence at least about 80% identical to the amino acid sequence of any one of SEQ ID NOs: 1 to 7.
  • E48 A vector comprising the nucleic acid of E47.
  • E49 The vector of E48, wherein the vector is a DNA vector, an RNA vector, a plasmid, a cosmid, or a viral vector.
  • E50 A cell comprising the nucleic acid of E47 or the vector of any one of E48 to E49.
  • a method of producing a multispecific binding protein comprising culturing the cell of E50 and collecting the multispecific binding protein from the cell and/or the culture medium.
  • a pharmaceutical composition comprising the multispecific binding protein of any one of E1 to E46, the nucleic acid of E47, the vector of any one of E48 to E49, or the cell of E50, and a pharmaceutically acceptable carrier and/or diluent.
  • E54 The multispecific binding protein of any one of E1 to E46, the nucleic acid of E47, the vector of any one of E48 to E49, the cell of E50, or the pharmaceutical composition of E52 for use as a medicament.
  • E56 A method of selectively depleting or ablating a hematopoietic stem cell (HSC) or progenitor cell population in a subject in need thereof, the method comprising administering to said subject an effective amount of the multispecific binding protein of any one of E1 to E46, the nucleic acid of E47, the vector of any one of E48 to E49, the cell of E50, or the pharmaceutical composition of E52.
  • HSC hematopoietic stem cell
  • a method of engrafting stem cells in a subject comprising: (a) administering to the subject an effective amount of the multispecific binding protein of any one of E1 to E46, the nucleic acid of E47, the vector of any one of E48 to E49, the cell of E50, or the pharmaceutical composition of E52, thereby selectively depleting or ablating hematopoietic stem cell or progenitor cell population in a target tissue of the subject; and (b) administering a stem cell population to the target tissue of the subject, wherein the administered stem cell population engrafts in the target tissue of the subject.
  • E59 The multispecific binding protein for use according to E54, the nucleic acid for use according to E54, the vector for use according to E54, the cell for use according to E54, or the pharmaceutical composition for use according to E54 or the methods of any one of E55 to E57, wherein the subject has a malignant, pre-malignant or non-malignant disorder.
  • FIG. 1 An exemplary multispecific binding protein comprising HSA-binding agent (a-HSA), CD16a binding agent (a-CD16a), CD117 binding agent (a-CD117), 2-in-1 binding domain comprising a first binding region for CD117 (dotted) and a second binding region for the CD47-binding agent (“mask”, striped), and CD47 binding agent (a-CD47).
  • a-HSA HSA-binding agent
  • CD16a binding agent a-CD16a
  • a-CD117 CD117 binding agent
  • 2-in-1 binding domain comprising a first binding region for CD117 (dotted) and a second binding region for the CD47-binding agent (“mask”, striped), and CD47 binding agent (a-CD47).
  • the CD117-binding agent (a-CD117) binds CD117 on a target cell, for example a hematopoietic stem cell (HSC).
  • HSC hematopoietic stem cell
  • Figure 2 NK cell degranulation assay.
  • DARPin proteins at different concentrations (10 nM (hatched), 1 nM (striped), 0.1 nM (dotted) and 0 nM (black) with binding specificity for CD117 and CD16a were tested for their capacity to mediate degranulation (% of CD107a) of isolated primary NK cells (gated as live CD56+) in the presence of Kasumi-1 cells.
  • Figure 3 DARPin dependent cellular phagocytosis assay (DDCP).
  • DDCP DARPin dependent cellular phagocytosis assay
  • DARPin proteins at different concentrations (10 nM (hatched), 1 nM (striped), 0.1 nM (dotted) and 0 nM (black) with binding specificity for CD117 and CD16a were tested for their capacity to induce M0 macrophages (C11 b+) to phagocytose cell-tracker labelled Kasumi-1 target cells. Shown are % of double positive CD11 b+Cell Trace+ cells.
  • CD47 blockade eliminates the inhibitory signal, while CD16a activation amplifies the phagocytic and/or cytotoxic response, leading to increased clearance of target cells by enhancing the ability of phagocytes to recognize and engulf these cells and by enhancing cytotoxicity.
  • target refers to an individual molecule such as a nucleic acid molecule, a polypeptide or protein, a carbohydrate, or any other naturally occurring molecule, including any part of such individual molecule, or to complexes of two or more of such molecules, or to a whole cell or a tissue sample, or to any non-natural compound.
  • a target is a naturally occurring or nonnatural polypeptide or protein, or a polypeptide or protein containing chemical modifications, for example, naturally occurring or non-natural phosphorylation, acetylation, or methylation.
  • CD47 refers to a transmembrane polypeptide, which belongs to the immunoglobulin superfamily.
  • Human CD47 (Cluster of Differentiation 47) is identified by UniProt Ref. No. Q08722 and is also known as integrin associated protein (IAP), OA3 or MER6.
  • CD47 partners with membrane integrins and also binds ligands including signal-regulatory protein alpha (SIRPalpha).
  • SIRPalpha signal-regulatory protein alpha
  • CD47 functions as a marker of “self and transmits a "don't-eat-me” signal by binding to SIRPalpha expressed by myeloid cells, macrophages, dendritic cells and neutrophils.
  • peptide also encompasses peptides modified by, e.g., glycosylation, and proteins comprising two or more polypeptide chains, each of length of 4 to 600 amino acids long, cross-linked by, e.g., disulfide bonds, such as, e.g., insulin and immunoglobulins.
  • binding protein refers to a protein comprising at least one binding domain.
  • a binding protein may also comprise two, three, four, five or more binding domains.
  • said binding protein is a recombinant binding protein.
  • any such binding protein may comprise additional polypeptides (such as e.g., polypeptide tags, peptide linkers, fusion to other proteinaceous domains with binding specificity, cytokines, hormones, or antagonists), or chemical modifications (such as coupling to polyethylene-glycol, toxins (e.g., DM1), small molecules, antibiotics and alike) known to the person skilled in the art.
  • binding domain refers to a protein domain with binding specificity for a target.
  • said binding domain is a recombinant binding domain.
  • Patent application W02002/020565 and Forrer et al., (FEBS Letters 539, 2-6, 2003)) contain a general description of repeat protein features and repeat domain features, techniques and applications.
  • the term “repeat protein” refers to a protein comprising one or more repeat domains.
  • a repeat protein comprises one, two, three, four, five or six repeat domains.
  • said repeat protein may comprise additional non-repeat protein domains, polypeptide tags and/or peptide linkers.
  • the repeat domains can be binding domains.
  • 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.
  • a repeat domain further comprises an N-terminal and/or a C-terminal capping module.
  • a capping module can be a repeat module.
  • repeat domains Such repeat domains, repeat modules, and capping modules, sequence motives, as well as structural homology and sequence homology are known from examples of ankyrin repeat domains (W02002/020565), leucine-rich repeat domains (W02002/020565), tetratricopeptide repeat domains (Main, E.R., et al., Structure 11 (5), 497-508, 2003), and armadillo repeat domains (W02009/040338). It is further known that such repeat domains are different from proteins comprising repeated amino acid sequences, where every repeated amino acid sequence is able to form an individual domain (for example FN3 domains of Fibronectin).
  • an ankyrin repeat domain refers to a repeat domain comprising two or more consecutive ankyrin repeat modules as structural units.
  • Ankyrin repeat domains may be modularly assembled into larger ankyrin repeat proteins, optionally with half-life extension domains, using standard recombinant DNA technologies (see, e.g., Forrer, P., et al., FEBS letters 539, 2-6, 2003); W02002/020565, WO2016/156596; WO2018/054971).
  • an ankyrin repeat domain comprises an N-terminal capping module, a C-terminal capping module, and at least one internal repeat module.
  • Capping modules are located at the N-and/or C-terminal end of an ankyrin repeat domain, typically forming tight tertiary interactions (i.e., tertiary structure interactions) with the ankyrin repeat module(s) in between, thereby providing a cap that shields the hydrophobic core of the ankyrin repeat domain at the side from exposure to the solvent.
  • the N-and/or C-terminal capping modules may be derived from a capping unit or other structural unit found in a naturally occurring repeat protein adjacent to a repeat unit. Examples of capping sequences are described in International Patent Publication Nos. WO 2002/020565 and WO 2012/069655, in U.S. Patent Publication No. US2013/0296221 , and by Interlandi et al., J Mol Biol. 2008 Jan 18;375(3):837-54.
  • construct refers to a binding protein comprising one or more designed ankyrin repeat domain and optionally a peptide linker and/or tag sequence.
  • An example of a peptide linker is provided in SEQ ID NO: 21 and an example of a tag sequence is provided in SEQ ID NO: 20.
  • the term “designed” as used in “designed repeat protein”, “designed repeat domain” and the like refers to the property that such repeat proteins and repeat domains, respectively, are man-made and do not occur in nature.
  • the multispecific binding proteins disclosed herein are designed repeat proteins and they comprise at least one designed ankyrin repeat domain.
  • the designed repeat domain is a designed ankyrin repeat domain. All of the amino acid sequences described herein may be substituted by one or more amino acids, suitably up to 15, up to 14, up to 13, up to 12, up to 11 , up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 substitution or deletion is made in any of the amino acid sequences described herein.
  • the multispecific binding protein may be a variant of a multispecific binding protein described herein, provided such a variant retains binding specificity for the ligands of the parent multispecific binding protein.
  • Variants may be made using the methods of protein engineering and site-directed mutagenesis known in the art using the recombinant polynucleotides (see example, see Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2001 , Cold Spring Harbor Laboratory Press, which is incorporated herein by reference). “Variants” of the multispecific binding protein includes insertions, deletions and substitutions, either conservative or non-conservative. Included are variants of the sequence of the multispecific binding protein where such variations do not substantially alter the activity and/or binding specificity for the ligands of the multispecific binding protein.
  • Individual binding domains may be linked covalently with a peptide linker.
  • the linker is of sufficient length to enable the domains to fold in such a way as to permit binding of the multispecific binding protein to its target(s).
  • a suitable linker may comprise from 1 to 50 amino acids, for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50 amino acids, suitably from 6 to 38 amino acids.
  • binding proteins comprising ankyrin repeat domains with binding specificity for CD47 are described in patent application with number WO2024/251695, which is incorporated by reference in its entirety. These binding proteins have been shown to block the interaction of CD47/SIRPa, thereby disrupting the "don’t eat me signal".
  • said ankyrin repeat domain with binding specificity for CD47 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 8.
  • said ankyrin repeat domain with binding specificity for CD47 comprises or consists of the amino acid sequence of SEQ ID NO: 8.
  • said ankyrin repeat domain with binding specificity for CD47 binds human soluble CD47 in PBS with a dissociation constant (KD) of or below about 10 -7 M, or of or below about 10 -8 M, or of or below about 10 -9 M, or of or below about 1 O -10 M, or of or below about 10 -11 M.
  • KD dissociation constant
  • the dissociation constant is determined by surface plasmon resonance (SPR), suitably in PBS, e.g. as described in Example 1 .
  • said ankyrin repeat domain with binding specificity for CD47 is capable of blocking the interaction of CD47 with signal-regulatory protein alpha (SIRPa). In one embodiment, said ankyrin repeat domain with binding specificity for CD47 blocks or reduces the interaction of CD47 with signal-regulatory protein alpha (SIRPa).
  • SIRPa signal-regulatory protein alpha
  • a suitable assay to assess whether said ankyrin repeat domain with binding specificity for CD47 is capable of blocking the interaction of CD47 with signal- regulatory protein alpha (SIRPa) is described in Example 1.
  • said ankyrin repeat domain with binding specificity for CD47 inhibits ligand binding with an IC50 of at most about 500 nM or less, at most about 400 nM or less, at most about 300 nM or less, at most about 200 nM or less, at most about 100 nM or less, at most about 90 nM or less, at most about 80 nM or less, at most about 70 nM or less, at most about 60 nM or less, at most about 50 nM or less.
  • said ankyrin repeat domain with binding specificity for CD47 reduces the interaction of CD47 with signal- regulatory protein alpha (SIRPa) by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% compared to a binding protein, suitably an ankyrin repeat domain, which does not have binding specificity for CD47.
  • SIRPa signal- regulatory protein alpha
  • CD16a also known as FcyRllla, is a receptor expressed primarily on immune cells such as natural killer (NK) cells, macrophages, and neutrophils. This receptor plays a pivotal role in antibodydependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP).
  • ADCC antibody dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • CD16a Upon engagement, CD16a triggers signaling cascades within immune cells, leading to the activation of various effector functions.
  • NK cells it initiates cytotoxic activities against target cells, releasing cytotoxic granules and inducing cell death.
  • CD16a activation stimulates phagocytosis, enabling these cells to engulf and eliminate target cells.
  • binding proteins comprising ankyrin repeat domains with binding specificity for CD16a are described in patent application with number WO2024/251628, which is incorporated by reference in its entirety. These binding proteins have been shown to stimulate effector cells.
  • said ankyrin repeat domain with binding specificity for CD16a comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 9.
  • said ankyrin repeat domain with binding specificity for CD16a comprises or consists of the amino acid sequence of SEQ ID NO: 9.
  • said ankyrin repeat domain with binding specificity for CD16a binds human soluble CD16a in PBS with a dissociation constant (KD) of or below about 10 -7 M, or of or below about 10 -8 M, or of or below about 10 -9 M, or of or below about 10 -10 M, or of or below about 10 -11 M.
  • KD dissociation constant
  • the dissociation constant is determined by surface plasmon resonance (SPR), suitably in PBS, e.g. as described in Example 2.
  • said ankyrin repeat domain with binding specificity for CD16a is capable of stimulating effector cells, suitably innate immune cells, such as NK cells, monocytes and macrophages.
  • innate immune cells such as NK cells, monocytes and macrophages.
  • a suitable assay to assess whether said ankyrin repeat domain with binding specificity for CD16a is capable of stimulating effector cells is described in Example 4.
  • said ankyrin repeat domain with binding specificity for CD16a stimulates effector cells with an EC50 of at most about 200 pM or less, at most about 150 pM or less, at most about 100 pM or less, at most about 50 pM or less.
  • said ankyrin repeat domain with binding specificity for CD117 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 10.
  • said ankyrin repeat domain with binding specificity for CD117 comprises or consists of the amino acid sequence of SEQ ID NO:
  • said ankyrin repeat domain with binding specificity for CD117 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 11 .
  • said ankyrin repeat domain with binding specificity for CD117 comprises or consists of the amino acid sequence of SEQ ID NO:
  • said ankyrin repeat domain with binding specificity for CD117 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 26.
  • said ankyrin repeat domain with binding specificity for CD117 comprises or consists of the amino acid sequence of SEQ ID NO: 26.
  • said ankyrin repeat domain with binding specificity for CD117 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 27.
  • said ankyrin repeat domain with binding specificity for CD117 comprises or consists of the amino acid sequence of SEQ ID NO:
  • said ankyrin repeat domain with binding specificity for CD117 comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 28.
  • said ankyrin repeat domain with binding specificity for CD117 comprises or consists of the amino acid sequence of SEQ ID NO:
  • said ankyrin repeat domain with binding specificity for CD117 binds human soluble CD117 in PBS with a dissociation constant (KD) of or below about 10 -7 M, or of or below about 10 -8 M, or of or below about 10 -9 M, or of or below about 1 O -10 M, or of or below about 10 -11 M.
  • KD dissociation constant
  • the dissociation constant is determined by surface plasmon resonance (SPR), suitably in PBS, e.g. as described in Example 3.
  • Such dual-specific repeat domains can be created by combining repeat modules of two parental repeat domains. The binding specificity to the respective targets of the parental repeat domains therefore confers the binding specificity for each of the two targets.
  • Such dual-specific repeat domains with a first binding specificity for CD117 and a second binding specificity for CD47-binding DARPins have been described in EP24150569.2.
  • said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about
  • said ankyrin repeat domain comprises an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about
  • said ankyrin repeat domain comprises an amino acid sequence at least about
  • said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said ankyrin repeat domain to the first and second targets is mutually exclusive, is capable of competing for binding with stem cell factor (SCF).
  • said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said ankyrin repeat domain to the first and second targets is mutually exclusive, competes for binding with stem cell factor (SCF).
  • said ankyrin repeat domain binds to CD117 with a KD value of, or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM.
  • said ankyrin repeat domain binds to CD117 with a KD value of less than or equal to about 1 nM. In another embodiment, said ankyrin repeat domain binds to CD117 with a KD value of less than or equal to about 100 pM. In another embodiment, said ankyrin repeat domain binds to CD117 with a KD value of less than or equal to about 10 pM. In yet another embodiment, said ankyrin repeat domain binds to CD117 with a KD value of less than or equal to about 1 pM.
  • said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said ankyrin repeat domain to the first and second targets is mutually exclusive, binds to a CD47-binding ankyrin repeat domain with a dissociation constant (KD) below 10 -5 M.
  • KD dissociation constant
  • said ankyrin repeat domain binds to a CD47-binding ankyrin repeat domain with a KD of about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, from about 10 -5 M to about 10 -15 M, from about 10 -6 M to about 10 -15 M, from about 10 -7 M to about 10 -15 M, from about 10 -8 M to about 10 _ 15 M, from about 10 -9 M to about 10 -15 M, from about 10 -10 M to about 10 -15 M, from about 10 -11 M to about 10 -15 M, from about 10 -12 M to about 10 -15 M, from about 10 -5 M to about 10 -14 M, from about 10 -6 M to about 10 -14 M, from about 10 -7 M to about 10 -10 -9
  • said ankyrin repeat domain binds to a CD47-specific binding agent with a KD value of, or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM.
  • said ankyrin repeat domain binds to a CD47-specific binding agent with a KD value of less than or equal to about 1 nM. In another embodiment, said ankyrin repeat domain binds to a CD47-specific binding agent with a KD value of less than or equal to about 100 pM. In another embodiment, said ankyrin repeat domain binds to a CD47-specific binding agent with a KD value of less than or equal to about 10 pM. In yet another embodiment, said ankyrin repeat domain binds to a CD47-specific binding agent with a KD value of less than or equal to about 1 pM.
  • said ankyrin repeat domain binds to each of said first and second target with KD1 and KD2 being independently about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 1 O -10 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, from about 10 -5 M to about 10 -15 M, from about 10 -6 M to about 10 -15 M, from about 10 -7 M to about 10 -15 M, from about 10 -8 M to about 10 -15 M, from about 10 -9 M to about 10 -15 M, from about 1 O -10 M to about 10 -15 M, from about 10 -11 M to about 10 -15 M, from about 10 -12 M to about 10 -15 M, from about 10 -5 M to about 10 -14 M, from about 10 -6 M to about 10 -14 M, from about 10 -7 M to about 10 -10 -9 M
  • KD1 and KD2 are independently equal to or less than: about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM.
  • KD1 and KD2 are independently less than or equal to about 1 nM. In another embodiment, KD1 and KD2 are independently less than or equal to about 100 pM. In another exemplary embodiment, KD1 and KD2 are independently less than or equal to about 10 pM. In yet another embodiment, said first designed repeat domain binds to each of the first and second target with KD1 and KD2 being independently less than or equal to about 1 pM.
  • said ankyrin repeat domain binds to CD117 with a first binding affinity and to the CD47-binding ankyrin repeat domain with a second binding affinity, wherein the ratio of said first binding affinity and said second binding affinity is between about 1 :1 and about 1 :10 5 .
  • said ratio may be equal to about 1 :1 , about 1 :10, about 1 :10 2 , about 1 :10 3 , about 1 :10 4 or about 1 :10 5 .
  • said ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, wherein said first binding region binds to ankyrin repeat domain with binding specificity for CD47 with a dissociation constant (KD) of or below about 10' 5 M and/or to CD117 with a dissociation constant (KD) of or below about 10' 5 M.
  • KD dissociation constant
  • KD dissociation constant
  • the multispecific binding proteins provided herein further comprise one or more half-life extending moieties.
  • said half-life extending moiety binds to human serum albumin.
  • the half-life extending moiety comprises an immunoglobulin domain.
  • the immunoglobulin domain comprises an Fc domain.
  • the Fc domain is derived from any one of the known heavy chain isotypes: IgG (y), IgM (p), IgD (6), IgE (s), or IgA (a).
  • the Fc domain comprises an uninterrupted native sequence (i.e., wild type sequence) of an Fc domain.
  • the immunoglobulin Fc domain comprises a variant Fc domain resulting in altered biological activity. For example, at least one point mutation or deletion may be introduced into the Fc domain so as to reduce or eliminate the effector activity (e.g., International Patent Publication No. WO 2005/063815), and/or to increase the homogeneity during the production of the recombinant binding protein.
  • the Fc domain is the Fc domain of human IgGi and comprises one or more of the following effector-null substitutions: L234A, L235A, and G237A (Eu numbering).
  • the Fc domain does not comprise the lysine located at the C-terminal position of human lgG1 (i.e., K447 by Eu numbering). The absence of the lysine may increase homogeneity during the production of the recombinant binding protein. In some embodiments, the Fc domain comprises the lysine located at the C-terminal position (K447, Eu numbering).
  • the half-life extending moiety comprises an ankyrin repeat domain binding human serum albumin comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to SEQ ID NO: 17.
  • a multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for CD47, (2) a second ankyrin repeat domain with binding specificity for the CD47-binding ankyrin repeat domain, (3) a third ankyrin repeat domain with binding specificity for CD117, wherein binding of said second ankyrin repeat domain and said third ankyrin repeat domain is mutually exclusive, and (4) a fourth ankyrin repeat domain with binding specificity for CD16a.
  • the multispecific binding protein may comprise, instead of the second ankyrin repeat domain with binding specificity for the CD47-binding ankyrin repeat domain and the third ankyrin repeat domain with binding specificity for CD117, a single ankyrin repeat domain with a first binding specificity for the CD47-binding ankyrin repeat domain and a second binding specificity for CD117, wherein binding to CD47-binding ankyrin repeat domain and CD117 is mutually exclusive.
  • said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin.
  • multispecific binding proteins comprising (1) a first ankyrin repeat domain with binding specificity for CD47, (2) a second ankyrin repeat domain with a first binding specificity for the CD47-binding ankyrin repeat domain and a second binding specificity for CD117, wherein binding to CD47-binding ankyrin repeat domain and CD117 is mutually exclusive, (3) a third ankyrin repeat domain with binding specificity for CD16a, and optionally (4) a fourth ankyrin repeat domain with binding specificity for CD117.
  • said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin.
  • a multispecific binding protein comprising (1) a first ankyrin repeat domain with binding specificity for CD47, (2) a second ankyrin repeat domain with a first binding specificity for the CD47-binding ankyrin repeat domain and a second binding specificity for CD117, wherein binding to CD47-binding ankyrin repeat domain and CD117 is mutually exclusive, (3) a third ankyrin repeat domain with binding specificity for CD16a, and (4) a fourth ankyrin repeat domain with binding specificity for CD117.
  • said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin.
  • SEQ ID NO: 8 (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 12 to 16, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as at least about
  • said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin (HSA) comprising an amino acid sequence at least about 80%, at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 17 to 19.
  • HSA human serum albumin
  • a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of any one of SEQ ID NOs: 12 to 16, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and optionally (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 10 or 11.
  • said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of any one of SEQ ID NOs: 17 to 19.
  • an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 8
  • said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 17.
  • a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to
  • an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 8
  • an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about
  • an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about
  • said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 17.
  • a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of SEQ ID NO: 13, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 10.
  • said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of SEQ ID NO: 17.
  • a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to
  • an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 8
  • an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about
  • an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about
  • an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about
  • an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about
  • said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 17.
  • a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of SEQ ID NO: 15, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 10.
  • said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of SEQ ID NO: 17.
  • a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to
  • an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 8
  • an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about
  • a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of SEQ ID NO: 16, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 10.
  • said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of SEQ ID NO: 17.
  • an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about
  • an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence at least about 80% identical, such as at least about 81 %, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 11 .
  • said multispecific binding protein further comprises an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to SEQ ID NO: 17.
  • an ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence at least about 80% identical, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least
  • a multispecific binding protein comprising (1) an ankyrin repeat domain with binding specificity for CD47 comprising an amino acid sequence of SEQ ID NO: 8, (2) an ankyrin repeat domain comprising a first binding region with binding specificity for an ankyrin repeat domain with binding specificity for CD47 and a second binding region with binding specificity for CD117, wherein binding of said repeat domain to the first and second targets is mutually exclusive, comprising an amino acid sequence of SEQ ID NO: 16, (3) an ankyrin repeat domain with binding specificity for CD16a comprising an amino acid sequence of SEQ ID NO: 9, and (4) an ankyrin repeat domain with binding specificity for CD117 comprising an amino acid sequence of SEQ ID NO: 11 .
  • said multispecific binding protein further comprises a fifth ankyrin repeat domain with binding specificity for human serum albumin comprising an amino acid sequence of SEQ ID NO: 17.
  • the multispecific binding protein comprises said first, second, and third binding agents, and wherein said multispecific recombinant protein is capable of binding the respective targets of said first, second, and third binding agents simultaneously.
  • the multispecific binding protein comprises said first, second, third, and fourth binding agents, and wherein said multispecific recombinant protein is capable of binding the respective targets of said first, second, third and fourth binding agents simultaneously.
  • the multispecific binding protein comprises said first, second, third, fourth, and fifth binding agents, and wherein said multispecific recombinant protein is capable of binding the respective targets of said first, second, third, fourth, and fifth binding agents simultaneously.
  • said multispecific binding protein is capable of conditionally blocking the interaction of CD47 with signal-regulatory protein alpha (SIRPa).
  • SIRPa signal-regulatory protein alpha
  • said multispecific binding protein conditionally inhibits ligand binding with an IC50 of at most about 500 nM or less, at most about 400 nM or less, at most about 300 nM or less, at most about 200 nM or less, at most about 100 nM or less, at most about 90 nM or less, at most about 80 nM or less, at most about 70 nM or less, at most about 60 nM or less, at most about 50 nM or less.
  • said multispecific binding protein conditionally blocks or reduces the interaction of CD47 with signal-regulatory protein alpha (SIRPa).
  • said multispecific binding protein conditionally reduces the interaction of CD47 with signal-regulatory protein alpha (SIRPa) by at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99% compared to a binding protein, suitably an ankyrin repeat domain, which does not have binding specificity for CD47.
  • SIRPa signal-regulatory protein alpha
  • said multispecific binding protein inhibits ligand binding with an IC50 of at most about 5 nM or less, at most about 4 nM or less, at most about 3 nM or less, at most about 2 nM or less, at most about 1 nM or less, at most about 0.9 nM or less, at most about 0.8 nM or less, at most about 0.7 nM or less, at most about 0.6 nM or less, at most about 0.5 nM or less.
  • E. coli expression strains were used for cloning and protein production, e.g. E. coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA). If appropriate, proteins were produced with an N-terminal His-tag (such as SEQ ID NO: 20) for ease of purification.
  • ankyrin repeat protein libraries having randomized ankyrin repeat modules and/or randomized capping modules can be constructed.
  • libraries can be assembled based on a fixed N-terminal capping module or a randomized N-terminal capping module, and a fixed C-terminal capping module or a randomized C-terminal capping module.
  • 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.
  • randomized modules in such libraries may comprise additional polypeptide loop insertions with randomized amino acid positions.
  • polypeptide loop insertions are complementarity determining region (CDR) loop libraries of antibodies or de novo generated peptide libraries.
  • CDR complementarity determining region
  • 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.
  • ankyrin repeat proteins libraries may contain randomized loops (with fixed and randomized positions) of variable length (e.g. 1 to 20 amino acids) inserted in one or more beta-turns of an ankyrin repeat domain.
  • Any such N-terminal capping module of an ankyrin repeat protein library suitably comprises the RILLAA, RILLKA or RELLKA motif and any such C-terminal capping module of an ankyrin repeat protein library suitably comprises the KLN, KLA or KAA motif.
  • ankyrin repeat protein library may be guided by known structures of an ankyrin repeat domain interacting with a target.
  • Examples of such structures identified by their Protein Data Bank (PDB) unique accession or identification codes (PDB-IDs), are 1 WDY, 3V31 , 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1 N11 , 1 S70 and 2ZGD.
  • PDB Protein Data Bank
  • 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.
  • Example 1 Selection of binding proteins comprising an ankyrin repeat domain with binding specificity for CD47
  • CD47-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Pluckthun, loc. cit.) using part of the extracellular domain of CD47 (UniProt Ref. No: Q08722, residues 19 to 141) as target protein, libraries of ankyrin repeat proteins as described above, and established protocols (see, e.g., Zahnd, C., Amstutz, P. and Pluckthun, A., Nat. Methods 4, 69-79, 2007), and is further described in WO2024/251695.
  • DARPin proteins were expressed in E. coli cells with a Flag-tag and a His-tag. Crude extracts thereof were prepared to test binding of the Flag-tagged DARPin proteins to human CD47 recombinant protein in an HTRF assay.
  • the binding affinity of a purified ankyrin repeat protein to biotinylated recombinant human CD47-Fc target was analyzed using a ProteOn XPR 36 instrument (NAHLC200M, Xantec ProteOn Sensor Chip) and the measurement was performed according to standard procedures.
  • the CD47-binding ankyrin repeat domain with SEQ ID NO: 8 (“aCD47”) was subcloned into a derivative of the pQE30 (Qiagen) expression vector, containing an N-terminal His-tag, and expressed and purified as described above.
  • Biotinylated human CD47-Fc target was diluted in PBST (PBS, pH 7.4 containing 0.005% Tween 20®) and coated on an NAHLC200M chip (BioRad) to a level of around 350 and 800 resonance units (RU).
  • the interaction of ankyrin repeat protein and human CD47 was then measured by injecting 200 pl running buffer (PBS, pH 7.4 containing 0.005% Tween 20®) containing serial dilutions of ankyrin repeat proteins covering a concentration range between 16.7 nM, 5.6 nM, 1.85 nM and 0.62 nM for multi-trace SPR measurements, followed by a running buffer flow for at least 20 minutes at a constant flow rate of 100 pl/min (off-rate measurement).
  • the regeneration was performed using 30 pl of 10 mM Glycine pH 2.5.
  • the signals i.e. resonance unit (RU) values
  • RU resonance unit
  • a reference injection i.e. injection of running buffer only
  • Binding parameters KD, on-rate, off-rate against CD47 were as shown in Table 1.
  • Dissociation constants KD were calculated from the estimated on- and off-rates using standard procedures.
  • Variants with increased affinity to and/or reduced off-rate from target protein were generated using affinity maturation. Thereby, one initially identified binding protein was selected for affinity maturation.
  • the affinity maturation procedure entailed saturation mutagenesis of each randomized position and one non-randomized position (i.e. position 5 in 2 nd internal repeat) of the ankyrin repeat domain used as a starting point. Sequences generated by the affinity maturation procedure were screened for lower off-rates by competition HTRF, resulting in the ankyrin repeat domain with SEQ ID NO: 9 (“aCD16a”). Determination of dissociation constants (KD) of recombinant ankyrin repeat proteins with binding specificity for CD16a by Surface Plasmon Resonance (SPR) analysis
  • a cell binding competition assay was performed. Briefly, Kasumi-3 cells (CD117 expressing cell line; ATCC CRL2725) were seeded on a 96 well plate. After adding Fc block for 10 minutes at 4°C, the cells were washed and 1 nM of biot-hSCF was added to the cells for 30 minutes at 4°C, followed by centrifugation (350g, 4 minutes) and washed twice by adding 100 pl of FACS buffer.
  • DARPins or of a control benchmark monoclonal anti-CD117 antibody which does not compete with SCF (clone 104D2, Invitrogen, #MA1 -10072) were added to the cells (from 100 nM or 50 nM, 4-fold dilution) for 30 minutes at 4°C.
  • the cells were then washed twice as described above and incubated with streptavidin-AF647 in order to detect bound SCF to the CD117 receptor on cells. After 20 minutes of incubation at 4°C, cells were washed twice and incubated with cell fix buffer for 20 min at 4°C.
  • Table 4 IC50 values corresponding to the cell binding competition against SCF.
  • CD117 binding was confirmed by a cell binding assay using Kasumi-1 cancer cell line expressing CD117.
  • the CD117 expressing cells were washed Ix with 500 pl PBS, spun at 350g for 5 minutes and the supernatant was discarded.
  • a titration of CD117 designed ankyrin repeat proteins diluted in FACS buffer was added to the pellet of cells, resuspended and incubated for 30 minutes at 4°C. The cells were washed twice with 200 pl cold PBS, spun at 350g for 5 minutes and the supernatant was discarded.
  • Selected CD117-specific ankyrin repeat proteins were formatted as bispecific immune cell engagers with an anti-CD16a specific ankyrin repeat domain (see Table 6).
  • the cytotoxic activity of these engagers was tested in a DARPin-dependent cellular cytotoxicity (DDCC) assay using Jurkat-NFAT- CD16a reporter cells. Upon binding of DARPin to CD16a, these cells express the Lucia luciferase reporter gene. The extent of induced DDCC can therefore be quantified by a luminescent readout. Kasumi-1 cells, which express the CD117 receptor, were used as target cells in this assay.
  • Kasumi-1 target cells were plated in 96 well U-bottom plates one day prior to the assay at a density of 10,000 cells per well in DMEM medium supplemented with 10% FBS and were incubated overnight at 37°C.
  • DARPin proteins serially diluted in assay medium were added to the Kasumi-1 cells.
  • Jurkat-Lucia NFAT-CD16a Reporter cells were added at a concentration of 50,000 cells per well, so as to achieve an E:T ratio of 5:1.
  • the assay incubation duration was 24h after which the assay plates were centrifuged at 300g for 5min.
  • Table 6 EC50 values and maximal signal (RLU-relative light units) obtained in the DDCC reporter assay for CD16a-CD117 bispecific constructs.
  • the bispecific constructs further comprised an N- terminal His tag (SEQ ID NO: 20) for ease of purification.
  • PBMCs Peripheral Blood Mononuclear cells
  • Kasumi-1 target cells, NK cells, titrations of the tested proteins and the anti-CD107a BV421 antibody were added to a 96 well plate and incubated for 2 hours at 37°C.
  • Cells were then centrifuged at 350g for 4 min at 4°C and washed with PBS, followed by cell resuspension with human Fc block (BD Biosciences) and incubation for 15 min at 4°C. After centrifugation, an antibody mix (CD56 BV605 BD and CD16 FITC Biolegend) was added to the cells in FACS buffer which were incubated for 30 min at 4°C.
  • Live NK cells (CD56+) were gated and levels of degranulation were obtained by assessing the degranulation marker CD107a. Levels of CD16 were also assessed since engagement of CD16 can lead to CD16 downregulation. Figure 2 shows that all tested DARPin proteins mediated efficient NK cell degranulation through CD16a engagement.
  • Macrophage induced cellular phagocytosis The activity of the bispecific ankyrin repeat proteins, aCD117-aCD16a-1 , aCD117-aCD16a-2, and aCD117-aCD16a-3, was also tested in a DARPin-dependent cellular phagocytosis (DDCP) assay by flow cytometry. Macrophages derived from monocytes isolated from cryopreserved Peripheral Blood Mononuclear cells (PBMCs) from healthy donors were used as effector cells. Kasumi-1 cells, which express CD117, were used as target cells.
  • PBMCs Peripheral Blood Mononuclear cells
  • monocytes were isolated from cryopreserved PBMCs using a CD14 ultrapure isolation kit (Miltenyi Biotec, 130-118-906), according to manufacturer’s instructions. After isolation, monocytes were differentiated into MO-like macrophages by in vitro culture with M-CSF (Miltenyi Biotec, 130-093-866) in Cellgenix GMP DC medium for six days (final well concentration of 0.05 ug/ml). Macrophages were then harvested, washed and seeded overnight in an assay plate.
  • Kasumi-1 target cells were harvested, labelled with a proliferation dye (Cell Trace TM Yellow cells, ThermoFisher) according to the manufacturer’s instructions and pre-incubated with the tested proteins. Kasumi-1 cells were then added on top of the pre-seeded macrophages at an effector to target ratio of 5:1 for 4 hours at 37°C. After co-culture, plates were centrifuged and cells were harvested using dissociation buffer (Cell dissociation buffer, Gibco, 13151014). Cells were washed and stained for viability (Zombie Aqua viability dye, Biolegend, 30 min at 4°C).
  • a proliferation dye Cell Trace TM Yellow cells, ThermoFisher
  • Example 5 Selection of binding proteins comprising a 2-i n-1 ankyrin repeat domain with binding specificity for CD117 and CD47-binding ankyrin repeat domain
  • ankyrin repeat proteins with binding specificity for CD47-binding DARPins were selected from DARPin libraries essentially as described by Binz et al. 2004 (loc. cit.).
  • a CD47-binding DARPin comprising the amino acid sequence with SEQ ID NO: 8 described in Example 1 was used as target material for the ribosome display selection.
  • Generation of this ankyrin repeat protein with binding specificity for CD47- binding DARPins as well as generation of 2-in-1 binding domains is further described in EP24150569.2.
  • HTRF Homogeneous Time Resolved Fluorescence
  • Selected ankyrin repeat proteins with binding specificity for CD47-binding DARPins were then fused with the CD117-binding ankyrin repeat domains described in Example 3.
  • the fusion proteins were further engineered, including replacement of the “RILLA” motif was replaced with “RELLK” in the N- terminal capping module of CD117-binding ankyrin repeat domains, mutation of position 15 in the N- terminal capping module, transformation of a terminal capping module into an internal repeat module, or vice versa as well as engineering of potential target interaction residues of some repeat modules to create a range of binding affinities for the CD47-binding ankyrin repeat domain.
  • 2-in-1 domains were cloned following standard procedures and expressed with a N-terminal Flag-tag of SEQ ID NO: 22.
  • Selected 2-in-1 domains were then assessed for their binding behavior to CD117 or CD47-binding DARPin compared to the binding behavior of their respective parental DARPins.
  • a titration of the 2-in- 1 domains to evaluate their binding response to targets CD117 or CD47-binding DARPin was performed in an ELISA.
  • Biotinylated human CD117 target material was purchased from AcroBiosystems (Catalogue num. CD7-H82E6) which also comprises a His and Avi-tag.
  • CD47-binding DARPin of SEQ ID NOs: 25 and 26 were tagged with a N-terminal His-tag of SEQ ID NO: 20 and with a C-terminal Avi-tag of SEQ ID NO: 23, and biotinylated.
  • Targets were coated at 20nM on neutravidin coated Nunc Maxisorp 96-well plates (Thermo 442404). Serial dilutions (1 :3) were performed. Signal was detected using an anti-Flag-tag-HRP antibody (SIGMA, A8592) and a Tecan sunrise reader (OD 450 nm, ref 620 nm). BC50 (half-maximum binding concentration) values are shown in Table 7.
  • Table 7 BC50 (half-maximum binding concentration) values of selected 2-in-1 binding domains with binding specificity to CD117 and CD47-binding ankyrin repeat domain.
  • an optimal concentration of each tested 2-in-1 domain was determined by ELISA on the CD117 target (Fc-tagged human CD117, AcroBiosystems CD7-H5255), which was coated on 96 well plates (F96 MaxiSorp NUNC Immuno plate). 2-in-1 domains were titrated and the BC90 concentration (i.e. 90% maximum binding concentration) was selected at a fixed concentration of each 2-in-1 domain for the subsequent challenge with the competitor.
  • the competitor was the CD47-binding DARPin of SEQ ID NO: 8, further comprising an N-terminal His-tag of SEQ ID NO: 20 and a C- terminal Avi-tag of SEQ ID NO: 23.
  • a titration of the competitor (5000, 1250, 312.50, 78.13, 19.53, 4.88, 1 .22 and 0.31 nM) was added to the 2-in-1 domain (used at the corresponding fixed BC90 cone.) and incubated for 1 hour prior to detection.
  • the signal was detected via the flag-tag (HRP Anti-DDDDK tag, Abeam, ab2493) on each 2-in-1 domain, which allowed to visualize the extent of competition of the competitor with the interaction of the 2-in-1 domain and the coated CD117 target.
  • a reduction of the signal in function of the increasing competitor concentration therefore indicates a mutually exclusive binding character of the 2-in-1 domain.
  • Figure 4B shows the resulting measures with fitted curves, where the plots on the left show the 2-in-1 titration (points) and the evolution of the signal at the fixed BC90 concentration (triangles) where the titrated competitor is applied. The plots on the right in Figure 4B show the corresponding evolution of the signal in function of the competitor concentration. Accordingly, mutual binding exclusivity to CD117 and CD47-binding DARPin was observed for all tested 2-in- 1 domains. Curves represent a fitted four-parameters sigmoid model.
  • Multispecific binding proteins in various formats were generated (see Table 8) alongside appropriate controls (see Table 9).
  • the order of the different domains as indicated in the tables reflects the actual sequence of the domains from N-terminus to C-terminus in the molecular structure of the proteins.
  • the individual domains were linked with a linker with SEQ ID NO: 25, e.g., in the format of CD47- binding domain (“aCD47”) - linker - 2-in- 1 domain (“2-in- 1 ”) - linker - CD117-binding domain (“aCD117”) - linker - CD16a-binding domain (“aCD16a”) - linker - HSA binding domain (“aHSA”).
  • All proteins additionally comprised a His-tag (SEQ ID NO: 20) at the N-terminus for ease of purification. Constructs were expressed and purified using their His-tag according to standard protocols. Briefly, E. coli BL21 cells were transformed with the ankyrin repeat proteins, plated on LB-agar (containing 1% glucose and 50 pg/ml ampicillin) and then incubated overnight at 37°C. For each construct, a single colony was picked into 50 ml of TB medium (containing 1% glucose and 50 pg/ml of ampicillin) and incubated overnight at 37°C, shaking at 220 rpm.
  • TB medium containing 1% glucose and 50 pg/ml of ampicillin
  • the stationary overnight cultures were used to inoculate TB medium (containing 50 pg/ml ampicillin) and incubated at 37°C, shaking at 220 rpm. At an absorbance of 1 .0 to 1 .5 at 600 nm, the cultures were induced with 0.5 mM IPTG and incubated further for 4 to 5 hrs. The cultures were centrifuged, and the resulting pellets were re-suspended in 25 ml of TBS500 (50 mM Tris-HCI, 500 mM NaCI, pH 8) and lysed (sonication).
  • TBS500 50 mM Tris-HCI, 500 mM NaCI, pH 8
  • 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 AKTAxpressTM system according to standard protocols.
  • multispecific proteins all comprised a CD47-binding ankyrin repeat domain, a 2-in-1 ankyrin repeat domain comprising (i) a binding region binding the CD47-binding ankyrin repeat domain (“mask”) and (ii) a CD117-binding region, a CD16a-binding ankyrin repeat domain, as well as an HSA-binding ankyrin repeat domain. Some variants also comprised a further CD117-binding ankyrin repeat domain. All proteins additionally comprised a His-tag (SEQ ID NO: 20) at the N-terminus for ease of purification.
  • Table 8 Multispecific proteins in various domain formats.
  • the individual domains were linked with a linker with SEQ ID NO: 25 in the format of CD47-binding domain (“aCD47”) - linker - 2-in-1 domain (“2-in- 1 ”) - linker - CD117-binding domain (“aCD117”) - linker - CD16a-binding domain (“aCD16a”) - linker - HSA binding domain (“aHSA”).
  • Ni2C non-binding randomised control
  • HSA human serum albumin
  • anti- aCD47 ankyrin repeat domain binding ankyrin repeat domain with binding specificity for CD47 (“aCD47”);
  • a[target] ankyrin repeat domains with binding specificity for [target].
  • Individual domains were linked with peptide linker with SEQ ID NO: 25.
  • Cell binding assays were carried out on the AML cell line Kasumi-1 (DSMZ ACC 220) (CD1177CD47 + ) as well as engineered cell lines CHO-k1 hCD117 (CD117 + ) (lentiviral transduction of CHO-k1 cell line to express human CD117), CHO-k1 hCD47 (CD47 + ) (lentiviral transduction of CHO- k1 cell line to express human CD47) to address the question whether the constructs bind to CD117, and selectively release the CD47-binding agent in the presence of CD117.
  • Kasumi-1 DSMZ ACC 220
  • CD1177CD47 + engineered cell lines
  • CHO-k1 hCD117 CD117 +
  • CHO-k1 hCD47 CD47 +
  • lentiviral transduction of CHO- k1 cell line to express human CD47 to address the question whether the constructs bind to CD117, and selectively release the CD47-binding agent in the presence of CD117.
  • 50,000 cells were seeded, spun down at 350g for 4 minutes at 4 °C and the supernatant decanted.
  • Multispecific binding proteins and controls were prepared in FACS buffer (PBS + 2 % FBS + 10 pM HSA) and added to the cell pellets. Cells and binding proteins were incubated for 30 min at 4°C, and washed by adding 150 pl FACS buffer and centrifuging at 350g for 4 min. Supernatant was decanted, and cells washed with 200 pl FACS buffer, centrifuged at 350g for 4 min and the supernatant decanted.
  • cells were stained with 50 pl per well of live-dead green (1 :3000) and anti-penta his-AF647 (1 :200) diluted in FACS buffer. Cells were incubated for 30 min at 4°C and washed twice as described above. Finally, cells were resuspended in 50 pL/well of 1 :10 diluted cell fix buffer in water, incubated at 4°C for 20 min, followed by the addition of 150 pl PBS/2mM EDTA. Cells were centrifuged at 400g for 4 min, supernatant aspirated, and the cells resuspended in 200 pL PBS/2mM EDTA, and acquired on an Attune NxT flow cytometer.
  • the CD47-binding agent If the CD47-binding agent is not released from the conditional mask in the presence of CD117, the CD47-binding agent cannot bind to CD47 on the target cells, and therefore does not compete with biotinylated CD47-binding ankyrin repeat domain.
  • the lack of competition on CHO-k1 hCD47 cells confirms CD117 mediated release of the conditional mask.
  • Kasumi-1 or CHO-K1 hCD47 cells were plated at a density of 50,000 cells per 96-well plate. Cells were collected by centrifugation at 350g for 4 min at 4°C and removal of the supernatant. 50 pl of increasing concentrations of multispecific binding proteins diluted in FACS buffer (PBS + 2 % FBS + 10 pM HSA) were added to the cells and incubated for 30 min at 4°C and washed twice in FACS buffer. Biotinylated CD47-binding ankyrin repeat domain comprising the amino acid sequence with SEQ ID NO: 8 was added to the cells at a concentration of 10 nM in FACS buffer and incubated for 30 min at 4°C.
  • the capacity of the multispecific binding proteins to compete with biotinylated CD47-binding ankyrin repeat domain for binding to CD47 target is higher in the presence of CD117. This shows that the presence of CD117 is necessary and sufficient for efficient unmasking of the CD47-binding ankyrin repeat domain comprised in the multispecific binding proteins disclosed herein. Shown are the measured median fluorescent intensity (MFI AF647). Curves represent a fitted four-parameters sigmoid model.
  • the CD47-binding ankyrin repeat domain is released from its binding to the anti-CD47-binding ankyrin repeat domain, it was assessed whether the CD47/SIRPa pathway was inhibited.
  • the multispecific binding proteins were tested for SIRPIa competition using the PathHunter Jurkat SIRPa Signaling Bioassay Kit (Eurofins DiscoverX products LLC). Ligand engagement through co-culture of Jurkat CD47 presenting cells (CD477CD117 ) or Kasumi-3 cells (CD477CD117 + ) and Jurkat SIRPIa signaling cells results in phosphorylation and intracellular pathway activation leading to a chemiluminescence signal.
  • Multispecific binding proteins #1 to 7 showed specific and dose dependent cytotoxicity in the presence of CD117 and CD47, while DDCC was significantly lower or not detectable for the multispecific binding proteins in the absence of CD117 as shown in Figure 8A and Table 12.
  • Table 12 in the absence of a masking moiety (Control #1), potent DDCC is obtained in the presence but also absence of target cells showing unspecific activity. This unspecific activity is abolished or significantly decreased in the absence of a CD47-specific ankyrin repeat domain (Control #2) or in the presence of a CD47-mask which is released in a CD117-dependent manner.
  • Table 12 EC50 values obtained in the DDCC reporter assay for multispecific binding proteins and controls. N.d.: not determinable.
  • NK cells were isolated from PBMC using established protocols and CD16a expression was confirmed by flow cytometry analysis. Mobilised CD34+ cells from healthy donors were commercially obtained (Allcells). Target expression of CD117 and CD47 on CD34+ cells was confirmed by flow cytometry analysis.
  • the assay was performed essentially as described above in Example 4.
  • the multispecific binding protein was applied at four different concentrations (1 nM, 0.2 nM, 0.04 nM, and 0.008 nM) to Kasumi- 1 target cells, and at two different concentrations (1 nM, 0.2 nM) to CHO-hCD47 target cells.
  • Multispecific binding proteins #1 to 7 induced specific and dose dependent phagocytosis in the presence of CD117 and CD47, while DDCP was at background level in the absence of CD117 as shown in Figure 9.
  • DDCP was at background level in the absence of CD117 as shown in Figure 9.
  • Control#4 higher levels of unspecific DDCP of CHO-hCD47 cells were obtained, while this was significantly reduced in the presence of a CD47- mask (see Control #3).
  • Lack of CD117 conditional release of the CD47-mask results in lower DDCP levels compared to constructs where conditional unmasking occurs (multispecific binding proteins #1 to 7).
  • Titrations (5 nM, 1.2, 0.3, 0.08 and 0.02 nM) of multispecific binding protein MSC#2 or control antibodies (Fc-active (lgG1) anti-CD117 Ab based on JSP-191 , magrolimab (anti-CD47 antibody lgG4) or a combination of both as well as a non-binding control DARPin containing a CD16a binding domain (SEQ ID NO: 9) were added to the co-culture. Cells were then observed for 48h using Incucyte scan using phase and red fluorescent channel at 10x magnification and scanned every hour for the first 24 hours and every other hour thereafter. 2 images/well were captured per timepoint.
  • Figure 9C demonstrates higher macrophage-mediated phagocytosis activity of MSC#2 compared to an Fc-active anti-CD117 antibody, magrolimab (anti-CD47 antibody) or a combination of both.
  • MSC #2 was demonstrated by its ability to induce phagocytosis of pHrodo-labelled CD117+CD47+ (Kasumi-1 cell line), but not of CD117-CD47+ (Raji cell line) by M0- like macrophages.
  • the experimental setup was as described above.
  • MSC #2 was tested at different concentrations (100 nM, 33 nM, 11 nM).
  • levels of phagocytosis of CD117- target cells were comparable to untreated cells (D), whereas MSC #2 stimulated significant levels of phagocytosis of Kasumi-1 target cells by MO-like macrophages (E).
  • the assay was performed according to manufacturer’s instructions. Briefly, U2OS reporter cells were seeded and cultured for 48 hours at 37°C /5% CO2. Titrations of multispecific binding proteins and control constructs were prepared, and added to the cells, which were incubated for 1 hr. Human SCF was added and incubated for 3 hrs at room temperature. Detection agent was added, cells incubated for 1 hr at room temperature, followed by detection of the luminescent signal on a Tecan plate reader.
  • the multispecific binding proteins MSC #1 to 7 block signaling by SCF. Further, no agonist activity was observed by the multispecific binding proteins.
  • Table 13 IC50 values obtained in the c-Kit reporter assay for multispecific binding proteins and an antibody control. N.d. not determinable.
  • Example 7 Pharmacokinetic analysis of recombinant proteins in female BALB/c mice
  • MSC#2 SEQ ID NO: 2
  • MSC#5 SEQ ID NO: 5
  • MSC#6 SEQ ID NO: 6
  • MSC#7 SEQ ID NO: 7
  • Each fusion protein was administered as a single intravenous bolus injection in phosphate-buffered saline (PBS) solution into the tail vein of 6 mice.
  • the target dose level was 1 mg/kg with an application volume of 5 mL/kg.
  • Pharmacokinetic data analysis was performed using Version 8.3 of the WinNonlin program as part of Phoenix 64, Pharsight, North Carolina. Calculation of the pharmacokinetic parameters based on the mean concentration-time data of the animals dosed via intravenous bolus injection was performed with non-compartmental analysis (NCA model 200-202, IV bolus, linear trapezoidal linear interpolation). The following pharmacokinetic parameters were calculated: AUCinf, AUCIast, AUC_%extrapol, Cmax, Tmax, Cl_pred, Vss_pred, t1/2
  • Vss i.v. dose • AUMCinf / (AUCinf)2.
  • AUMCinf denotes the total area under the first moment of drug concentration-time curve extrapolated to infinity using the same extrapolation procedure as described for calculation of AUCinf.
  • PK parameters based on concentrations given in nmol/L dose values given as mg/kg were converted to nmol/kg by using the molecular weight of the ankyrin repeat proteins.
  • Table 14 shows the summary of pharmacokinetic characteristics of the four tested ankyrin repeat proteins MSC #2, MSC #5, MSC #6 and MSC #7 following single intravenous administration of 1 mg/kg.
  • Each ankyrin repeat protein comprises a C-terminal human serum albumin binding ankyrin repeat domain (SEQ ID NO: 17).
  • Example 8 In Vivo efficacy evaluation of exemplary multi-specific binding proteins in CD34+ humanized mice.
  • NSG mice 50 female NSG mice, age of animals at study initiation 21 to 22 weeks (provider Jackson laboratories). NSG mice were humanized by engraftment of human cord blood-derived CD34+ stem cells isolated from two donors. 25 NSG mice were engrafted per donor.
  • Table 15 Flow cytometry panel used for humanization assessment in blood.
  • Treatment groups 50 mice were enrolled in the study. All animals were randomly allocated to the 5 different study groups (ten mice per group with five each for the two cord blood donors). The treatment start date is denoted as day 0 (DO).
  • Table 17 Flow cytometry panel used for bone marrow and blood analysis on the day of termination.
  • CD117+ cells within a CD34- compartment in the bone marrow were also reduced in frequency upon treatment with multispecific binding protein #2, with significantly greater reduction in comparison than with the anti-CD117 antibody-treated group (Figure 11C).

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Abstract

La présente divulgation concerne des protéines de liaison multispécifiques comprenant des domaines de liaison ayant une spécificité de liaison à un marqueur de cellule souche, une protéine immunorégulatrice et un antigène associé à une cellule immunitaire. De plus, la divulgation concerne des acides nucléiques codant pour de telles protéines de liaison multispécifiques, des compositions pharmaceutiques comprenant de telles protéines de liaison multispécifiques ou des acides nucléiques, et l'utilisation de telles protéines de liaison multispécifiques, acides nucléiques ou compositions pharmaceutiques dans des procédés de conditionnement d'un sujet avant la réception d'une greffe de cellules souches.
PCT/EP2025/050108 2024-01-05 2025-01-03 Protéines de liaison multispécifiques Pending WO2025146487A1 (fr)

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EP24181677.6 2024-06-12

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