Bispecific anti-c-kit and anti-CD203c antigen binding molecules and uses thereof

Abstract

Provided herein are bispecific antigen binding molecules that bind to c-Kit and CD203c. Further provided herein are therapeutic uses of such molecules and related pharmaceutical compositions, nucleic acid molecules, expression vectors and host cells and methods of producing them.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 393,036, filed on July 28, 2022, the entire contents of which are hereby incorporated by reference.

[0003] Reference to an electronic sequence listing

[0004] The entire contents of the electronic sequence listing (UHFL_002_01WO_SeqList_ST26.xml; size: 71,462 bytes; and creation date: July 24, 2023) are incorporated herein by reference. Technical Field

[0005] The present disclosure relates to bispecific antigen binding molecules and therapeutic uses of such molecules. Background Art

[0006] Granulocytes, such as mast cells and basophils, are core drivers of the pathology of many chronic inflammatory diseases. Existing approaches for treating such diseases target the mast cell survival receptor c-Kit. While this approach has been clinically validated to effectively deplete mast cells and improve associated inflammation, indiscriminate inhibition of c-Kit may result in on-target dose-limiting bone marrow suppression. These adverse events are difficult to avoid with high-affinity target engagement driven by bivalent engagement with standard antibodies. Granulocyte-selective drugs are very scarce, leaving patients with mast cell-driven diseases with a huge unmet medical need. Summary of the invention

[0007] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit heavy chain variable (VH) domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c.

[0008] Provided herein is an anti-c-Kit and anti-CD203c bispecific protein, wherein the bispecific protein according to claim 1, wherein the anti-c-Kit VH domain comprises a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 4; the anti-c-CD203c VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 73; and the VL domain comprises a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 6, a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 7, and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 8.

[0009] Provided herein is an anti-c-Kit and anti-CD203c bispecific protein, wherein the anti-c-Kit VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO:2, a HCDR2 comprising the amino acid sequence of SEQ ID NO:3, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:4; the anti-c-CD203c VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO:10, a HCDR2 comprising the amino acid sequence of SEQ ID NO:11, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:12; and the VL domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO:6, a LCDR2 comprising the amino acid sequence of SEQ ID NO:7, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:8.

[0010] Provided herein is an anti-c-Kit and anti-CD203c bispecific protein, wherein the anti-c-Kit VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO:2, a HCDR2 comprising the amino acid sequence of SEQ ID NO:3, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:4; the anti-c-CD203c VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO:14, a HCDR2 comprising the amino acid sequence of SEQ ID NO:15, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:12; and the VL domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO:6, a LCDR2 comprising the amino acid sequence of SEQ ID NO:7, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:8.

[0011] Provided herein is an anti-c-Kit and anti-CD203c bispecific protein, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 65; the anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 71; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0012] Provided herein is an anti-c-Kit and anti-CD203c bispecific protein, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 9; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0013] Provided herein is an anti-c-Kit and anti-CD203c bispecific protein, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 13; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0014] Provided herein is an anti-c-Kit and anti-CD203c bispecific protein, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises the amino acid sequence of any one of SEQ ID NO: 16-51; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0015] Provided herein is an anti-c-Kit and anti-CD203c bispecific protein, wherein the anti-c-Kit VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 63-67; the anti-c-CD203c VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 68-72; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0016] In some embodiments, the bispecific protein comprises an immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin constant region. In some embodiments, the immunoglobulin constant region is an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 immunoglobulin constant region.

[0017] In some embodiments, the immunoglobulin constant region is an immunologically inert constant region.

[0018] In some embodiments, the first polypeptide chain comprises a first immunoglobulin constant region and the second polypeptide chain comprises a second immunoglobulin constant region, and wherein the first immunoglobulin constant region and the second immunoglobulin constant region comprise a knob-hole mutation.

[0019] In some embodiments, the first immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions S354C and T366W, and wherein the second immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions Y349C, T366S, L368A, and Y407V, wherein numbering is according to the EU index as in Kabat.

[0020] In some embodiments, the first immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions Y349C, T366S, L368A, and Y407V, and wherein the second immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions S354C and T366W, wherein numbering is according to the EU index as in Kabat.

[0021] Provided herein is a bispecific protein, wherein (a) the first immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:52, and the second immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:53; or (b) the first immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:53, and the second immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:52.

[0022] Provided herein are bispecific proteins that bind c-Kit and CD203c, wherein c-Kit and CD203c are located on the surface of the same cell.

[0023] In some embodiments, the bispecific protein is a designed ankyrin repeat protein (DARPin), a tandem VHH, or a tandem immunoglobulin new antigen receptor (IgNAR).

[0024] Provided herein are immunoconjugates comprising a bispecific protein disclosed herein linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulator, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an antiproliferative agent, or a pro-apoptotic agent.

[0025] Provided herein is a pharmaceutical composition comprising a bispecific protein or immunoconjugate disclosed herein, and a pharmaceutically acceptable carrier, diluent or excipient.

[0026] Provided herein are nucleic acid molecules encoding the bispecific proteins disclosed herein. Provided herein are nucleic acid molecules encoding the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain of the bispecific proteins disclosed herein.

[0027] Provided herein are expression vectors comprising the nucleic acid molecules disclosed herein.

[0028] Provided herein is a recombinant host cell comprising a nucleic acid molecule or an expression vector disclosed herein.

[0029] Provided herein is a method for producing a bispecific protein, the method comprising: culturing a recombinant host cell disclosed herein under conditions whereby a nucleic acid molecule is expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

[0030] Provided herein is a method for treating an inflammatory disease or neoplasm in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0031] Provided herein is a method for improving symptoms of an inflammatory disease or neoplasm in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a bispecific protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0032] In some embodiments of the methods provided herein, the inflammatory disease is a chronic inflammatory disease. In some embodiments of the methods provided herein, the inflammatory disease is a mast cell driven disease. In some embodiments of the methods provided herein, the inflammatory disease is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis or eosinophilic esophagitis.

[0033] In some embodiments of the methods provided herein, the neoplasm is a mast cell driven neoplasm. In some embodiments of the methods provided herein, the neoplasm is systemic mastocytosis or mast cell leukemia.

[0034] Provided herein are bispecific proteins, immunoconjugates or pharmaceutical compositions disclosed herein for use as medicaments.

[0035] Provided herein are bispecific proteins, immunoconjugates or pharmaceutical compositions disclosed herein for use in treating inflammatory diseases or neoplasms.

[0036] In some embodiments of the uses disclosed herein, the inflammatory disease is a chronic inflammatory disease. In some embodiments of the uses disclosed herein, the inflammatory disease is a mast cell driven disease. In some embodiments of the uses disclosed herein, the inflammatory disease is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis or eosinophilic esophagitis.

[0037] In some embodiments of the uses disclosed herein, the neoplasm is a mast cell driven neoplasm. In some embodiments of the uses disclosed herein, the neoplasm is systemic mastocytosis or mast cell leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Depicted are graphs showing that the bispecific format ensures specificity for activated mast cells. Monovalent anti-c-Kit has low functional affinity for c-Kit on hematopoietic stem cells (HSC). Without the binding stabilization provided by co-engagement with CD203c, the monovalent binding is transient and results in low c-Kit inhibitory potency that cannot effectively compete with the high affinity binding of the endogenous dimeric SCF ligand. However, on dual receptor expressing cells, bivalent binding can occur, SCF can no longer compete for receptor occupancy, and c-Kit signaling is abolished.

[0039] Figure 2A schematic representation of a knob-hole bispecific format is depicted. To enable simplified production and purification, a common light chain bispecific format was used for dual c-Kit / CD203c targeting. This was combined with engineered knob-hole mutations of cysteine ​​residues in the CH3 Fc domain that drive heterodimeric heavy chain pairing. Tables 1-3 provide exemplary common light chain and heterodimeric heavy chain sequences.

[0040] Figure 3A – Figure 3C Depicted are the results of characterization of purified IgG with common light chain (CLC). Purified anti-CD203c IgG null antibody was assayed together with anti-c-kit MH1 light chain in a direct binding enzyme-linked immunosorbent assay (ELISA) against purified recombinant human ( Figure 3A ) and rhesus monkeys ( Figure 3B )CD203c protein was titrated (in nM). 1.27 was subsequently titrated in a flow-based assay on the basophil cell line KU812, which expresses CD203c ( Figure 3C ).

[0041] Figure 4 Describe the design of 1.27 heavy chain CDR1 and CDR2 variant optimization library. Show the heavy chain amino acid sequence of parent clone 1.27. CDR1 and CDR2 sequences are described in gray. Compared with the classical Kabat nomenclature, the CDR definition used throughout the text has been expanded. The CDR positions targeted by mutagenesis are highlighted in brackets. The table illustrates that each targeted position is replaced by any other amino acid (except cysteine), and all possible single mutations and double mutations are sampled.

[0042] Figure 5 Depicted is the design of the 1.27 heavy chain CDR3 variant optimized library.The heavy chain amino acid sequence of parental clone 1.27 is shown.The CDR3 sequence is depicted in grey, and the CDR3 positions targeted for mutagenesis are highlighted in brackets.The table illustrates that each targeted position is replaced by any other amino acid (except cysteine), and all possible single and double mutations are sampled.

[0043] Fig. 6A – Figure 6C Depicted are the results of characterization of antibody variants from the 1.27 optimized library. Antibody variants isolated from the 1.27 optimized library and parental 1.27 clones were compared to human ( Fig. 6A ) and rhesus monkeys ( Figure 6B )CD203c and compared the binding of this antibody variant to the parental 1.27 clone to KU812 cells by flow-based assay ( Figure 6C ).

[0044] Figure 7 Depicted are results from an assay of binding of CLC bispecific antibodies to KU812 cells. Bispecific molecules composed of anti-c-Kit MH1 and anti-CD203c 1.27 (parental) or anti-CD203cF6 (affinity optimized) were compared to anti-c-Kit IgG, anti-c-Kit one-armed antibodies, and anti-CD203c IgG for binding to KU812 cells. The EC50 of this bispecific molecule was similar to that of anti-c-Kit IgG, but its Bmax was much higher due to the synergistic binding of both arms on dual target expressing cells.

[0045] Figure 8 Depicted are results from an assay for inhibition of c-Kit receptor phosphorylation by the F6 / MH1 bispecific antibody. The F6 / MH1 bispecific molecule exhibited potent inhibition of c-Kit receptor phosphorylation compared to the single-armed MH1 c-Kit antibody.

[0046] Figure 9A-9B Depicted are results from binding assays performed using alternative common light chains containing CDRs grafted from MH1 and paired with CD203c VH domains. The results indicate that not all common light chain / heavy chain pairs retain dual target binding. Alternative common light chains containing CDRs grafted from MH1 and paired with CD203c VH domains retain c-Kit binding ( Fig. 9A ), but lost CD203c(ENPP3) binding ( Fig. 9B ).

[0047] Fig.10 Depicted are the results from an ELISA comparing the binding of the F6 / MH1 bispecific molecule to human and cynomolgus CD203c. The results demonstrate that the F6 / MH1 bispecific molecule retains equivalent binding to human and cynomolgus CD203c.

[0048] Fig.11A – Fig.11D Depicted are the results of SPR characterization of affinity-optimized CD203c VH binding domains. The parent anti-CD203c binding domain 1.27 ( Fig.11A ) and affinity-optimized variant F6 ( Fig. 11B ) and F6.12( Fig. 11C ) and their comparative affinities to human and cynomolgus monkey CD203c were assessed using single cycle kinetics. The results are summarized in the attached table ( Fig.11D )middle.

[0049] Fig. 12A – Fig. 12BDepicted are results from analysis of binding of affinity optimized CD203c VH binding domains to KU812 cells. Production of parental anti-CD203c binding domain 1.27 ( Fig. 12A ) and compared its binding to KU812 cells with that of affinity-optimized F6 IgG ( Fig. 12B ).

[0050] Fig.13 Depicted are substitution mutations generated from analysis of pi engineering of anti-c-Kit and anti-CD203c VH domains. The table summarizes the top 5 double mutation combinations selected for MH1 VH (anti-c-Kit) and F6.12 VH (anti-CD203c) domain engineering to maximize the pi difference between the heavy chains.

[0051] Fig.14 Depicted are the preparative CEX (cation exchange) and analytical SEC (size exclusion) chromatograms of the MH1_A / F6.12_B bispecific. Preparative CEX was used to assess the efficiency of separation of heterodimer and homodimer species during bispecific purification. The table summarizes the peak compositions obtained under these conditions. The monomer purity of the samples was also analyzed by SEC.

[0052] Fig.15 Preparative CEX and analytical SEC chromatograms of the MH1_C / F6.12_D bispecific are depicted. Preparative CEX was used to assess the efficiency of separation of heterodimeric and homodimeric species during bispecific purification. The table summarizes the peak compositions obtained under these conditions. The monomer purity of the samples was also analyzed by SEC.

[0053] Fig.16 Depicted are the results of a comparison of c-Kit target binding before and after pi engineering using SPR. Bispecific binding to human and cynomolgus c-Kit was compared before (MH1 / F6.12) and after (MH1_C / F6.12_D) heavy chain pi engineering using a single cycle kinetic protocol.

[0054] Fig.17 Depicted are the results of a comparison of CD203c target binding before and after pi engineering using SPR. A single cycle kinetic protocol was used to compare bispecific binding to human and cynomolgus CD203c before (MH1 / F6.12) and after (MH1_C / F6.12_D) heavy chain pi engineering.

[0055] Fig.18Depicted are the results of a comparison of dual target binding before and after pI engineering using SPR. The table summarizes the kinetic data obtained using a single cycle kinetic protocol comparing human and cynomolgus c-Kit and human and cynomolgus CD203c binding to a number of bispecific constructs before (MH1 / F6.12) and after (MH1_A / F6.12_B, MH1_A / F6.12_C, MH1_C / F6.12_B and MH1_C / F6.12_D).

[0056] Fig.19A – Fig.19C Depicted are the results of a comparison of binding to KU812 cells before and after pI engineering using flow cytometry. Fig.19A ) and after (MH1_C / F6.12_B( Fig.19B ) and MH1_C / F6.12_D( Fig.19C )) binding of many bispecific constructs to dual targets expressed on KU812 cells. DETAILED DESCRIPTION

[0057] Studies have shown that both IgE-dependent and IgE-independent activation of mast cells and basophils results in robust upregulation of cell surface CD203c. CD203c is unique to the mast cell / basophil lineage and, unlike c-Kit, is not expressed on other hematopoietic stem cell precursors. Studies have shown that CD203c distinguishes between the erythroid and granulocyte lineages, therefore, targeting granulocytes via CD203c may avoid myelosuppression. Anti-c-kit / anti-CD203c bispecific antibodies have the potential to maintain the efficacy of mast cell depletion via c-Kit inhibition, but only on cells that co-express activated CD203c. This is in Figure 1 It is schematically shown in .

[0058] Advances in molecular engineering have enabled the development of bispecific molecules, which allow single-dose administration but are still associated with considerable complexity in manufacturing. Problems with the correct pairing of the two light chains can lead to severe heterogeneity, which requires additional downstream processing. This problem can be solved by making the individual antibodies separately and forming the bispecific antibody after production. However, this requires the development of two manufacturing cell lines. The bispecific anti-c-Kit / anti-CD203c protein described herein uses a common light chain format, which has the advantage of simplifying downstream processing.

[0059] Protein molecules

[0060] Provided herein are bispecific proteins that bind c-Kit and CD203c in cis, ie, where the c-Kit and CD203c molecules are located on the surface of the same cell.

[0061] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit heavy chain variable (VH) domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c.

[0062] Provided herein are VL domain sequences that form (1) a binding domain that specifically binds c-Kit when paired with an anti-c-Kit VH domain; and (2) a binding domain that specifically binds CD203c when paired with an anti-CD203c VH domain.

[0063] In some embodiments, the protein comprises one or more amino acid sequences provided in Tables 1-7.

[0064] Table 1. Exemplary anti-c-Kit heavy chain binding domain sequences

[0065]

[0066] In the VH domain sequences, the CDR sequences are underlined.

[0067] Table 2. Exemplary anti-c-Kit / anti-CD203c light chain binding domain sequences

[0068]

[0069]

[0070] In the VL domain sequences, the CDR sequences are underlined.

[0071] Table 3. Exemplary anti-CD203c heavy chain binding domain sequences

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] In the VH domain sequence, the CDR sequences are underlined. The residues in bold font are altered from the corresponding residues in the 1.27 sequence.

[0078] Table 4. Exemplary Fc sequences

[0079]

[0080] Anti-c-Kit / anti-CD203c protein designs may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA, and may or may not have effector function capabilities.

[0081] In some embodiments, the proteins disclosed herein comprise domains and regions of antibody molecules. The term "antibody" broadly refers to an immunoglobulin (Ig) molecule that typically comprises four polypeptide chains (two heavy (H) chains and two light (L) chains), or any functional fragment, mutant, variant or derivative thereof that retains the necessary target binding characteristics of an Ig molecule. Such mutants, variants or derivative antibody formats are known in the art.

[0082] In a full-length antibody, each heavy chain comprises a heavy chain variable domain (abbreviated herein as VH domain) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. The IgG, IgA and IgD constant regions comprise a flexible hinge region between the CH1 domain and the CH2 domain. Each light chain comprises a light chain variable domain (abbreviated herein as VL domain) and a light chain constant region. The light chain constant region comprises one domain, CL. The VH and VL domains can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which are interspersed with more conserved regions, called framework regions (FRs). Each VH domain and VL domain consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0083] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be an original sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain may be different, the human IgG heavy chain Fc region is generally defined as extending from the amino acid residue at the Cys226 position or from Pro230 to its carboxyl terminus. The numbering of residues in the Fc region is based on the EU index as in Kabat. The Fc region of an immunoglobulin generally comprises two constant domains, CH2 and CH3. The Fc region may exist in the form of a dimer or a monomer. The Fc region is combined with various cell receptors (such as Fc receptors) and other immune molecules (such as complement proteins). In some embodiments, the bispecific protein provided herein comprises an Fc region.

[0084] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) and class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2) or subclass. IgG, IgD, and IgE antibodies typically contain two identical heavy chains and two identical light chains and two antigen-binding domains, each consisting of a VH and a VL. Typically, an IgA antibody is composed of two monomers, each consisting of two heavy chains and two light chains (this is also true for IgG, IgD, and IgE antibodies); thus, an IgA molecule has four antigen-binding domains, each consisting of a VH and a VL. Some IgA antibodies are monomeric in that they are composed of two heavy chains and two light chains. Secretory IgM antibodies typically consist of five monomers, each consisting of two heavy chains and two light chains (this is also true for IgG and IgE antibodies). Thus, an IgM molecule has ten antigen binding domains, each of which is composed of a VH and a VL. The cell surface form of IgM has a two heavy chain / two light chain structure similar to IgG, IgD, and IgE antibodies.

[0085] As used herein, the terms "immunobinding" and "immunobinding properties" refer to the type of non-covalent interaction that occurs between an immunoglobulin molecule (e.g., an antibody or antigen-binding portion thereof) or a protein comprising one or more immunoglobulin-derived binding domains and an antigen for which the immunoglobulin or protein is specific. The strength or affinity of the immunological binding interaction can be measured using the dissociation constant (K) of the interaction. d ) is used to represent the d The smaller the value, the greater the affinity. The immunological binding properties of a selected polypeptide can be quantified using methods well known in the art. One such method requires measuring the rates of formation and dissociation of the antigen binding site / antigen complex, where these rates depend on the concentration of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates equally in both directions. Thus, the "binding rate constant" (Kon ) and the “dissociation rate constant” (K off ) can be determined by calculating the concentration and the actual association and dissociation rates. (See, Malmqvist, Nature 361: 186-187 (1993)). Koff / Kon The ratio of 2 is such that all affinity-independent parameters are offset and is equal to the dissociation constant Kd (See Davies et al. (1990) Annual Rev Biochem 59:439-473). When measured as by an assay such as a radioligand binding assay or similar assays known to those skilled in the art, the equilibrium binding constant (K d ) is ≤10 μM, preferably ≤10 nM, more preferably ≤10 nM, most preferably ≤100 pM to about 1 pM, then the antibodies or antigen-binding portions provided herein are considered to specifically bind to PD-L1 or CD3. A method for determining the K of an antibody d The method is through the use of surface plasmon resonance (SPR), usually using biosensor systems, such as system.

[0086] Functionally, the binding affinity of the proteins provided herein can be -5 M to 10 -12 For example, the binding affinity of the protein provided herein is 10 -6 M to 10 -12 M, 10 -7 M to 10 -12 M, 10 -8 M to 10 -12 M, 10 -9 M to 10 - 12 M, 10 -5 M to 10 -11 M, 10 -6 M to 10 -11 M, 10 -7 M to 10 -11 M, 10 -8 M to 10 -11 M, 10 -9 M to 10 -11 M, 10 -10 M to 10 - 11 M, 10 -5 M to 10 -10 M, 10 -6 M to 10 -10 M, 10 -7 M to 10 -10 M, 10-8 M to 10 -10 M, 10 -9 M to 10 -10 M, 10 -5 M to 10 -9 M, 10 -6 M to 10 -9 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -9 M, 10 -5 M to 10 -8 M, 10 -6 M to 10 -8 M, 10 -7 M to 10 -8 M, 10 -5 M to 10 -7 M, 10 -6 M to 10 -7 M or 10 -5 M to 10 -6 M.

[0087] Provided herein is a bispecific protein, the bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain, the first immunoglobulin heavy chain comprising an anti-c-Kit heavy chain variable (VH) domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain, the second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain, the immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 4; the anti-c-CD203c VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 16. NO:73; and the VL domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO:6, a LCDR2 comprising the amino acid sequence of SEQ ID NO:7, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:8.

[0088] Provided herein is a bispecific protein, the bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain, the first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain, the second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain, the immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 4; the anti-c-CD203c VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 5. NO:12; and the VL domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO:6, LCDR2 comprising the amino acid sequence of SEQ ID NO:7, and LCDR3 comprising the amino acid sequence of SEQ ID NO:8.

[0089] Provided herein is a bispecific protein, the bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain, the first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain, the second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain, the immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 4; the anti-c-CD203c VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 16. NO:12; and the VL domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO:6, LCDR2 comprising the amino acid sequence of SEQ ID NO:7, and LCDR3 comprising the amino acid sequence of SEQ ID NO:8.

[0090] Table 5. Exemplary anti-c-Kit heavy chain binding domain sequences (MH1 VH domain variants)

[0091]

[0092]

[0093] The CDR sequences are underlined. The residues in bold font are altered from the corresponding residues in the MH1 VH domain sequence.

[0094] Table 6. Exemplary anti-CD203c heavy chain binding domain sequences (F6.12 VH domain variants)

[0095]

[0096] The CDR sequences are underlined. The residues in bold font are altered from the corresponding residues in the F6.12 VH domain sequence.

[0097] Table 7. Sequences of the bispecific molecule MH1_C / F6.12_D

[0098]

[0099] In the variable domains, the CDR sequences are underlined.

[0100] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 65; the anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 71; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0101] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 65; the anti-c-CD203c VH domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 71; and the VL domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 72. An amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of NO:5.

[0102] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 9; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0103] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 9; and the VL domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10. An amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of NO:5.

[0104] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 13; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0105] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit heavy chain variable (VH) domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 13; and the VL domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14. An amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of NO:5.

[0106] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 51; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0107] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit heavy chain variable (VH) domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 51; and the VL domain comprises an amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 52. An amino acid sequence that is at least about 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence of NO:5.

[0108] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 16-51; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0109] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a light chain variable VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 63-67; the anti-c-CD203c VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 68-72; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0110] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 54; and the VL domain comprises the amino acid sequence of SEQ ID NO: 58.

[0111] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 55; and the VL domain comprises the amino acid sequence of SEQ ID NO: 59.

[0112] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit heavy chain variable (VH) domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 56; and the VL domain comprises the amino acid sequence of SEQ ID NO: 60.

[0113] Provided herein is a bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit VH domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 57; and the VL domain comprises the amino acid sequence of SEQ ID NO: 61.

[0114] In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence provided herein with 1, 2 or 3 conservative amino acid substitutions (e.g., in Tables 1, 3 or 5-7). In some embodiments, the immunoglobulin light chain comprises an amino acid sequence provided herein with 1, 2 or 3 conservative amino acid substitutions (e.g., in Tables 2 or 7). In some embodiments, conservative amino acid substitutions are made only in FR sequences and not in CDR sequences.

[0115] In some embodiments, the bispecific protein comprises an anti-c-Kit VH domain MH1_C (or the corresponding CDR sequence), an anti-c-CD203c VH domain F6.12_D (or the corresponding CDR sequence), and a VL domain MH1 (or the corresponding CDR sequence).

[0116] In some embodiments, the bispecific protein comprises anti-c-Kit VH domain MH1 (or the corresponding CDR sequence), anti-c-CD203c VH domain 1.27 (or the corresponding CDR sequence), and VL domain MH1 (or the corresponding CDR sequence).

[0117] In some embodiments, the bispecific protein comprises anti-c-Kit VH domain MH1 (or the corresponding CDR sequence), anti-c-CD203c VH domain F6 (or the corresponding CDR sequence), and VL domain MH1 (or the corresponding CDR sequence).

[0118] In some embodiments, the bispecific protein comprises an anti-c-Kit VH domain MH1 (or the corresponding CDR sequence), an anti-c-CD203c VH domain F6.12 (or the corresponding CDR sequence), and a VL domain MH1 (or the corresponding CDR sequence).

[0119] In some embodiments, the bispecific protein provided herein comprises an immunoglobulin heavy chain constant region located at the C-terminus of an immunoglobulin heavy chain. In some embodiments, the immunoglobulin heavy chain constant region is IgG, IgE, IgM, IgD, IgA or IgY. In some embodiments, the immunoglobulin heavy chain constant region is IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2. In some embodiments, the immunoglobulin heavy chain constant region is IgG1. In some embodiments, the immunoglobulin heavy chain constant region is immunologically inert. In some embodiments, the immunoglobulin heavy chain constant region comprises one or more mutations to reduce or prevent FcγR binding, antibody-dependent cell-mediated cytotoxicity (ADCC) activity, antibody-dependent cellular phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC) activity. In some embodiments, the immunoglobulin heavy chain constant region is a wild-type human IgG1 constant region, a wild-type human IgG2 constant region, a wild-type human IgG4 constant region, a human IgG1 constant region comprising amino acid substitutions L234A, L235A and G237A, a human IgG1 constant region comprising amino acid substitutions L234A, L235A, G237A and P331S, or a human IgG4 constant region comprising amino acid substitutions S228P, wherein numbering is according to the EU index as in Kabat. In some embodiments, the positions of amino acid residues in the constant region of an immunoglobulin molecule are numbered according to the EU index as in Kabat (Ward et al., 1995 Therap. Immunol. 2: 77-94).

[0120] In some embodiments of the proteins provided herein, the first polypeptide chain comprises a first immunoglobulin constant region, and the second polypeptide chain comprises a second immunoglobulin constant region, and wherein the first immunoglobulin constant region and the second immunoglobulin constant region comprise a knob-hole mutation. In some embodiments, the first immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions S354C and T366W, and wherein the second immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions Y349C, T366S, L368A and Y407V, wherein numbering is according to the EU index as in Kabat. In some embodiments, the first immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions Y349C, T366S, L368A and Y407V, and wherein the second immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions S354C and T366W, wherein numbering is according to the EU index as in Kabat. In some embodiments, (a) the first immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:52, and the second immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:53; or (b) the first immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:53, and the second immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:52.

[0121] In some embodiments, the protein provided herein may include any heterodimerization mutation or heterodimerization technology. In some embodiments, the protein provided herein may not include any heterodimerization mutation or heterodimerization technology. In such embodiments, purification techniques may be used to isolate the protein.

[0122] In some embodiments, the protein provided herein can comprise an immunoglobulin light chain constant region that is a kappa light chain. In some embodiments, the kappa light chain comprises SEQ ID NO:62.

[0123] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYA CEVTHQGLSSPVTKSFNRGEC(SEQ ID NO:62)

[0124] In some embodiments, the proteins provided herein can comprise an immunoglobulin light chain constant region that is a lambda light chain.

[0125] In some embodiments, the bispecific proteins provided herein are designed ankyrin repeat proteins (DARPins). In some embodiments, the bispecific proteins provided herein are tandem VHHs. In some embodiments, the bispecific proteins provided herein are tandem immunoglobulin new antigen receptors (IgNARs).

[0126] Provided herein are immunoconjugates comprising a protein disclosed herein linked to a therapeutic agent. In some embodiments, the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulator, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an antiproliferative agent, or a pro-apoptotic agent.

[0127] Examples of suitable therapeutic agents include, but are not limited to, immunomodulators, cytotoxins, radioisotopes, chemotherapeutic agents, antiangiogenic agents, antiproliferative agents, pro-apoptotic agents, and cytostatic and lytic enzymes (e.g., RNA enzymes). Additional therapeutic agents include therapeutic nucleic acids, such as genes encoding immunomodulators, antiangiogenic agents, antiproliferative agents, or pro-apoptotic agents. These drug description words are not mutually exclusive, and therefore therapeutic agents may be described using one or more of the above terms.

[0128] Examples of suitable therapeutic agents for immunoconjugates include, but are not limited to, JAK kinase inhibitors, taxanes, maytansine, CC-1065 and multicarmycin, calicheamicin and other enediynes and auristatins. Other examples include antifolates, vinca alkaloids and anthracyclines. Plant toxins, other biologically active proteins, enzymes (i.e., ADEPT), radioactive isotopes, photosensitizers can also be used for immunoconjugates. In addition, secondary carriers can be used as cytotoxic agents (such as liposomes or polymers) to prepare conjugates, and suitable cytotoxins include agents that inhibit or prevent cell function and / or cause cell destruction. Representative cytotoxins include antibiotics, tubulin polymerization inhibitors, alkylating agents that bind to and destroy DNA, and agents that destroy protein synthesis or essential cellular proteins (such as protein kinases, phosphatases, topoisomerases, enzymes, and cell cycle proteins).

[0129] Representative cytotoxins include, but are not limited to, doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, nogalamycin, menogaril, pitarubicin, valrubicin, cytarabine, gemcitabine, mcitabine), trifluridine, ancitabine, enocitabine, azacitidine, doxifluhdine, pentostatin, broxuhdine, capecitabine, cladhbine, decitabine, floxuhdine, fludarabine, gougerotin, puromycin ( puromycin, tegafur, tiazofuhn, adhamycin, cisplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine zine), methotrexate, flurouracil, etoposide, paclitaxel, paclitaxel analogs, platinums (such as cisplatin and carboplatin), mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, authramycin, azaserine, bleomycin,Tamoxifen, idarubicin, dolastatin / auristatin, hemiasterlin, esperamicin, and maytansinoid.

[0130] Suitable immunomodulators include antihormonal drugs that block the effects of hormones on tumors and immunosuppressants that inhibit cytokine production, downregulate self-antigen expression, or mask MHC antigens.

[0131] Pharmaceutical composition

[0132] The bispecific proteins provided herein (also referred to herein as "active compounds") can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically comprise the protein (or an immunoconjugate comprising the protein) and a pharmaceutically acceptable carrier, diluent, or excipient. Such materials should be non-toxic and will not interfere with the efficacy of the protein. The exact nature of the carrier or other material will depend on the route of administration, which can be by injection, bolus, infusion, or any other suitable route, as discussed below.

[0133] As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that do not generally cause allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions for animals (especially humans) approved by U.S. federal or state government regulatory agencies or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias are considered "pharmaceutically acceptable". As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delaying agents that are compatible with pharmaceutical administration. Suitable carriers are described in the latest edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Some examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is known in the art. Unless any conventional media or agents are incompatible with the active compound, it is expected to be used in the composition. Supplementary effective compounds may also be incorporated into the composition. A pharmaceutically acceptable carrier, diluent or excipient may be a compound or combination of compounds that does not cause adverse reactions and allows, for example, facilitating administration of the protein, increasing its lifespan and / or efficacy in the body, or increasing its solubility in solution.

[0134] Pharmaceutical compositions disclosed herein can be formulated to be compatible with their intended route of administration. Examples of route of administration include parenteral administration, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions for parenteral, intradermal, or subcutaneous applications may include the following components: sterile diluents, such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methyl parabens; antioxidants, such as ascorbic acid or sodium bisulfate; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates, and agents for adjusting tension, such as sodium chloride or dextrose. pH can be adjusted with an acid or alkali (such as hydrochloric acid or sodium hydroxide). Parenteral preparations may be encapsulated in ampoules, disposable syringes, or multidose vials made of glass or plastic.

[0135] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In some cases, the composition is sterile and is a fluid that reaches the degree of easy injection. In some cases, the composition is stable under manufacturing and storage conditions and is preserved for the contamination of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium, which contains, for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.) and their suitable mixtures. For example, by using a coating such as lecithin, in the case of a dispersion, by maintaining the required particle size, and by using a surfactant, appropriate fluidity can be maintained. Preventing microbial action can be achieved by various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it is preferred to include isotonic agents, such as sugars, polyols such as mannitol, sorbitol, sodium chloride in the composition. The extended absorption of the composition can be achieved by including an agent (for example, aluminum monostearate and gelatin) that extends absorption in an injectable composition.

[0136] Sterile injectable solutions can be prepared by incorporating the required amount of active compound with one of the ingredients listed above or a combination of the ingredients listed above in an appropriate solvent, followed by filtration sterilization as required. Typically, dispersions are prepared by incorporating the active compound into a sterile vehicle containing an alkaline dispersion medium and other ingredients required from those ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method is vacuum drying and freeze drying, which produces a powder of the active ingredient and any additional required ingredients from a solution thereof previously aseptically filtered.

[0137] Oral compositions generally contain an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be admixed with excipients and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using fluid carriers for use as mouthwashes, wherein the compound in the fluid carrier is orally administered and gargled and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of a similar nature: binders such as microcrystalline cellulose, tragacanth or gelatin; excipients such as starch or lactose, disintegrants such as alginic acid, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetener such as sucrose or saccharin; or a flavoring such as mint, methyl salicylate or orange flavor.

[0138] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0139] Systemic administration can also be carried out by transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant suitable for the barrier to be penetrated is used in the formulation. Such penetrants are generally known in the art, and include, for example, detergents, bile salts and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into an ointment, salves, gel or cream as generally known in the art.

[0140] The agents may also be formulated in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0141] In some embodiments, the active compound is prepared with a carrier that prevents the compound from being rapidly excreted from the body, such as a controlled release formulation comprising an implant and a microencapsulated delivery system. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are apparent to those skilled in the art. These materials can also be purchased commercially. Liposomal suspensions can also be used as pharmaceutically acceptable carriers.

[0142] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unit dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are dictated by and directly dependent on the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for treating individuals.

[0143] In some embodiments, the protein may be provided in a lyophilized form for reconstitution prior to administration. For example, a lyophilized antibody molecule may be reconstituted in sterile water and mixed with saline prior to administration to an individual.

[0144] The pharmaceutical compositions provided herein may be included in a container, pack, or dispenser together with instructions for administration.

[0145] Nucleic acid molecule, vector, host cell and method for producing protein

[0146] Provided herein are nucleic acid molecules encoding the bispecific proteins disclosed herein. Provided herein is a nucleic acid molecule (e.g., an isolated nucleic acid molecule) encoding the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain of the bispecific protein disclosed herein (or the amino acid sequence of (i) VH domain, (ii) VL domain, or (iii) VH domain and VL domain of the protein). In some embodiments, the nucleic acid molecule encoding the VH domain, VL domain, or polypeptide chain comprises a signal sequence (or encodes a leader peptide). In some embodiments, the nucleic acid molecule encoding the VH domain, VL domain, or polypeptide chain does not comprise a signal sequence (or does not encode a leader peptide).

[0147] Also provided herein is an expression vector comprising nucleic acid molecules described herein. In some vectors, the nucleic acid molecules are operably connected to one or more regulatory sequences suitable for expressing nucleic acid fragments in host cells. In some cases, the expression vector comprises a sequence that mediates replication and comprises one or more selective markers. As used herein, "vector" means a construct that can deliver and preferably express one or more genes or sequences of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, clays or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and some eukaryotic cells, such as production cells.

[0148] A recombinant host cell is provided herein, comprising an expression vector or nucleic acid molecule disclosed herein."Host cell" includes a single cell, cell line or cell culture, which can be or has become a recipient of one or more vectors for incorporating a polynucleotide insert.Host cell includes the offspring of a single host cell. Due to natural, accidental or intentional mutations, offspring may not be identical (morphologically or on genomic DNA complementary sequences) with the original parent cell. Expression vectors can be transfected into host cells by standard techniques. Non-limiting examples include electroporation, calcium phosphate precipitation, DEAE-dextran transfection, etc. In some embodiments, the recombinant host cell comprises a single vector or a single nucleic acid molecule encoding the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the bispecific protein disclosed herein. In some embodiments, the recombinant host cell comprises a plurality of vectors or a plurality of nucleic acid molecules encoding the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the bispecific protein disclosed herein.

[0149] The protein molecules of the present invention or parts thereof can be produced using techniques known in the art, such as recombinant technology, phage display technology, synthetic technology, computational technology, or a combination of such techniques or other techniques known in the art.

[0150] Further provided herein is a method for producing a protein disclosed herein, the method comprising: culturing a recombinant host cell comprising an expression vector described herein under conditions whereby the nucleic acid molecule is expressed, thereby producing the protein. The protein can then be isolated from the host cell or culture. Provided herein is a method for producing a protein, the method comprising: culturing a recombinant host cell comprising an expression vector disclosed herein under conditions whereby the nucleic acid molecule is expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

[0151] Protein disclosed herein can be produced by any of a variety of methods known to those skilled in the art. In certain embodiments, protein disclosed herein can be produced by recombinant production. For example, one or more nucleic acid sequences or parts thereof encoding the heavy chain or light chain provided herein can be introduced into bacterial cells (e.g., Escherichia coli, Bacillus subtilis (B.subtilis)) or eukaryotic cells (e.g., yeast such as Saccharomyces cerevisiae (S.cerevisiae), or mammalian cells such as CHO cell lines, various Cos cell lines, HeLa cells, HEK293 cells, various myeloma cell lines or transformed B cells or hybridomas), or introduced into an in vitro translation system, and the translated polypeptide can be separated. In some embodiments, light chain proteins and heavy chain proteins are produced in cells with signal sequences, which are removed after producing the mature protein disclosed herein.

[0152] One skilled in the art will be able to determine whether a protein comprising a given polypeptide sequence binds to c-Kit protein and / or CD203c protein using standard methods (eg, Western blot, ELISA, etc.).

[0153] Medical uses of bispecific proteins

[0154] Provided herein are methods and uses of the bispecific proteins, immunoconjugates, and pharmaceutical compositions disclosed herein for providing therapeutic benefit to subjects with inflammatory diseases. Provided herein are methods and uses of the bispecific proteins, immunoconjugates, and pharmaceutical compositions disclosed herein for providing therapeutic benefit to subjects with neoplasms.

[0155] The activatable proteins, immunoconjugates or pharmaceutical compositions disclosed herein can be used for human or animal treatment methods, including preventive or prophylactic treatment (e.g., treatment performed before the onset of a subject's disease to reduce the risk of the subject developing the disease; delay its onset; or reduce its severity after onset). The treatment method may include administering a protein, immunoconjugate or pharmaceutical composition to a subject in need thereof.

[0156] Provided herein is a method for treating an inflammatory disease or neoplasm in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein. Provided herein is a bispecific protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in treating an inflammatory disease or neoplasm.

[0157] Provided herein is a method for improving symptoms of an inflammatory disease or neoplasm in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0158] Provided herein are bispecific proteins, immunoconjugates or pharmaceutical compositions disclosed herein for use as medicaments.

[0159] In some embodiments of the methods and uses disclosed herein, the inflammatory disease is a chronic inflammatory disease. In some embodiments of the methods and uses disclosed herein, the inflammatory disease is a mast cell driven disease. In some embodiments of the methods and uses disclosed herein, the inflammatory disease is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.

[0160] In some embodiments of the methods and uses disclosed herein, the neoplasm is a mast cell driven neoplasm. In some embodiments of the methods and uses disclosed herein, the neoplasm is systemic mastocytosis or mast cell leukemia.

[0161] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an agent (e.g., a protein, an immunoconjugate, or a pharmaceutical composition) disclosed herein that is sufficient to reduce or improve the severity and / or duration of an inflammatory disease or neoplasm, or one or more symptoms thereof, prevent the development of the disease, cause regression of the disease, prevent the recurrence, development, onset, or progression of one or more symptoms associated with the disease, or enhance or improve the preventive or therapeutic effect of another related therapy (e.g., a preventive or therapeutic agent) on an inflammatory disease or neoplasm.

[0162] The actual amount of administration and the rate and time course of administration will depend on the nature and severity of the disease being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disease, the delivery site of the composition, the method of administration, the schedule of administration, and other factors known to the practicing physician. Therapeutic prescriptions (e.g., dosage determinations, etc.) are within the scope of the duties of general practitioners and other doctors, and may depend on the severity of the symptoms and / or the progression of the disease being treated. Appropriate doses of antibody-based protein molecules are well known in the art (Ledermann JA et al., 1991, Int. J. Cancer 47: 659-664; Bagshawe KD et al., 1991, Antibody, Immunoconjugates and Radiopharmaceuticals 4: 915-922). This article or Physician's Desk Reference (2003) may indicate a specific dose according to the type of drug used. The therapeutically effective amount or suitable dose can be determined by comparing the in vitro activity of antibody-based protein molecules and the in vivo activity in animal models. Methods for extrapolating effective doses in mice and other test animals to humans are known. The exact dose will depend on many factors, including whether the antibody-based protein is used for prevention or treatment, the size and location of the area to be treated, the exact nature of the antibody-based protein, and the nature of any detectable label or other molecule attached to the antibody-based protein.

[0163] Typical protein dosages will be in the range of 100 μg to 1 g for systemic application and in the range of 1 μg to 1 mg for intradermal injection. An initial higher loading dose may be administered, followed by one or more lower doses. In some embodiments, the protein is of IgG1 or IgG4 isotype. The dosage for a single treatment of an adult subject may be proportionally adjusted for children and infants. Treatment may be repeated daily, twice a week, weekly or monthly, at the discretion of the physician. The treatment regimen for a subject may depend on the pharmacokinetic and pharmacodynamic properties of the protein composition, the route of administration and the nature of the disorder being treated.

[0164] Treatment can be cyclical, and the period between administrations can be about two weeks or longer, such as about three weeks or longer, about four weeks or longer, about once a month or longer, about five weeks or longer, or about six weeks or longer. For example, treatment can be performed every two to four weeks or every four to eight weeks. Treatment can be performed before and / or after surgery, and / or can be directly administered or applied to the anatomical site of surgical treatment or invasive procedure. Suitable formulations and routes of administration are described above.

[0165] In some embodiments, the proteins, immunoconjugates or pharmaceutical compositions disclosed herein can be administered as subcutaneous injections. Subcutaneous injections can be administered using an automatic injector, for example, for long-term prevention / treatment.

[0166] In some embodiments, the therapeutic effect of a protein, immunoconjugate, or pharmaceutical composition disclosed herein may last for several half-lives, depending on the dose. For example, the therapeutic effect of a single dose of a protein, immunoconjugate, or pharmaceutical composition disclosed herein may last for 1 month or longer, 2 months or longer, 3 months or longer, 4 months or longer, 5 months or longer, or 6 months or longer in a subject.

[0167] In some embodiments, a subject can be treated with a protein, immunoconjugate or pharmaceutical composition disclosed herein and an additional therapeutic agent or therapy for treating cancer, or symptoms or complications of an inflammatory disease or neoplasm. The protein, immunoconjugate or pharmaceutical composition disclosed herein and the additional therapeutic agent or therapy can be administered simultaneously or sequentially.

[0168] In some embodiments, the subject is a human, non-human primate, pig, horse, cow, dog, cat, guinea pig, mouse, or rat. In some embodiments, the subject is a human adult. In some embodiments, the subject is a human child.

[0169] Further provided herein are proteins, immunoconjugates or pharmaceutical compositions disclosed herein for use in treating a disease or disorder.

[0170] Provided herein are proteins, immunoconjugates or pharmaceutical compositions disclosed herein for use as medicaments.

[0171] definition

[0172] Unless otherwise indicated, the terms used herein have the definitions commonly used in the art.Some terms are defined below, and additional definitions can be found in the remainder of the detailed description.

[0173] The term "a" or "an" refers to one or more of the entity, i.e., it may refer to plural referents. Therefore, the terms "a", "one", "one or more", and "at least one" are used interchangeably herein. In addition, an "element" referred to by the indefinite article "a" or "an" does not exclude the possibility that more than one element is present, unless the context clearly requires the presence of one and only one element.

[0174] Unless otherwise indicated or apparent from the context, the term "about" means within 10% above or below the reported value (unless the number is more than 100% or less than 0% of the possible value). When used in conjunction with a range of values ​​or a series of values, the term "about" applies to the endpoints of the range or each value in the recited values ​​in the series unless otherwise indicated. As used in this application, the terms "about" and "approximately" are used as equivalents.

[0175] As used herein, the term "sequence identity" refers to the degree that the polynucleotide or polypeptide sequence of two optimal comparisons is constant in the entire comparison window of residues (e.g., nucleotides or amino acids). The "identity score" of the compared segments of the test sequence and the reference sequence is the number of identical residues shared by the two compared sequences divided by the total number of residues in the reference sequence segments (i.e., the smaller defined part of the whole reference sequence or the reference sequence). "Identity percentage" is the identity score multiplied by 100. The alignment program ClustalOmega available at ebi.ac.uk / Tools / msa / clustalo can be used to calculate the identity percentage using default parameters. See Sievers et al., "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega" (2011October 11) Molecular Systems Biology 7:539. For the purpose of calculating the identity with the sequence, extensions such as tags are not included.

[0176] As used herein, the term "HCDR" refers to the heavy chain complementarity determining region. As used herein, the term "LCDR" refers to the light chain complementarity determining region.

[0177] As used herein, the terms "amino terminus," "N-terminus," "carboxyl terminus," and "C-terminus" refer to positions within a polypeptide chain. When the context permits, these terms are used with reference to a particular sequence or portion of a polypeptide to indicate proximity or relative position. For example, a sequence within a polypeptide that is located at the carboxyl terminus of a reference sequence is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily located at the carboxyl terminus of the complete polypeptide.

[0178] As used herein, the term "conservative substitution" refers to replacing an amino acid with another amino acid that does not significantly and deleteriously change the functional activity. A preferred example of a "conservative substitution" is replacing an amino acid with another amino acid that has a value of ≥0 in the following BLOSUM 62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89: 10915-10919):

[0179]

[0180] The terms "antibody-drug conjugate" and "immunoconjugate" refer to a protein of the disclosure conjugated to a cytotoxic, cytostatic and / or therapeutic agent.

[0181] The term "isolated molecule" (where the molecule is, for example, a protein, polypeptide, polynucleotide, antibody, or antigen-binding molecule) is a molecule that, depending on its origin or source of derivation, (1) is not associated with naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not exist in nature. Thus, a chemically synthesized molecule or a molecule expressed in a cellular system different from the cell from which it naturally originates will be "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by separation using purification techniques well known in the art. Molecular purity or heterogeneity can be determined by a number of methods known in the art. For example, the purity of a polypeptide sample can be determined by using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide. For some purposes, higher resolution can be provided by using high performance liquid chromatography (HPLC) or other purification means known in the art.

[0182] c-Kit (also known as KIT, cluster of differentiation 117 (CD117), PBT, SCFR, KIT proto-oncogene receptor tyrosine kinase) is a transmembrane protein that belongs to the immunoglobulin superfamily and binds to the soluble factor SCF (stem cell factor). c-Kit is a receptor tyrosine kinase type III that is highly expressed by hematopoietic stem cells as well as a variety of other cell types, such as mature mast cells.

[0183] CD203c (also known as ENPP3, B10, NPP3, PDNP3, and PD-IBETA) is a type II transmembrane protein that belongs to the ectonucleotide pyrophosphatase / phosphodiesterase 3 (E-NPP3) family of enzymes involved in the hydrolysis of oligonucleotides and nucleoside phosphates. CD203c is unique to the mast cell / basophil lineage of hematopoietic effector cells.

[0184] The terms "inhibit," "block," or "neutralize," as used herein with respect to the biological activity of a protein disclosed herein, mean the ability of the protein to significantly antagonize, prohibit, prevent, suppress, slow, destroy, eliminate, stop, reduce, or reverse, for example, the progression, intensity, or severity of an inhibited substance, including but not limited to the binding of c-Kit to SCF, or the binding of CD203c to a binding partner, including but not limited to an enzyme inhibitor.

[0185] As used herein, the terms "treat," "treating," or "treatment of" (and grammatical variations thereof) mean that the severity of a subject's condition is reduced, at least partially improved or stabilized and / or some relief, alleviation, reduction or stabilization of at least one clinical symptom is achieved, and / or there is a delay in the progression of a disease or disorder.

[0186] As used herein, the terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to preventing and / or delaying the onset of and / or reducing the severity of a disease, disorder, and / or one or more clinical symptoms in a subject relative to what would occur in the absence of the compositions and / or methods described herein. Prevention can be complete, such as the complete absence of the disease, disorder, and / or one or more clinical symptoms. Prevention can also be partial, such that the occurrence and / or onset of the disease, disorder, and / or one or more clinical symptoms in a subject is less severe than would occur in the absence of the compositions and / or methods described herein.

[0187] As used herein, a "therapeutically effective amount" is an amount of a protein or pharmaceutical composition provided herein that is effective in treating a disease or condition in a subject or in improving its signs or symptoms. A "therapeutically effective amount" may vary according to, for example, the disease and / or disease symptoms, the severity of the disease and / or the symptoms of the disease or condition, the age, weight and / or health of the patient to be treated, and the judgment of the prescribing physician.

[0188] In the present specification, unless otherwise indicated, any concentration range, percentage range, ratio range or integer range should be understood to include the value of any integer within the range, and include fractions thereof (such as one tenth and one hundredth of an integer) where appropriate.

[0189] Use of alternatives (eg, "or") should be understood to mean either, both, or any combination of the alternatives.

[0190] All references, articles, publications, patents, patent publications and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the reference to any reference, article, publication, patent, patent publication and patent application cited herein does not and should not be taken as an admission or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0191] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0192] Numbered implementation plan

[0193] Notwithstanding the appended claims, the present disclosure sets forth the following numbered embodiments:

[0194] Embodiment 1. A bispecific protein, comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain, the first immunoglobulin heavy chain comprising an anti-c-Kit heavy chain variable (VH) domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain, the second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) identical third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain, the immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c.

[0195] Embodiment 2. The bispecific protein of embodiment 1, wherein the anti-c-Kit VH domain comprises a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:2, a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO:3, and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO:4; the anti-c-CD203c VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO:14, a HCDR2 comprising the amino acid sequence of SEQ ID NO:15, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:73; and the VL domain comprises a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO:6, a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO:7, and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO:8.

[0196] Embodiment 3. The bispecific protein of embodiment 1, wherein the anti-c-Kit VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO:2, a HCDR2 comprising the amino acid sequence of SEQ ID NO:3, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:4; the anti-c-CD203c VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO:10, a HCDR2 comprising the amino acid sequence of SEQ ID NO:11, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:12; and the VL domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO:6, a LCDR2 comprising the amino acid sequence of SEQ ID NO:7, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:8.

[0197] Embodiment 4. The bispecific protein of embodiment 1, wherein the anti-c-Kit VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO:2, a HCDR2 comprising the amino acid sequence of SEQ ID NO:3, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:4; the anti-c-CD203c VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO:14, a HCDR2 comprising the amino acid sequence of SEQ ID NO:15, and a HCDR3 comprising the amino acid sequence of SEQ ID NO:12; and the VL domain comprises a LCDR1 comprising the amino acid sequence of SEQ ID NO:6, a LCDR2 comprising the amino acid sequence of SEQ ID NO:7, and a LCDR3 comprising the amino acid sequence of SEQ ID NO:8.

[0198] Embodiment 5. The bispecific protein of embodiment 2, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 65; the anti-c-CD203cVH domain comprises the amino acid sequence of SEQ ID NO: 71; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0199] Embodiment 6. The bispecific protein of embodiment 3, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203cVH domain comprises the amino acid sequence of SEQ ID NO: 9; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0200] Embodiment 7. The bispecific protein of embodiment 4, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203cVH domain comprises the amino acid sequence of SEQ ID NO: 13; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0201] Embodiment 8. The bispecific protein of embodiment 1, wherein the anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; the anti-c-CD203cVH domain comprises the amino acid sequence of any one of SEQ ID NOs: 16-51; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0202] Embodiment 9. The bispecific protein of embodiment 1, wherein the anti-c-Kit VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 63-67; the anti-c-CD203c VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 68-72; and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0203] Embodiment 10. The bispecific protein of any one of embodiments 1-9, wherein the bispecific protein comprises an immunoglobulin constant region.

[0204] Embodiment 11. The bispecific protein of embodiment 10, wherein the immunoglobulin constant region is an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin constant region.

[0205] Embodiment 12. The bispecific protein of embodiment 10, wherein the immunoglobulin constant region is an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 immunoglobulin constant region.

[0206] Embodiment 13. The bispecific protein of Embodiment 10, wherein the immunoglobulin constant region is an immunologically inert constant region.

[0207] Embodiment 14. A bispecific protein as described in any one of embodiments 1-9, wherein the first polypeptide chain comprises a first immunoglobulin constant region and the second polypeptide chain comprises a second immunoglobulin constant region, and wherein the first immunoglobulin constant region and the second immunoglobulin constant region comprise a knob-hole mutation.

[0208] Embodiment 15. The bispecific protein of embodiment 14, wherein the first immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions S354C and T366W, and wherein the second immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions Y349C, T366S, L368A, and Y407V, wherein numbering is according to the EU index as in Kabat.

[0209] Embodiment 16. The bispecific protein of embodiment 14, wherein the first immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions Y349C, T366S, L368A, and Y407V, and wherein the second immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions S354C and T366W, wherein numbering is according to the EU index as in Kabat.

[0210] Embodiment 17. A bispecific protein as described in embodiment 14, wherein (a) the first immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:52, and the second immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:53; or (b) the first immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:53, and the second immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:52.

[0211] Embodiment 18. A bispecific protein that binds c-Kit and CD203c, wherein said c-Kit and CD203c are located on the surface of the same cell.

[0212] Embodiment 19. The bispecific protein of embodiment 18, wherein the bispecific protein is a designed ankyrin repeat protein (DARPin), a tandem VHH, or a tandem immunoglobulin neoantigen receptor (IgNAR).

[0213] Embodiment 20. An immunoconjugate comprising the bispecific protein of any one of embodiments 1-19 linked to a therapeutic agent.

[0214] Embodiment 21. The immunoconjugate of embodiment 20, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulatory agent, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

[0215] Embodiment 22. A pharmaceutical composition comprising the bispecific protein of any one of Embodiments 1-19 or the immunoconjugate of Embodiments 20 or 21, and a pharmaceutically acceptable carrier, diluent or excipient.

[0216] Embodiment 23. A nucleic acid molecule encoding the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the bispecific protein of any one of embodiments 1-17.

[0217] Embodiment 24. A nucleic acid molecule encoding the bispecific protein of embodiment 18 or 19.

[0218] Embodiment 25. An expression vector comprising the nucleic acid molecule of embodiment 22 or 23.

[0219] Embodiment 26. A recombinant host cell comprising the nucleic acid molecule of embodiment 23 or 24 or the expression vector of embodiment 25.

[0220] Embodiment 27. A method for producing a bispecific protein, the method comprising: culturing the recombinant host cell of embodiment 26 under conditions whereby the nucleic acid molecule is expressed, thereby producing the protein; and isolating the protein from the host cell or culture.

[0221] Embodiment 28. A method for treating an inflammatory disease or neoplasm in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the bispecific protein of any one of Embodiments 1-19, the immunoconjugate of Embodiment 20 or 21, or the pharmaceutical composition of Embodiment 22.

[0222] Embodiment 29. A method for improving symptoms of an inflammatory disease or neoplasm in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the bispecific protein of any one of Embodiments 1-19, the immunoconjugate of Embodiment 20 or 21, or the pharmaceutical composition of Embodiment 22.

[0223] Embodiment 30. The method of embodiment 28 or 29, wherein the inflammatory disease is a chronic inflammatory disease.

[0224] Embodiment 31. The method of embodiment 28 or 29, wherein the inflammatory disease is a mast cell driven disease.

[0225] Embodiment 32. The method of embodiment 28 or 29, wherein the inflammatory disease is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.

[0226] Embodiment 33. The method of embodiment 28 or 29, wherein the neoplasm is a mast cell driven neoplasm.

[0227] Embodiment 34. The method of embodiment 28 or 29, wherein the neoplasm is systemic mastocytosis or mast cell leukemia.

[0228] Embodiment 35. The bispecific protein of any one of embodiments 1-19, the immunoconjugate of embodiment 20 or 21, or the pharmaceutical composition of embodiment 22 for use as a medicament.

[0229] Embodiment 36. The bispecific protein of any one of embodiments 1-19, the immunoconjugate of embodiment 20 or 21, or the pharmaceutical composition of embodiment 22, for use in treating an inflammatory disease or a neoplasm.

[0230] Embodiment 37. The bispecific protein, immunoconjugate, or pharmaceutical composition for use as described in Embodiment 36, wherein the inflammatory disease is a chronic inflammatory disease.

[0231] Embodiment 38. The bispecific protein, immunoconjugate, or pharmaceutical composition for use as described in Embodiment 36, wherein the inflammatory disease is a mast cell driven disease.

[0232] Embodiment 39. The bispecific protein, immunoconjugate, or pharmaceutical composition for use as described in Embodiment 36, wherein the inflammatory disease is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.

[0233] Embodiment 40. The bispecific protein, immunoconjugate, or pharmaceutical composition for use as described in Embodiment 36, wherein the neoplasm is a mast cell driven neoplasm.

[0234] Embodiment 41. The bispecific protein, immunoconjugate, or pharmaceutical composition for use as described in Embodiment 36, wherein the neoplasm is systemic mastocytosis or mast cell leukemia.

[0235] The present disclosure will be further clarified by the following examples, which are intended to be merely illustrative of the present disclosure and not limiting.

[0236] Example

[0237] Generation and analysis of anti-c-Kit and anti-CD203c bispecific proteins

[0238] Materials and methods

[0239] IgG / bispecific expression and purification

[0240] Mammalian codon-optimized synthetic genes encoding the heavy and light chain variable domains of the anti-CD203c antibody were cloned into mammalian expression vectors containing human IgG1 with null effector function ("IgG1 null"; human IgG1 containing L234A, L235A, G237A mutations in the lower hinge that abolish normal immunoglobulin ADCC, ADCP and CDC functions) and human Cκ domains, respectively. The Fc sequence is provided in Table 4. Co-transfection of vectors containing heavy and light chains was performed in a CHO mammalian expression system, followed by protein A-based purification of IgG, and quantification and quality control on denaturing and native SDS-PAGE and analytical SEC.

[0241] Using human IgG1 with the above effector function null mutations, Figure 2 The common light chain bispecific antibodies were expressed and purified in a similar manner using heavy chain constructs with either "knob" or "hole" mutations as described in . After protein A purification, the bispecific molecules were subjected to a second step of preparative cation exchange chromatography to achieve a single POI > 95%.

[0242] Direct Binding ELISA for IgG / Bispecific Antibodies

[0243] The binding and cross-reactivity of this molecule to human, rhesus and cynomolgus CD203c was initially assessed by binding ELISA. Human CD203c His-tagged recombinant protein and rhesus CD203c His-tagged recombinant protein were coated onto MaxiSorp TM Flat-bottom 96-well plate surface. Purified samples were titrated in 5-fold serial dilutions starting from 100 nM and allowed to bind to the coated antigen. IgG was detected using mouse anti-human IgG conjugated with horseradish peroxidase. Binding signals were visualized using 3,3',5,5'-tetramethylbenzidine substrate solution (TMB) and absorbance was measured at 450 nm.

[0244] Binding to cell surface CD203c on KU812 cells

[0245] All treatments are performed to cells seeded in T75 flasks at an inoculation density of 0.3e6 / ml. Cells are cultured in the presence of 80ng / ml SCF and treated with 20ng / ml IL3 24 hours before antibody staining. After SCF / IL3 treatment, cells are harvested and stained with live-dead stain (Zombie UV Biolegend 423108). The cells are then stained with the titration solution of anti-CD203c antibody / bispecific antibody or corresponding isotype control in a 5-fold dilution series starting with 50mg / mL, and detected with anti-human-Fc AF647 antibody (JIR 109-605-098). Subsequently, samples are run on Yeti analyzer, so that 10K live cells are gated. Subsequent analysis is performed using FlowJo software, and data are represented by MFI (median fluorescence intensity).

[0246] 1.27 Heavy chain optimized library design, cloning and selection

[0247] Using large-scale oligonucleotide assembly and high-fidelity oligonucleotide pool splicing, a 2-domain (HCDR1 and HCDR2) precisely defined complexity variant library with single and double mutations and a single-domain (HCDR3) precisely defined complexity variant library with single and double mutations were synthesized. All amino acids were represented at all positions except cysteine, avoiding the insertion of NG and DG developability risk motifs. These designs are summarized in Figure 4 and Figure 5 These libraries were cloned into a custom pCAT-Fab-MH1 phagemid vector containing anti-c-Kit-MH1 VL, transformed into E. coli TG-1 cells and rescued essentially as described in detail previously (Finlay et al., 2011, Methods Mol Biol 681:383-401).

[0248] Phage selection was performed by coating streptavidin magnetic microbeads with biotinylated CD203c protein (human or rhesus monkey), washing the beads three times with PBS and resuspending them in PBS pH 7.4 containing 5% skim milk protein. The beads were coated with 100 nM target protein in the first round of selection, and then the antigen concentration was reduced 4-5 times in three consecutive rounds. In each round, phages were first eluted using trypsin and then reinfected into TG1 cells.

[0249] Periplasmic extract production (small scale)

[0250] The production of soluble Fabs was performed for individual E. coli clones. E. coli TG1 cells in logarithmic growth phase were induced with isopropyl-1-thio-β-D-galactopyranoside. Periplasmic extracts containing soluble Fabs were generated by freeze / thaw cycles: bacterial cell pellets were frozen overnight at -20°C, then thawed and resuspended in PBS pH 7.4 at room temperature. The supernatant containing soluble Fabs was collected after shaking and centrifugation at room temperature. These Fabs were tested for binding to 1 mg / mL coated human / rhesus CD203c in a direct binding ELISA (starting with 85% periprep, using 1.5-fold dilutions, and 8-point titration). Periprep binding was detected using anti-HA-HRP.

[0251] C-kit receptor phosphorylation assay

[0252] KU812 cells were cultured in RPMI without SCF + 10% FCS for 48 hours. 6 / mL was inoculated in 24-well plates with a total volume of 1.5mL and maintained for 1 hour, followed by treatment with isotype control or test agent for 2 hours. Cells were stimulated with 40ng / mL SCF for 5 minutes, washed in PBS, and cell lysates were prepared using lysis buffer according to the manufacturer's instructions (Biotechne). Total c-kit levels and phosphorylated c-kit levels were detected using ELISA kits from Biotechne using 6.25mg and 50mg total protein, respectively.

[0253] Affinity determination of optimized CD203c binding domain using SPR

[0254] After affinity optimization, CD203c VH binding domains F6 and F6.12 were produced in Fab format and compared with the parental 1.27 Fab for binding to human and cynomolgus CD203c proteins. The biotinylated version of the CD203c protein was captured on a BiotinCap chip and the antibody was injected in solution in a 2-fold serial dilution. The SPR affinity determination assay was performed on a BIAcore T20 using the following conditions:

[0255] Biacore chip: CAP chip (Biotin CAPture kit; Cytiva; catalog number 28920234)

[0256] Surface preparation: Inject Biotin CAPture reagent (Biotin CAPture kit; Cytiva; catalog number 28920234) at 2 μl / min for 5 minutes

[0257] • Ligand capture—inject biotinylated antigens in 1xHBS-EP+ pH 7.4 at 20 nM and 10 nM respectively: bio-huCD203c (Acro, Cat. nr. H52H4) and bio-cyCD203c (Icosagen) at 10 μl / min over flow cell 2 for 1 min

[0258] Analyte binding: The three test antibodies were injected at 30 μl / min for 1 min in flow cell 1 and flow cell 2 in an eight-step two-fold dilution series (1000 nM - 7.81 nM for 1.27-Fab, F6 Fab and F6.12 Fab). A complete blank assay run without analyte antibodies was included as a blank reference.

[0259] Dissociation rate measurement: 600 seconds

[0260] Regeneration: Use the regeneration solution provided in the Biotin CAPture kit to restore baseline levels

[0261] SPR running buffer: 1xHBS-EP+pH 7.4 (Cytiva; catalog number: BR100669)

[0262] Analysis: Data were analyzed using the Biacore Insight evaluation software using either a steady-state affinity model or a 1:1 binding model. Sensorgrams were referenced to flow cell 1 and a 0 nM concentration cycle.

[0263] Fig.11A – Fig.11D The data for the three Fabs are summarized in .

[0264] pi engineering of bispecific heavy chains for facile purification

[0265] The MH1 anti-c-Kit heavy chain and the F6.12 anti-CD203c heavy chain were selected for pI engineering to allow for easy downstream purification after Protein A chromatography using cation exchange. A number of criteria were used to select mutations that drove the anti-c-Kit heavy chain to have a more negative pI, and the anti-CD203c heavy chain to have a more positive pI. These criteria included:

[0266] Distal to the antigen-interacting CDR

[0267] Analysis of NGS antibody libraries available in published literature

[0268] Alignment with germline homologs (VH1-46 for anti-c-Kit / VH5-51 for anti-CD203c)

[0269] Comparison with known and approved clinical-stage antibody therapies

[0270] Compare the deposited structures of the two germlines in the PDB to provide structural context for the selected mutations

[0271] Model all mutation candidates to predict effects on stability, dipole moment, developability, and immunogenicity

[0272] A series of five double mutants with improved or neutral stability changes were preferentially selected for each heavy chain while changing the pI. These double mutants do not exist within the same 12-mer sequence segment to reduce the risk of immunogenicity and do not create unfavorable charge / hydrophobic patches or post-translational modification sites. They maintain developability parameters within or close to those described for clinical-stage therapeutic antibodies. These double mutation combinations are summarized in Fig.13 middle.

[0273] Production, purification and characterization of lead pI-engineered bispecific antibodies

[0274] The 5 double mutants of the MH1 anti-c-Kit VH domain were combined with each of the 5 double mutants of the F6.12 CD203cVH domain to generate 25 bispecific antibodies, each produced in 50 mL of ExpiCHO cells. ExpiCHO cells were cultured for 9 days (depending on cell viability), after which they were centrifuged to clarify the supernatant and the supernatant was processed immediately. The HiTrapMabSelect Sure Protein A 5mL column (GE Healthcare, catalog number 11-0034-95) on the Pure 25FPLC system captures the antibodies produced from the clarified supernatant. Immediately neutralize the acidic fraction corresponding to the affinity-purified antibody with 30% 1M Tris pH 8.0. Analyze 2.5 μL of each sample using Protein clearHT chip (catalog number CLS1486695) under non-reducing and reducing conditions on LabChip according to the manufacturer's instructions. Analyze about 10 μg of each sample on ThermoVanquish Flex UHPLC system (Thermo Fisher) by SEC-HPLC. The sample is injected into Superdex 200Increase 5 / 150GL (catalog number 28-9909-45) column and the monomer peak % is quantified. In order to conduct a preliminary assessment of the peak separation via cation exchange, analytical CEX is performed on each sample. Approximately 10 μg of each sample was injected onto a TSKgel SP-STAT (7 μm, 4.6 mm ID x 10 cm L) column previously equilibrated in CEX buffer A (25 mM sodium phosphate (pH 6)). An increasing salt gradient was applied using CEX buffer B (25 mM sodium phosphate, 1 M sodium chloride (pH 6)).

[0275] To further characterize the preferred bispecific antibodies, the same process was repeated using ExpiCHO cells with an amplified volume of 200 mL. After Pro-A purification, many different preparative cation exchange methods were compared to evaluate the optimal resolution of the main heterodimeric bispecific peaks. Initially, the desalted sample was injected into a HiTrap SP HP 5mL CEX column (Cytiva, catalog number 17-1151-02). In order not to impair the resolution of the separation, a total of 2 runs were performed. 1-20% of buffer B (25mM NaH2PO4 / Na2HPO4 1M NaCl pH 6.3) from 40CV was tested, followed by a salt gradient of 20-100% of buffer B for 10CV. However, the resolution of this column was not optimal, and a RESOURCE S 6 mL CEX column (Cytiva, catalog number 17118001) was selected as an alternative using 40 CV of 5-12% buffer B, followed by 10 CV of 12-100% buffer B. Several column fractions were taken for analytical SDS-PAGE characterization by LabChip as described above, and LC-MS analysis. Each sample was deglycosylated using Rapid PNGase F (non-reducing format) (New England Biolabs, Cat. nr. P0711S) following the manufacturer's recommendations. Protein BEH C4 Intact deglycosylated samples were analyzed by reverse phase liquid chromatography on a BioAccord system using Waters (Cat. No. 186004495). Data were processed using MaxEnt1 software.

[0276] Results and discussion

[0277] Lead IgG generation and screening

[0278] Anti-CD203c antibodies carrying anti-c-kit MH1 light chain (described in Table 2) were readily expressed and purified from CHO cells using protein-A affinity chromatography. Analytical SEC showed that all clones were >95% monomeric and the preparations had low endotoxin content of <1 EU / mg.

[0279] Purified IgG was initially tested for binding to His-tagged recombinant forms of human and rhesus CD203c ( Figure 3A and Figure 3B ), and all clones that exhibited measurable binding to these two orthologs were then subjected to binding analysis using flow cytometry on KU812 cells endogenously expressing human CD203c. Of the clones tested, only 1.27 retained robust binding to CD203c expressed on the cell surface ( Figure 3C), and this clone was selected as the parent clone for subsequent heavy chain mutagenesis and variant library generation.

[0280] 1.27 Generation and screening of heavy chain library

[0281] Anti-CD203c parental clone 1.27 was used as a template to generate Figure 4 and Figure 5 The two heavy chain precision variant libraries described in . These libraries were not directed against the light chain, all variants maintained the common anti-c-kit MH1 light chain described in Table 2. The library was selected after 4 iterative rounds of phage selection on human and rhesus CD203c and was screened in periprepFab format by direct binding ELISA for the ability to bind to these two orthologs. Subsequent sequence analysis allowed the selection of a leader set of optimized clones with sequences derived from the HCDR1 and HCDR2 and HCDR3 libraries.

[0282] The 1.27 output clones summarized in Table 3 were expressed and purified in IgG format and retested for binding to human / rhesus CD203c by direct ELISA in a purified IgG format and to cell surface CD203c on KU812 cells using a flow-based assay. Based on the data summarized in Figure 6, the best performing clones were preferentially selected for common light chain bispecific generation. The clones generated included HCDR1 / HCDR2 variants, HCDR3 variants, and clones combining mutations from both libraries, which are summarized in Table 3.

[0283] Characterization of optimized common light chain bispecific antibodies

[0284] Common light chain bispecific molecules generated from the heavy and light chain sequences detailed in Tables 1-3 were expressed in CHO cells and purified by protein-A affinity chromatography followed by a preparative cation exchange chromatography step to obtain a >95% monomer fraction. The purified proteins were evaluated for synergistic binding to two targets (CD203c and c-Kit) on KU812 cells. Figure 7 The bispecific molecules that engaged the dual receptors (1.27 / MH1 and F6 / MH1) on KU812 cells demonstrated enhanced binding compared to bivalent CD203c IgG and bivalent and monovalent c-Kit IgG. This enhanced binding implies that the F6 / MH1 bispecific molecule has potent inhibition of c-Kit receptor phosphorylation compared to monovalent anti-c-kit, as shown in Figure 2. Figure 8 This inhibition suggests that monovalent c-Kit targeting can inhibit receptor phosphorylation and that co-engagement with CD203c greatly enhances the potency of this inhibition through enhanced receptor occupancy.

[0285] However, not all bispecific pairs retained binding to both targets. When an alternative common light chain derived from the anti-CD203c binding domain was grafted with the MH1 CDRs (Table 2; SEQ ID NOs: 58-61) and paired with the CD203c VH domain (Table 3; SEQ ID NOs: 54-57), c-Kit binding was retained, but binding to CD203c was lost. These data are summarized in Fig. 9A and Fig. 9B , indicating that successful bispecific pairings cannot be predicted a priori.

[0286] The F6 / MH1 bispecific molecules were tested for comparative binding to human and cynomolgus monkey CD203c by ELISA. Fig.10 The data shown in demonstrate equivalent binding to these two orthologs.

[0287] Affinity optimization of the anti-CD203c 1.27 binding domain

[0288] SPR was used to characterize variants from the 1.27 affinity optimized library. Binding of the parental 1.27 in Fab format (F6 with mutations in H-CDR1 and H-CDR2 and F6.12 with mutations in all three VH CDRs) to chips coated with biotinylated human or cynomolgus monkey CD203c was measured. 1:1 interaction kinetics assays showed that the KD of F6 was improved 10-fold to 74 nM, and the KD of F6.12 was further improved to 55 nM ( Fig.11A – Fig.11D This improvement in binding was further confirmed in KU812 cells, where F6 IgG (EC50 1.311 nM) showed a 75-fold increase in binding compared to the parental 1.27 IgG (EC50 98.23 nM). Fig. 12A – Fig. 12B Summarized in .

[0289] MH1 / F6.12 bispecific pI engineering

[0290] Both bispecific heavy chains were engineered for pI to improve downstream process development and optimize heterodimer formation. Five double mutants were generated for anti-c-Kit VH MH1 to drive toward a more negative pI, and five double mutants were generated for anti-CD203c VH F6.12 to drive toward a more positive pI. These mutations are summarized in Fig.13A matrix of all possible 25 variant bispecific antibodies was generated and their productivity, analytical SEC, and pilot heterodimer formation were compared by analytical CEX. Each bispecific variant differed in the CEX curve, with some showing multiple peaks that were not easily resolved. Fig.14 An example of such a curve is shown, where there are 3 overlapping peaks for MH1_A / F6.12_B. However, some variants showed clear resolution and preferential heterodimer formation, which could be purified by preparative CEX with robust yields. This is Fig.15 The case of MH1_C / F6.12_D described in.

[0291] MH1_C / F6.12_D was compared to the parental bispecific MH1 / F6.12 to ensure that interaction with both targets was maintained after pI engineering. Both variants exhibited binding to human and cynomolgus monkey c-Kit ( Fig.16 ) and human and cynomolgus monkey CD203c ( Fig.17 ) for which the kinetic data are summarized in Fig.18 This was also true for binding to cell surface targets, with equivalent binding exhibited before and after pI engineering on KU812 cells ( Fig.19A – Fig.19C ).

Claims

1. A bispecific protein comprising: (a) a first polypeptide chain comprising a first immunoglobulin heavy chain comprising an anti-c-Kit heavy chain variable (VH) domain; (b) a second polypeptide chain comprising a second immunoglobulin heavy chain comprising an anti-CD203c VH domain; and (c) the same third and fourth polypeptide chains, wherein the third polypeptide chain and the fourth polypeptide chain each comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c.

2. The bispecific protein of claim 1, wherein The anti-c-Kit VH domain comprises a heavy chain complementary determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain complementary determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain complementary determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 4; The anti-c-CD203c VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 73; and The VL domain comprises a light chain complementary determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO:6, a light chain complementary determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO:7, and a light chain complementary determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO:

8.

3. The bispecific protein of claim 1, wherein The anti-c-Kit VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 4; The anti-c-CD203c VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and The VL domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO:6, LCDR2 comprising the amino acid sequence of SEQ ID NO:7, and LCDR3 comprising the amino acid sequence of SEQ ID NO:

8.

4. The bispecific protein of claim 1, wherein The anti-c-Kit VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 4; The anti-c-CD203c VH domain comprises a HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, a HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and a HCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and The VL domain comprises LCDR1 comprising the amino acid sequence of SEQ ID NO:6, LCDR2 comprising the amino acid sequence of SEQ ID NO:7, and LCDR3 comprising the amino acid sequence of SEQ ID NO:

8.

5. The bispecific protein of claim 2, wherein The anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 65; The anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 71; and The VL domain comprises the amino acid sequence of SEQ ID NO:

5.

6. The bispecific protein of claim 3, wherein The anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; The anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 9; and The VL domain comprises the amino acid sequence of SEQ ID NO:

5.

7. The bispecific protein of claim 4, wherein The anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; The anti-c-CD203c VH domain comprises the amino acid sequence of SEQ ID NO: 13; and The VL domain comprises the amino acid sequence of SEQ ID NO:

5.

8. The bispecific protein of claim 1, wherein The anti-c-Kit VH domain comprises the amino acid sequence of SEQ ID NO: 1; The anti-c-CD203c VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 16-51; and The VL domain comprises the amino acid sequence of SEQ ID NO:

5.

9. The bispecific protein of claim 1, wherein The anti-c-Kit VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 63-67; The anti-c-CD203c VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 68-72; and The VL domain comprises the amino acid sequence of SEQ ID NO:

5.

10. The bispecific protein of any one of claims 1-9, wherein the bispecific protein comprises an immunoglobulin constant region.

11. The bispecific protein of claim 10, wherein the immunoglobulin constant region is an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin constant region.

12. The bispecific protein of claim 10, wherein the immunoglobulin constant region is an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 immunoglobulin constant region.

13. The bispecific protein of claim 10, wherein the immunoglobulin constant region is an immunologically inert constant region.

14. The bispecific protein of any one of claims 1-9, wherein the first polypeptide chain comprises a first immunoglobulin constant region and the second polypeptide chain comprises a second immunoglobulin constant region, and wherein the first immunoglobulin constant region and the second immunoglobulin constant region comprise a knob-hole mutation.

15. The bispecific protein of claim 14, wherein the first immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions S354C and T366W, and wherein the second immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions Y349C, T366S, L368A, and Y407V, wherein numbering is according to the EU index as in Kabat.

16. The bispecific protein of claim 14, wherein the first immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions Y349C, T366S, L368A, and Y407V, and wherein the second immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions S354C and T366W, wherein numbering is according to the EU index as in Kabat.

17. The bispecific protein of claim 14, wherein (a) the first immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO: 52, and the second immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO: 53; or (b) the first immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:53, and the second immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO:

52.

18. A bispecific protein that binds c-Kit and CD203c, wherein the c-Kit and CD203c are located on the surface of the same cell.

19. The bispecific protein of claim 18, wherein the bispecific protein is a designed ankyrin repeat protein (DARPin), a tandem VHH, or a tandem immunoglobulin neoantigen receptor (IgNAR).

20. An immunoconjugate comprising the bispecific protein of any one of claims 1-19 linked to a therapeutic agent.

21. The immunoconjugate of claim 20, wherein the therapeutic agent is a cytotoxin, a radioisotope, a chemotherapeutic agent, an immunomodulator, a cytostatic enzyme, a cytolytic enzyme, a therapeutic nucleic acid, an anti-angiogenic agent, an anti-proliferative agent, or a pro-apoptotic agent.

22. A pharmaceutical composition comprising the bispecific protein of any one of claims 1-19 or the immunoconjugate of claim 20 or 21, and a pharmaceutically acceptable carrier, diluent or excipient.

23. A nucleic acid molecule encoding the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain of the bispecific protein of any one of claims 1 to 17.

24. A nucleic acid molecule encoding the bispecific protein of claim 18 or 19.

25. An expression vector comprising the nucleic acid molecule of claim 22 or 23.

26. A recombinant host cell comprising the nucleic acid molecule of claim 23 or 24 or the expression vector of claim 25.

27. A method for producing a bispecific protein, the method comprising: Cultivating the recombinant host cell of claim 26 under conditions whereby the nucleic acid molecule is expressed, thereby producing the protein; as well as The protein is isolated from the host cell or culture.

28. A method for treating an inflammatory disease or neoplasm in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the bispecific protein of any one of claims 1-19, the immunoconjugate of claim 20 or 21, or the pharmaceutical composition of claim 22.

29. A method for improving symptoms of an inflammatory disease or neoplasm in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the bispecific protein of any one of claims 1-19, the immunoconjugate of claim 20 or 21, or the pharmaceutical composition of claim 22.

30. The method of claim 28 or 29, wherein the inflammatory disease is a chronic inflammatory disease.

31. The method of claim 28 or 29, wherein the inflammatory disease is a mast cell driven disease.

32. The method of claim 28 or 29, wherein the inflammatory disease is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.

33. The method of claim 28 or 29, wherein the neoplasm is a mast cell driven neoplasm.

34. The method of claim 28 or 29, wherein the neoplasm is systemic mastocytosis or mast cell leukemia.

35. The bispecific protein of any one of claims 1-19, the immunoconjugate of claim 20 or 21, or the pharmaceutical composition of claim 22 for use as a medicament.

36. The bispecific protein of any one of claims 1-19, the immunoconjugate of claim 20 or 21, or the pharmaceutical composition of claim 22 for use in treating an inflammatory disease or a neoplasm.

37. The bispecific protein, immunoconjugate, or pharmaceutical composition for use according to claim 36, wherein the inflammatory disease is a chronic inflammatory disease.

38. The bispecific protein, immunoconjugate, or pharmaceutical composition for use as claimed in claim 36, wherein the inflammatory disease is a mast cell driven disease.

39. The bispecific protein, immunoconjugate, or pharmaceutical composition for use as claimed in claim 36, wherein the inflammatory disease is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis or eosinophilic esophagitis.

40. The bispecific protein, immunoconjugate, or pharmaceutical composition for use according to claim 36, wherein the neoplasm is a mast cell driven neoplasm.

41. The bispecific protein, immunoconjugate, or pharmaceutical composition for use according to claim 36, wherein the neoplasm is systemic mastocytosis or mast cell leukemia.

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