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 and methods of producing such molecules, as well as related pharmaceutical compositions, nucleic acid molecules, expression vectors, and host cells.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 393,036, filed July 28, 2022, the disclosure of which is incorporated herein by reference in its entirety.

[0002] Electronic Sequence Listing Reference The contents of the electronic sequence listing (UHFL_002_01WO_SeqList_ST26.xml, size: 71,462 bytes, created on: July 24, 2023) are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to bispecific antigen-binding molecules and therapeutic uses of such molecules. [Background technology]

[0004] Granulocytes (e.g., mast cells and basophils) are central contributors to the pathology of many chronic inflammatory diseases. Existing approaches to treat such diseases target the mast cell survival receptor c-Kit. While this approach has been clinically validated to effectively deplete mast cells and ameliorate associated inflammation, indiscriminate c-Kit inhibition can result in on-target, dose-limiting myelosuppression. These adverse events are difficult to avoid due to the high-affinity target binding facilitated by bivalent binding with standard antibodies. Granulocyte-selective drugs are lacking, remaining a significant unmet medical need for patients suffering from mast cell-driven diseases. Summary of the Invention

[0005] 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 and fourth polypeptide chains comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specific binding to c-Kit and CD203c, respectively.

[0006] Provided herein is an anti-c-Kit and anti-CD203c bispecific protein, the bispecific protein according to claim 1, wherein the anti-c-Kit VH domain comprises a heavy chain complementarity-determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain complementarity-determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain complementarity-determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 4; the anti-c-CD203c VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 73; and the VL domain comprises a light chain complementarity-determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 6, a light chain complementarity-determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 7, and a light chain complementarity-determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 8.

[0007] Provided herein is an anti-c-Kit and anti-CD203c bispecific protein, wherein the anti-c-Kit VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 4; the anti-c-CD203c VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and the VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0008] Provided herein is an anti-c-Kit and anti-CD203c bispecific protein, wherein the anti-c-Kit VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 4; the anti-c-CD203c VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and the VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an 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 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.

[0010] 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.

[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: 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.

[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 any one of SEQ ID NOs: 16 to 51, 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 any one of SEQ ID NOs: 63 to 67, the anti-c-CD203c VH domain comprises the amino acid sequence of any one of SEQ ID NOs: 68 to 72, and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0014] 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.

[0015] In some embodiments, the immunoglobulin constant region is an immunologically inert constant region.

[0016] 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 the first immunoglobulin constant region and the second immunoglobulin constant region comprise knobs-in-holes mutations.

[0017] In some embodiments, the first immunoglobulin constant region comprises a CH3 domain comprising the amino acid substitutions S354C and T366W, and the second immunoglobulin constant region comprises a CH3 domain comprising the amino acid substitutions Y349C, T366S, L368A, and Y407V, where numbering is according to the EU index as in Kabat.

[0018] In some embodiments, the first immunoglobulin constant region comprises a CH3 domain comprising the amino acid substitutions Y349C, T366S, L368A, and Y407V, and the second immunoglobulin constant region comprises a CH3 domain comprising the amino acid substitutions S354C and T366W, where numbering is according to the EU index as in Kabat.

[0019] Provided herein are bispecific proteins, wherein (a) a first immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO: 52 and a second immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO: 53, or (b) a first immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO: 53 and a second immunoglobulin constant region comprises the amino acid sequence of SEQ ID NO: 52.

[0020] Provided herein are bispecific proteins that bind to c-Kit and CD203c, where the c-Kit and CD203c are present on the surface of the same cell.

[0021] In some embodiments, the bispecific protein is a designed ankyrin repeat protein (DARPin), a tandem VHH, or a tandem immunoglobulin neoantigen receptor (IgNAR).

[0022] 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 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.

[0023] Provided herein are pharmaceutical compositions comprising a bispecific protein or immunoconjugate disclosed herein and a pharmaceutically acceptable carrier, diluent, or excipient.

[0024] 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.

[0025] Provided herein are expression vectors comprising the nucleic acid molecules disclosed herein.

[0026] Provided herein are recombinant host cells comprising the nucleic acid molecules or expression vectors disclosed herein.

[0027] Provided herein are methods of producing a bispecific protein, comprising culturing a recombinant host cell disclosed herein under conditions to express the nucleic acid molecules, thereby producing the protein, and isolating the protein from the host cell or culture.

[0028] Provided herein are methods of treating an inflammatory disease or neoplasm in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a bispecific protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0029] Provided herein are methods for ameliorating the symptoms of an inflammatory disease or neoplasia in a subject in need thereof, the methods comprising administering to the subject a therapeutically effective amount of a bispecific protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0030] 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-induced 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.

[0031] In some embodiments of the methods provided herein, the neoplasm is a mast cell-induced neoplasm. In some embodiments of the methods provided herein, the neoplasm is systemic mastocytosis or mast cell leukemia.

[0032] Provided herein is a bispecific protein, immunoconjugate, or pharmaceutical composition disclosed herein for use as a medicament.

[0033] Provided herein is a bispecific protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in treating an inflammatory disease or neoplasm.

[0034] 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-induced 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.

[0035] In some embodiments of the uses disclosed herein, the neoplasm is a mast cell-induced neoplasm. In some embodiments of the uses disclosed herein, the neoplasm is systemic mastocytosis or mast cell leukemia. [Brief explanation of the drawings]

[0036] [Figure 1] Figure 1 shows 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 (HSCs). Without the stabilization of binding provided by co-binding to CD203c, monovalent binding is transient, resulting in low c-Kit inhibitory potency that cannot effectively compete with the high-affinity binding of endogenous dimeric SCF ligands. However, in dual receptor-expressing cells, bivalent binding can occur, SCF can no longer compete for receptor occupancy, and c-Kit signaling is eliminated. [Figure 2] A schematic diagram of the knobs-in-holes bispecific format is shown. A common light chain bispecific format was used for dual c-Kit / CD203c targeting to allow for streamlined production and purification. This is combined with knobs-in-holes mutations of engineered cysteine residues in the CH3 Fc domain to direct heterodimeric heavy chain pairing. Exemplary common light chain and heterodimeric heavy chain sequences are shown in Tables 1-3. [Figure 3] Figures 3A-3C show the results of characterization of purified IgG with the common light chain (CLC). Purified anti-CD203c IgG null antibody with the anti-c-kit MH1 light chain was titrated (nM) in a direct-linked enzyme-linked immunosorbent assay (ELISA) against purified recombinant human (Figure 3A) and rhesus monkey (Figure 3B) CD203c proteins. Subsequently, 1.27 was titrated in a flow-based assay against the basophilic cell line KU812, which expresses CD203c on its cell surface (Figure 3C). [Figure 4] Figure 1 shows the design of the 1.27 heavy chain CDR1 and CDR2 variant optimized library. The heavy chain amino acid sequence of the parent clone 1.27 is shown. The CDR1 and CDR2 sequences are shown in gray. This CDR definition used throughout this document is expanded compared to the classic Kabat nomenclature. The CDR positions targeted by mutagenesis are highlighted in brackets. The table shows that each target position was replaced with any other amino acid (except cysteine) and that all potential single and double mutations were sampled. [Figure 5] Figure 1 shows the design of the 1.27 heavy chain CDR3 variant optimized library. The heavy chain amino acid sequence of the parent clone 1.27 is shown. The CDR3 sequence is shown in gray, and the CDR3 positions targeted for mutagenesis are highlighted in brackets. The table indicates that each targeted position was replaced with any other amino acid (except cysteine) and that all potential single and double mutations were sampled. [Figure 6] Figures 6A-6C show the results of characterization of antibody variants from the 1.27-optimized library. Antibody variants isolated from the 1.27-optimized library were compared to the parental 1.27 clone for binding to human (Figure 6A) and rhesus monkey (Figure 6B) CD203c by direct binding ELISA and for binding to KU812 cells by a flow-based assay (Figure 6C). [Figure 7] Figure 1 shows the results of a binding assay of bispecific CLCs to KU812 cells. Bispecific molecules composed of anti-c-Kit MH1 and anti-CD203c 1.27 (parent) or anti-CD203c F6 (affinity-optimized) were compared with anti-c-Kit IgG, anti-c-Kit one-armed antibody, and anti-CD203c IgG for binding to KU812 cells. The bispecific molecules had a similar EC50 to anti-c-Kit IgG but a much higher Bmax due to cooperative binding of both arms to dual target-expressing cells. [Figure 8] 1 shows the results of an assay for inhibition of c-Kit receptor phosphorylation by the F6 / MH1 bispecific. The F6 / MH1 bispecific molecule showed potent inhibition of c-Kit receptor phosphorylation when compared to the one-arm MH1 c-Kit antibody. [Figure 9]Figures 9A and 9B show the results of binding assays using an alternative common light chain containing CDRs grafted from MH1 paired with the CD203c VH domain. The results indicate that not all common light chain / heavy chain pairs retain dual target binding. An alternative common light chain containing CDRs grafted from MH1 paired with the CD203c VH domain retains c-Kit binding (Figure 9A) but loses CD203c (ENPP3) binding (Figure 9B). [Figure 10] 1 shows the results of an ELISA comparing the binding of the F6 / MH1 bispecific molecule to human and cynomolgus monkey CD203c. The results demonstrate that the F6 / MH1 bispecific molecule retains equivalent binding to both human and cynomolgus monkey CD203c. [Figure 11A]

[0049] Figure 11 shows results from SPR characterization of affinity-optimized CD203c VH binding domains. The parent anti-CD203c binding domain 1.27 (Figure 11A), and affinity-optimized variants F6 (Figure 11B) and F6.12 (Figure 11C) were prepared as Fabs and comparatively evaluated for affinity to human and cynomolgus CD203c using single-cycle kinetics. The results are summarized in the accompanying table (Figure 11D). [Figure 11B]

[0049] Figure 11 shows results from SPR characterization of affinity-optimized CD203c VH binding domains. The parent anti-CD203c binding domain 1.27 (Figure 11A), and affinity-optimized variants F6 (Figure 11B) and F6.12 (Figure 11C) were prepared as Fabs and comparatively evaluated for affinity to human and cynomolgus CD203c using single-cycle kinetics. The results are summarized in the accompanying table (Figure 11D). [Figure 11C]

[0049] Figure 11 shows results from SPR characterization of affinity-optimized CD203c VH binding domains. The parent anti-CD203c binding domain 1.27 (Figure 11A), and affinity-optimized variants F6 (Figure 11B) and F6.12 (Figure 11C) were prepared as Fabs and comparatively evaluated for affinity to human and cynomolgus CD203c using single-cycle kinetics. The results are summarized in the accompanying table (Figure 11D). [Figure 11D]

[0049] Figure 11 shows results from SPR characterization of affinity-optimized CD203c VH binding domains. The parent anti-CD203c binding domain 1.27 (Figure 11A), and affinity-optimized variants F6 (Figure 11B) and F6.12 (Figure 11C) were prepared as Fabs and comparatively evaluated for affinity to human and cynomolgus CD203c using single-cycle kinetics. The results are summarized in the accompanying table (Figure 11D). [Figure 12] Figures 12A and 12B show the results of an analysis of the binding of affinity-optimized CD203c VH binding domains to KU812 cells. The parental anti-CD203c binding domain 1.27 (Figure 12A) was produced as an IgG and compared to the binding of affinity-optimized F6 IgG (Figure 12B) to KU812 cells. [Figure 13] Figure 1 shows substitution mutations generated from analysis of pI engineering of anti-c-Kit and anti-CD203c VH domains. The table summarizes the top five double mutant combinations selected for engineering the MH1 VH (anti-c-Kit) and F6.12 VH (anti-CD203c) domains to maximize the pI difference between the heavy chains. [Figure 14] Preparative CEX (cation exchange) and analytical SEC (size exclusion) chromatograms of the MH1_A / F6.12_B bispecific are shown. 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. Samples were also analyzed by SEC for monomeric purity. [Figure 15] Preparative CEX and analytical SEC chromatograms of MH1_C / F6.12_D bispecific are shown. 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. Samples were also analyzed by SEC for monomeric purity. [Figure 16] 1 shows the results of a comparison of c-Kit target binding before and after pI engineering using SPR. A single-cycle kinetic protocol was used to compare bispecific binding to human and cynomolgus c-Kit before (MH1 / F6.12) and after (MH1_C / F6.12_D) heavy chain pI engineering. [Figure 17-1]

[0033] Figure 1 shows the results of comparing 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. [Figure 17-2]

[0033] Figure 1 shows the results of comparing 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. [Figure 18] The results of a comparison of dual target binding before and after pI engineering using SPR are shown. The table summarizes kinetic data obtained using a single-cycle kinetic protocol to compare human and cynomolgus c-Kit and human and cynomolgus CD203c binding to several 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). [Figure 19] Figures 19A-19C show the results of a comparison of binding to KU812 cells before and after pI engineering using flow cytometry. Binding to dual targets expressed in KU812 cells was compared for several bispecific constructs before (MH1 / F6.12) (Figure 19A) and after (MH1_C / F6.12_B (Figure 19B) and MH1_C / F6.12_D (Figure 19C)) pI engineering. DETAILED DESCRIPTION OF THE INVENTION

[0037] Both IgE-dependent and IgE-independent activation of mast cells and basophils has been shown to result in robust upregulation of cell surface CD203c. CD203c is specific to the mast cell / basophil lineage and, unlike c-Kit, is not expressed on other hematopoietic stem cell precursors. CD203c has been shown to direct differentiation of erythroid and granulocyte lineages, and thus CD203c-mediated targeting of granulocytes may circumvent bone marrow suppression. Anti-c-Kit / anti-CD203c bispecific antibodies may maintain the efficacy of c-Kit inhibition-mediated mast cell depletion, but only on cells co-expressing activating CD203c. This is shown schematically in Figure 1.

[0038] Advances in molecular engineering have enabled the development of bispecific molecules that allow for single-drug administration but involve significant manufacturing complexity. Problems with the correct pairing of both light chains can result in significant heterogeneity, requiring additional downstream processing. This can be overcome by producing the individual antibodies separately and forming the bispecific 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, offering the advantage of streamlined downstream processing.

[0039] protein molecules Provided herein are bispecific proteins that bind c-Kit and CD203c in cis, i.e., the c-Kit and CD203c molecules are present on the surface of the same cell.

[0040] 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 and fourth polypeptide chains comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specific binding to c-Kit and CD203c, respectively.

[0041] 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.

[0042] In some embodiments, the protein comprises one or more amino acid sequences presented in Tables 1-7.

[0043] [Table 1]

[0044] [Table 2]

[0045] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0046] [Table 4]

[0047] The anti-c-Kit / anti-CD203c protein design may be based on sequences derived from IgG1, IgG2, IgG3, IgG4, IgE, IgM, or IgA, and may or may not have effector function capabilities.

[0048] In some embodiments, the proteins disclosed herein comprise domains and regions of an antibody molecule. The term "antibody" broadly refers to an immunoglobulin (Ig) molecule, generally comprising four polypeptide chains: two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof that retains the essential target-binding characteristics of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

[0049] In full-length antibodies, 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. IgG, IgA, and IgD constant regions comprise a flexible hinge region between the CH1 and CH2 domains. 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 regions of hypervariability called complementarity-determining regions (CDRs), which are interspersed with more conserved regions called framework regions (FRs). The VH and VL domains each comprise three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0050] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" may be a native-sequence Fc region or a variant Fc region. Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is usually defined to stretch from the amino acid residue at position Cys226 or Pro230 to the carboxyl terminus. The numbering of residues in the Fc region is according to the EU index as in Kabat. The Fc region of an immunoglobulin generally contains two constant domains, CH2 and CH3. The Fc region may exist in a dimeric or monomeric form. The Fc region binds to various cellular receptors, e.g., Fc receptors, and other immune molecules, e.g., complement proteins. In some embodiments, the bispecific proteins provided herein comprise an Fc region.

[0051] 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 generally contain two identical heavy chains and two identical light chains, and two antigen-binding domains, each composed of a VH and a VL. IgA antibodies generally consist of two monomers, each composed of two heavy chains and two light chains (as in IgG, IgD, and IgE antibodies); in this configuration, the IgA molecule has four antigen-binding domains, each also composed of a VH and a VL. Certain IgA antibodies are monomers composed of two heavy chains and two light chains. Secretory IgM antibodies are generally composed of five monomers, each of which consists of two heavy chains and two light chains (similar to IgG and IgE antibodies). Thus, an IgM molecule has 10 antigen-binding domains, each of which also consists of a VH and a VL. Cell-surface IgM has a two heavy chain / two light chain structure similar to IgG, IgD, and IgE antibodies.

[0052] As used herein, the terms "immunological binding" and "immunological binding properties" refer to the types of non-covalent interactions that occur between an immunoglobulin molecule (e.g., an antibody or antigen-binding portion thereof), or a protein containing an immunoglobulin-derived binding domain(s), and an antigen for which the immunoglobulin or protein is specific. The strength or affinity of an immunological binding interaction is determined by the dissociation constant (K d ) and can be expressed in units of smaller K d indicates a greater affinity. The immunological binding properties of selected polypeptides can be quantified using methods well known in the art. One such method involves measuring the rates of formation and dissociation of antigen-binding site / antigen complexes, which depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that affect the rates in both directions equally. Thus, the "on rate constant" (Kon ) and "off rate constant" (K off ) can be determined by calculating the concentration and the actual rates of association and dissociation. (See Malmqvist, Nature 361:186-187 (1993)). K off / K on The ratio of α to β allows for the cancellation of all parameters not related to affinity and gives the dissociation constant K d (See Davies et al. (1990) Annual Rev Biochem 59:439-473.) The antibodies or antigen-binding portions provided herein have an equilibrium binding constant (K d An antibody is said to specifically bind to PD-L1 or CD3 when its K is ≦10 μM, preferably ≦10 nM, more preferably ≦10 nM, and most preferably ≦100 pM to about 1 pM. d One way to measure is by using surface plasmon resonance (SPR), usually a biosensor system such as a Biacore® system.

[0053] Functionally, the binding affinity of the proteins provided herein is 10 -5 M~10 -12 For example, the binding affinity of the proteins provided herein can be in the range of 10 -6 M~10 -12 M, 10 -7 M~10 -12 M, 10 -8 M~10 -12 M, 10 -9 M~10 -12 M, 10 -5 M~10 -11 M, 10 -6 M~10 -11 M, 10 -7 M~10 -11 M, 10 -8 M~10 -11 M, 10 -9 M~10 -11M、10 -10 M~10 -11 M、10 -5 M~10 -10 M、10 -6 M~10 -10 M、10 -7 M~10 -10 M、10 -8 M~10 -10 M、10 -9 M~10 -10 M、10 -5 M~10 -9 M、10 -6 M~10 -9 M、10 -7 M~10 -9 M、10 -8 M~10 -9 M、10 -5 M~10 -8 M、10 -6 M~10 -8 M、10 -7 M~10 -8 M、10 -5 M~10 -7 M、10 -6 M~10 -7 Mまたは10 -5 M~10 -6 Mである。

[0054] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c, and the anti-c-Kit VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 4; The VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 73, and the VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0055] 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 and fourth polypeptide chains 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 HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 4; the anti-c-CD203c VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and the VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0056] 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 and fourth polypeptide chains 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 HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 4; the anti-c-CD203c VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and the VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0057] [Table 5]

[0058] [Table 6]

[0059] [Table 7]

[0060] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, and 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.

[0061] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, and 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; and The 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: 5.

[0062] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, and 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.

[0063] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, respectively, and 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; and The 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:5.

[0064] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, and 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.

[0065] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c, respectively, and 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; and The 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: 5.

[0066] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, and 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.

[0067] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c, respectively, and 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; and The 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:5.

[0068] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, and 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 to 51, and the VL domain comprises the amino acid sequence of SEQ ID NO: 5.

[0069] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a light chain variable VL domain capable of specifically binding to c-Kit and CD203c, and 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.

[0070] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, and 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.

[0071] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, and 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.

[0072] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c, and 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.

[0073] 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 and fourth polypeptide chains each comprise an immunoglobulin light chain comprising a VL domain capable of specifically binding to c-Kit and CD203c, and 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.

[0074] In some embodiments, the immunoglobulin heavy chain comprises an amino acid sequence provided herein (e.g., in Tables 1, 3, or 5-7) with one, two, or three conservative amino acid substitutions. In some embodiments, the immunoglobulin light chain comprises an amino acid sequence provided herein (e.g., in Table 2 or Table 7) with one, two, or three conservative amino acid substitutions. In some embodiments, conservative amino acid substitutions are made only in the FR sequences and not in the CDR sequences.

[0075] In some embodiments, the bispecific protein comprises the anti-c-Kit VH domain MH1_C (or the corresponding CDR sequences), the anti-c-CD203c VH domain F6.12_D (or the corresponding CDR sequences), and the VL domain MH1 (or the corresponding CDR sequences).

[0076] In some embodiments, the bispecific protein comprises the anti-c-Kit VH domain MHI (or the corresponding CDR sequences), the anti-c-CD203c VH domain 1.27 (or the corresponding CDR sequences), and the VL domain MHI (or the corresponding CDR sequences).

[0077] In some embodiments, the bispecific protein comprises the anti-c-Kit VH domain MH1 (or corresponding CDR sequences), the anti-c-CD203c VH domain F6 (or corresponding CDR sequences), and the VL domain MH1 (or corresponding CDR sequences).

[0078] In some embodiments, the bispecific protein comprises the anti-c-Kit VH domain MH1 (or corresponding CDR sequences), the anti-c-CD203c VH domain F6.12 (or corresponding CDR sequences), and the VL domain MH1 (or corresponding CDR sequences).

[0079] In some embodiments, the bispecific proteins provided herein comprise an immunoglobulin heavy chain constant region at the C-terminus of the 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 the amino acid substitutions L234A, L235A, and G237A, a human IgG1 constant region comprising the amino acid substitutions L234A, L235A, G237A, and P331S, or a human IgG4 constant region comprising the amino acid substitution S228P, numbering according to the EU index as in Kabat. In some embodiments, amino acid residue positions 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).

[0080] 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, wherein the first immunoglobulin constant region and the second immunoglobulin constant region comprise knobs-in-holes mutations. In some embodiments, the first immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions S354C and T366W, and the second immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions Y349C, T366S, L368A, and Y407V, numbering 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 the second immunoglobulin constant region comprises a CH3 domain comprising amino acid substitutions S354C and T366W, numbering 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.

[0081] In some embodiments, the proteins provided herein may comprise any heterodimerization mutation or heterodimerization technique. In some embodiments, the proteins provided herein may not comprise any heterodimerization mutation or heterodimerization technique. In such embodiments, purification techniques may be used to isolate the protein.

[0082] In some embodiments, the proteins 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. RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 62)

[0083] In some embodiments, the proteins provided herein can comprise an immunoglobulin light chain constant region that is a lambda light chain.

[0084] 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 neoantigen receptors (IgNARs).

[0085] 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 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.

[0086] Examples of suitable therapeutic agents include, but are not limited to, immunomodulators, cytotoxins, radioisotopes, chemotherapeutic agents, antiangiogenic agents, antiproliferative agents, proapoptotic agents, and cytostatic and cytolytic enzymes (e.g., RNAses). Additional therapeutic agents include therapeutic nucleic acids, e.g., genes encoding immunomodulators, antiangiogenic agents, antiproliferative agents, or proapoptotic agents. These drug descriptive names are not mutually exclusive, and thus, therapeutic agents may be described using one or more of the above terms.

[0087] Examples of suitable therapeutic agents for use in immunoconjugates include, but are not limited to, JAK kinase inhibitors, taxanes, maytansines, CC-1065 and duocarmycins, calicheamicins and other enediynes, and auristatins. Other examples include antifolates, vinca alkaloids, and anthracyclines. Plant toxins, other biologically active proteins, enzymes (i.e., ADEPT), radioisotopes, and photosensitizers may also be used in immunoconjugates. Additionally, conjugates can be made using secondary carriers, such as liposomes or polymers, as cytotoxic agents. Suitable cytotoxins include agents that inhibit or block cellular function and / or cause cell destruction. Representative cytotoxins include antibiotics, inhibitors of tubulin polymerization, alkylating agents that bind to and destroy DNA, and agents that disrupt protein synthesis or the function of essential cellular proteins, such as protein kinases, phosphatases, topoisomerases, enzymes, and cyclins.

[0088] Representative cytotoxins include doxorubicin, daunorubicin, idarubicin, aclarubicin, zorubicin, mitoxantrone, epirubicin, carubicin, nogalamycin, menogaril, pitarubicin, valrubicin, cytarabine, gemcitabine, trifluridine, ancitabine, enocitabine, azacitidine, doxifluhidine, pentostatin, broxuridine, capecitabine, cladribine, decitabine, floxuridine, fludarabine, gougerotin, puromycin, tegafur, tiazofurin, adriamycin, and citabine. These include, but are not limited to, suplatin, carboplatin, cyclophosphamide, dacarbazine, vinblastine, vincristine, mitoxantrone, bleomycin, mechlorethamine, prednisone, procarbazine, methotrexate, flurouracil, etoposide, taxol, taxol analogs, platins such as cisplatin and carboplatin, mitomycin, thiotepa, taxanes, vincristine, daunorubicin, epirubicin, actinomycin, authamycin, azaserine, bleomycin, tamoxifen, idarubicin, dolastatins / auristatins, hemiasterin, esperamicin, and maytansinoids.

[0089] Suitable immunomodulatory agents include antihormonal drugs that block hormone action on tumors, and immunosuppressants that suppress cytokine production, downregulate self-antigen expression, or mask MHC antigens.

[0090] Pharmaceutical Composition The bispecific proteins provided herein (also referred to herein as "active compounds") can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include the protein (or an immunoconjugate comprising the protein) and a pharmaceutically acceptable carrier, diluent, or excipient. Such materials should be non-toxic and should not interfere with the effectiveness of the protein. The precise nature of the carrier or other material will depend on the route of administration, as discussed below, which may be by injection, bolus, infusion, or any other suitable route.

[0091] As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that generally do not produce allergic or other serious adverse reactions when administered using routes well known in the art. Molecular entities and compositions that have been approved by a regulatory agency of the U.S. federal or state government or are listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans, 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 and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field (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 vehicles, such as fixed oils, may also be used. The use of such media and agents for pharmaceutical active substances is known in the art. Any conventional media or agent is contemplated for use in the composition, provided that it is not incompatible with the active compound. Supplementary active compounds can also be incorporated into the composition. A pharmaceutically acceptable carrier, diluent, or excipient is a compound or a combination of compounds that does not induce secondary reactions and can, for example, facilitate the administration of a protein, increase its shelf life and / or its effectiveness in the body, or increase its solubility in solution.

[0092] The pharmaceutical compositions disclosed herein can be formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent, e.g., water for injection, saline solution, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent, e.g., benzyl alcohol or methylparaben; an antioxidant, e.g., ascorbic acid or sodium bisulfate; a chelating agent, e.g., ethylenediaminetetraacetic acid (EDTA); a buffer, e.g., acetate, citrate, or phosphate, and an agent for adjusting isotonicity, e.g., sodium chloride or dextrose. pH can be adjusted using acids or bases, e.g., hydrochloric acid or sodium hydroxide. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple-dose vials made of glass or plastic.

[0093] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL® (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). In some cases, the composition is sterile and fluid to the extent that easy syringability exists. In some cases, the composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of microbial action can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it will be preferable to include isotonic agents, polyalcohols, for example, sugars, mannitol, sorbitol, sodium chloride in the composition. Prolonged absorption of injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin.

[0094] Sterile injectable solution can be prepared by incorporating the required amount of active compound into suitable solvent with one or combination of the above-listed components as needed, and then sterilize by filtration.Generally, dispersion is prepared by incorporating active compound into sterile vehicle that contains basic dispersion medium and other components required from above-listed.For the sterile powder that is used to prepare sterile injectable solution, preparation method is vacuum drying and freeze-drying, by which powder of active ingredient and any additional desired component can be obtained from the solution that has been previously sterilized and filtered.

[0095] Oral compositions generally contain an inert diluent or an edible carrier. They can be sealed in gelatin capsules or compressed into tablets. For oral therapeutic administration, the active compound can be incorporated with an excipient and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, in which the compound in the fluid carrier is orally administered, swirled in the mouth, and expectorated or swallowed. Pharmaceutically compatible binding agents and / or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primojel®, or corn starch; a lubricant such as magnesium stearate; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0096] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0097] Systemic administration can also be via transmucosal or transdermal means. For transmucosal or transdermal administration, a penetrant appropriate to the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be achieved by using nasal sprays or suppositories. For transdermal administration, the active compound is formulated into ointments, salves, gels, or creams as generally known in the art.

[0098] The medications can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0099] In some embodiments, the active compounds are prepared using carriers that protect the compounds from rapid elimination from the body, such as controlled-release formulations, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations will be apparent to those skilled in the art. Materials can also be commercially available. Liposomal suspensions can also be used as pharmaceutically acceptable carriers.

[0100] For ease of administration and uniform dosage, it is particularly advantageous to formulate oral or parenteral compositions in dosage unit form.As used herein, dosage unit form refers to a physically discrete unit suitable as a unit dose for the subject to be treated, and each unit contains a predetermined amount of active compound calculated to produce desired therapeutic effect in combination with required pharmaceutical carrier.The specification of dosage unit form of the present invention is determined by and directly depends on the inherent characteristics of active compound and the specific therapeutic effect to be achieved, and the inherent constraints in the technical field of compounding such active compound for treating individuals.

[0101] In some embodiments, the protein can be provided in lyophilized form for reconstitution prior to administration, for example, lyophilized antibody molecules can be reconstituted in sterile water and mixed with saline prior to administration to an individual.

[0102] The pharmaceutical compositions provided herein can be included in a container, pack, or dispenser together with instructions for administration.

[0103] Nucleic acid molecules, vectors, host cells and methods for producing proteins Provided herein are nucleic acid molecules encoding the bispecific proteins disclosed herein. Provided herein are nucleic acid molecules (e.g., isolated nucleic acid molecules) encoding the first polypeptide chain, second polypeptide chain, third polypeptide chain, and fourth polypeptide chain (or the amino acid sequence of (i) the VH domain, (ii) the VL domain, or both the VH domain and VL domain of a protein) of the bispecific proteins disclosed herein. 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 encodes a leader peptide).

[0104] Also provided herein are expression vectors containing the nucleic acid molecules described herein. In certain vectors, the nucleic acid molecule is operably linked to one or more control sequences suitable for expression of the nucleic acid segment in a host cell. In some cases, the expression vector includes a sequence that mediates replication and includes one or more selectable markers. As used herein, "vector" refers to a construct that can deliver, and preferably express, one or more gene(s) or sequence(s) of interest into a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid vectors, cosmid vectors, or phage vectors, DNA or RNA expression vectors bound to cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.

[0105] Provided herein are recombinant host cells comprising the expression vectors or nucleic acid molecules disclosed herein. A "host cell" includes an individual cell, cell line, or cell culture that can be or has been the recipient of a vector(s) for incorporation of a polynucleotide insert. A host cell includes the progeny of a single host cell. The progeny may not necessarily be completely identical (in morphology or genomic DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation. Expression vectors can be transfected into host cells by standard techniques. Non-limiting examples include electroporation, calcium phosphate precipitation, DEAE-dextran transfection, and the like. In some embodiments, the recombinant host cell comprises a single vector or a single nucleic acid molecule encoding the first, second, third, and fourth polypeptide chains of a bispecific protein disclosed herein. In some embodiments, the recombinant host cell comprises multiple vectors or multiple nucleic acid molecules encoding the first, second, third, and fourth polypeptide chains of a bispecific protein disclosed herein.

[0106] The protein molecules of the present invention, or portions thereof, can be produced using techniques known in the art, such as recombinant techniques, phage display techniques, synthetic techniques, computational techniques, or a combination of such techniques, or other techniques readily known in the art.

[0107] 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 to express the nucleic acid molecule, 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 to express the nucleic acid molecule, thereby producing the protein, and isolating the protein from the host cell or culture.

[0108] The proteins disclosed herein can be produced by any of a variety of methods known to those of skill in the art. In certain embodiments, the proteins disclosed herein can be recombinantly produced. For example, nucleic acid sequences encoding one or more heavy or light chains or portions thereof provided herein can be introduced into bacterial cells (e.g., E. coli, B. subtilis) or eukaryotic cells (e.g., yeast such as 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 into an in vitro translation system, and the translated polypeptides can be isolated. In some embodiments, the light and heavy chain proteins are produced intracellularly with signal sequences that are removed during production of the mature proteins disclosed herein.

[0109] One skilled in the art would be able to determine whether a protein comprising a given polypeptide sequence binds to c-Kit protein and / or CD203c protein using standard methods, e.g., Western blot, ELISA, etc.

[0110] Medical Uses of Bispecific Proteins Provided herein are methods and uses of the bispecific proteins, immunoconjugates, and pharmaceutical compositions disclosed herein for providing a therapeutic benefit to a subject having an inflammatory disease. Provided herein are methods and uses of the bispecific proteins, immunoconjugates, and pharmaceutical compositions disclosed herein for providing a therapeutic benefit to a subject having a neoplasia.

[0111] The activatable proteins, immunoconjugates, or pharmaceutical compositions disclosed herein can be used in methods of treatment of the human or animal body, including prophylactic or preventative treatment (e.g., treatment before the onset of a condition in a subject, to reduce the risk of the condition occurring in the subject, delay its onset, or reduce its severity after onset). The method of treatment can include administering the protein, immunoconjugate, or pharmaceutical composition to a subject in need thereof.

[0112] Provided herein are methods of treating an inflammatory disease or neoplasm in a subject, the methods comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein. Provided herein are bispecific proteins, immunoconjugates, or pharmaceutical compositions disclosed herein for use in treating an inflammatory disease or neoplasm.

[0113] Provided herein are methods for ameliorating symptoms of an inflammatory disease or neoplasia in a subject, the methods comprising administering to the subject a therapeutically effective amount of a protein, immunoconjugate, or pharmaceutical composition disclosed herein.

[0114] Provided herein is a bispecific protein, immunoconjugate, or pharmaceutical composition disclosed herein for use as a medicament.

[0115] 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-induced 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.

[0116] In some embodiments of the methods and uses disclosed herein, the neoplasm is a mast cell-induced neoplasm. In some embodiments of the methods and uses disclosed herein, the neoplasm is systemic mastocytosis or mast cell leukemia.

[0117] As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of a pharmaceutical agent, e.g., a protein, immunoconjugate, or pharmaceutical composition disclosed herein, sufficient to reduce or ameliorate the severity and / or duration of an inflammatory disease or neoplasm or one or more symptoms thereof, prevent the progression of the disease, cause regression of the disease, prevent the recurrence, onset, development, or progression of one or more symptoms associated with the disease, or enhance or improve the prophylactic or therapeutic effect(s) of another related therapeutic agent (e.g., a prophylactic or therapeutic agent) for an inflammatory disease or neoplasm.

[0118] The actual amount administered, as well as the rate and time course of administration, will vary depending on the nature and severity of what is being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the composition, the method of administration, the dosing schedule, and other factors known to medical professionals. Decisions regarding treatment prescription, e.g., dosage, etc., are within the responsibility of general practitioners and other physicians and may vary depending on the severity of the symptoms and / or progression of the disease being treated. Appropriate dosages for 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). Specific dosages may be provided herein, or specific dosages in the Physician's Desk Reference (2003) may be used, as appropriate, depending on the type of drug being administered. The therapeutically effective amount or suitable dosage of an antibody-based protein molecule can be determined by comparing its in vitro and in vivo activity in animal models. Methods for extrapolating effective dosages in mice and other test animals to humans are known. The exact dosage will depend on several factors, such as whether the antibody-based protein is for prophylaxis 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.

[0119] Typical protein doses range from 100 μg to 1 g for systemic administration and 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 an IgG1 or IgG4 isotype. Doses for single treatment of adult subjects may be adjusted proportionally for children and infants. At the physician's discretion, treatment may be repeated at daily, twice-weekly, weekly, or monthly intervals. A subject's treatment schedule may vary depending on the pharmacokinetic and pharmacodynamic properties of the protein composition, the route of administration, and the nature of the condition being treated.

[0120] Treatment may be administered periodically, with the period between administrations being about 2 weeks or more, e.g., about 3 weeks or more, about 4 weeks or more, about once a month or more, about 5 weeks or more, or about 6 weeks or more. For example, treatment may be administered every 2-4 weeks or every 4-8 weeks. Treatment may be administered before and / or after surgery and / or directly at the anatomical site where the surgical or invasive procedure will be performed. Suitable formulations and routes of administration are described above.

[0121] In some embodiments, a protein, immunoconjugate, or pharmaceutical composition disclosed herein can be administered by subcutaneous injection, for example, using an autoinjector for long-term prevention / treatment.

[0122] 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 administration of a protein, immunoconjugate, or pharmaceutical composition disclosed herein may last for 1 month or more, 2 months or more, 3 months or more, 4 months or more, 5 months or more, or 6 months or more in a subject.

[0123] In some embodiments, a subject may be treated with a protein, immunoconjugate, or pharmaceutical composition disclosed herein and an additional therapeutic agent or therapy used to treat a symptom or complication of cancer or an inflammatory disease or neoplasia. The protein, immunoconjugate, or pharmaceutical composition disclosed herein and the additional therapeutic agent or therapy may be administered simultaneously or sequentially.

[0124] In some embodiments, the subject is a human, a non-human primate, a pig, a horse, a cow, a dog, a cat, a guinea pig, a mouse, or a rat. In some embodiments, the subject is an adult human. In some embodiments, the subject is a pediatric human.

[0125] Further provided herein is a protein, immunoconjugate, or pharmaceutical composition disclosed herein for use in the treatment of a disease or disorder.

[0126] Provided herein is a protein, immunoconjugate, or pharmaceutical composition disclosed herein for use as a medicament.

[0127] definition Unless otherwise indicated, terms used herein have definitions commonly used in the art. Some terms are defined below, and additional definitions may be found in the remainder of the detailed description.

[0128] The term "a" or "an" can refer to one or more of that entity, i.e., to a plurality of referents. Thus, the terms "a," "an," "one or more," and "at least one" are used interchangeably herein. In addition, reference to "an element" with the indefinite article "a" or "an" does not exclude the possibility that there are more than one of that element, unless the context clearly requires that there be one, and only one, of that element.

[0129] Unless otherwise specified or clear from the context, the term "about" means within 10% of the reported numerical value (except where such number is greater than 100% or less than 0% of the possible values). When used in conjunction with a range or series of values, the term "about" applies to the endpoints of that range or each recited value in the series, unless otherwise indicated. As used in this application, the terms "about" and "approximately" are used as equivalent terms.

[0130] As used herein, the term "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant across a window of residue, e.g., nucleotide or amino acid, alignment. The "percent identity" for an aligned segment of a test sequence and a reference sequence is the number of identical residues shared by the two aligned sequences divided by the total number of residues in the reference sequence segment, i.e., the entire reference sequence or a smaller, defined portion of the reference sequence. The "percent identity" is the percent identity multiplied by 100. The percent identity can be calculated using the alignment program Clustal Omega, available at ebi.ac.uk / Tools / msa / clustalo, with default parameters. See Sievers et al., "Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega" (2011 October 11) Molecular Systems Biology 7:539. For purposes of calculating identity to a sequence, extensions, such as tags, are not included.

[0131] As used herein, the term "HCDR" refers to a heavy chain complementarity determining region. As used herein, the term "LCDR" refers to a light chain complementarity determining region.

[0132] The terms "amino terminus," "N terminus," "carboxyl terminus," and "C terminus" are used herein to denote positions within a polypeptide chain. Where the context permits, these terms are used with respect to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a particular sequence located at the carboxyl terminus of a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.

[0133] As used herein, the term "conservative substitution" refers to the replacement of an amino acid with another amino acid that does not significantly adversely change functional activity. A preferred example of a "conservative substitution" is the replacement of an amino acid with another amino acid that has a value of > 0 in the following BLOSUM62 substitution matrix (see Henikoff & Henikoff, 1992, PNAS 89:10915-10919):

[0134] [Table 8]

[0135] The terms "antibody-drug conjugate" and "immunoconjugate" refer to a protein of the present disclosure that is conjugated to a cytotoxic, cytostatic, and / or therapeutic agent.

[0136] The term "isolated molecule" (when the molecule is, for example, a protein, polypeptide, polynucleotide, antibody, or antigen-binding molecule) refers to a molecule that, in terms of its origin or source of derivation, (1) is not associated with naturally occurring binding components that accompany it in its natural state; (2) is substantially free from other molecules from the same species; (3) is expressed by cells from a different species; or (4) is not naturally occurring. Thus, a molecule that is chemically synthesized or expressed in a cellular system different from the cell from which it is naturally derived would be "isolated" from its naturally occurring binding components. A molecule can also be rendered substantially free of naturally occurring binding components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule can be assayed by several methods known in the art. For example, the purity of a polypeptide sample can be assayed using polyacrylamide gel electrophoresis and staining the gel to visualize the polypeptide. For certain purposes, higher resolution may be obtained using high-performance liquid chromatography (HPLC) or other means for purification known in the art.

[0137] c-Kit (also known as KIT, cluster of differentiation 117 (CD117), PBT, SCFR, and 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 type III receptor tyrosine kinase highly expressed in hematopoietic stem cells and several other cell types, including mature mast cells.

[0138] 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 specific for the basophil lineage of mast cells / hematopoietic effector cells.

[0139] As used herein with respect to the biological activity of a protein disclosed herein, the terms "inhibit," "block," or "neutralize" refer to the ability of the protein to substantially attenuate, hinder, prevent, suppress, slow down, interrupt, eliminate, stop, reduce, or reverse, e.g., the progression, intensity, or severity of, whatever is being inhibited, 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.

[0140] As used herein, the terms "treat," "treating," or "treatment of" (and grammatical variations thereof) mean to reduce the severity of, at least partially ameliorate, or stabilize a subject's condition, and / or achieve some relief, alleviation, reduction, or stabilization of at least one clinical symptom, and / or slow the progression of a disease or disorder.

[0141] As used herein, the terms "prevent," "preventing," and "prevention" (and grammatical variations thereof) refer to preventing and / or delaying the onset of a disease, disorder, and / or clinical symptom(s) in a subject and / or reducing the severity of the onset of a disease, disorder, and / or clinical symptom(s) compared to what would occur in the absence of the compositions and / or methods described herein. Prevention may be complete prevention, e.g., the complete absence of a disease, disorder, and / or clinical symptom(s). Prevention may also be partial prevention, such that the occurrence and / or severity of the onset of a disease, disorder, and / or clinical symptom(s) in a subject is less than what would occur in the absence of the compositions and / or methods described herein.

[0142] As used herein, a "therapeutically effective amount" is an amount of a protein or pharmaceutical composition provided herein effective to treat a disease or disorder or ameliorate a sign or symptom thereof in a subject. A "therapeutically effective amount" may vary depending on, for example, the disease and / or symptoms of the disease, the severity of the disease and / or symptoms of the disease or disorder, the age, weight, and / or health of the patient being treated, and the judgment of the prescribing physician.

[0143] In this description, any concentration range, percentage range, ratio range, or integer range should be understood to include any integer value within the stated range, and, where appropriate, fractions thereof (e.g., tenths and hundredths of integers), unless otherwise indicated.

[0144] The use of the alternative (eg, "or") should be understood to mean either one of the alternatives, either either of them, or any combination thereof.

[0145] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any reference, article, publication, patent, patent publication, or patent application cited herein is not, and should not be considered as, an admission or any indication that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0146] The headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0147] Numbered Embodiments While the claims are appended, the disclosure sets forth the following numbered embodiments.

[0148] Embodiment 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) identical third and fourth polypeptide chains, wherein said third and fourth polypeptide chains comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specific binding to c-Kit and CD203c, respectively.

[0149] Embodiment 2. The bispecific protein of embodiment 1, wherein the anti-c-Kit VH domain comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 2, a heavy chain complementarity determining region 2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 3, and a heavy chain complementarity determining region 3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 4; the anti-c-CD203c VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 73; and the VL domain comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 6, a light chain complementarity determining region 2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 7, and a light chain complementarity determining region 3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 8.

[0150] Embodiment 3. The bispecific protein of embodiment 1, wherein the anti-c-Kit VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 4; the anti-c-CD203c VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 10, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 11, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and the VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0151] Embodiment 4. The bispecific protein of embodiment 1, wherein the anti-c-Kit VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 3, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 4; the anti-c-CD203c VH domain comprises an HCDR1 comprising the amino acid sequence of SEQ ID NO: 14, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 15, and an HCDR3 comprising the amino acid sequence of SEQ ID NO: 12; and the VL domain comprises an LCDR1 comprising the amino acid sequence of SEQ ID NO: 6, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 7, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 8.

[0152] 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-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.

[0153] 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-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.

[0154] 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-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.

[0155] 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-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.

[0156] 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.

[0157] Embodiment 10. The bispecific protein of any one of embodiments 1 to 9, comprising an immunoglobulin constant region.

[0158] Embodiment 11. The bispecific protein of embodiment 10, wherein said immunoglobulin constant region is an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin constant region.

[0159] Embodiment 12 The bispecific protein of embodiment 10, wherein said immunoglobulin constant region is an IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2 immunoglobulin constant region.

[0160] Embodiment 13 The bispecific protein of embodiment 10, wherein said immunoglobulin constant region is an immunologically inert constant region.

[0161] Embodiment 14. The bispecific protein of any one of embodiments 1-9, wherein the first polypeptide chain comprises a first immunoglobulin constant region, the second polypeptide chain comprises a second immunoglobulin constant region, and the first immunoglobulin constant region and the second immunoglobulin constant region comprise knobs-in-holes mutations.

[0162] Embodiment 15. The bispecific protein of embodiment 14, wherein said first immunoglobulin constant region comprises a CH3 domain comprising the amino acid substitutions S354C and T366W, and said second immunoglobulin constant region comprises a CH3 domain comprising the amino acid substitutions Y349C, T366S, L368A, and Y407V, wherein numbering is according to the EU index as in Kabat.

[0163] Embodiment 16. The bispecific protein of embodiment 14, wherein said first immunoglobulin constant region comprises a CH3 domain comprising the amino acid substitutions Y349C, T366S, L368A, and Y407V, and said second immunoglobulin constant region comprises a CH3 domain comprising the amino acid substitutions S354C and T366W, wherein numbering is according to the EU index as in Kabat.

[0164] Embodiment 17. The bispecific protein of 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.

[0165] Embodiment 18. A bispecific protein that binds to c-Kit and CD203c, wherein said c-Kit and CD203c are present on the surface of the same cell.

[0166] Embodiment 19. The bispecific protein of embodiment 18, which is a designed ankyrin repeat protein (DARPin), a tandem VHH, or a tandem immunoglobulin neoantigen receptor (IgNAR).

[0167] Embodiment 20. An immunoconjugate comprising the bispecific protein of any one of embodiments 1 to 19 conjugated to a therapeutic agent.

[0168] 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.

[0169] Embodiment 22. A pharmaceutical composition comprising a bispecific protein according to any one of embodiments 1 to 19 or an immunoconjugate according to embodiment 20 or 21, and a pharmaceutically acceptable carrier, diluent or excipient.

[0170] 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 to 17.

[0171] Embodiment 24. A nucleic acid molecule encoding a bispecific protein according to embodiment 18 or 19.

[0172] Embodiment 25. An expression vector comprising the nucleic acid molecule of embodiment 22 or 23.

[0173] Embodiment 26. A recombinant host cell comprising a nucleic acid molecule according to embodiment 23 or 24, or an expression vector according to embodiment 25.

[0174] Embodiment 27. A method of producing a bispecific protein, comprising culturing a recombinant host cell of embodiment 26 under conditions to express the nucleic acid molecule, thereby producing the protein, and isolating the protein from the host cell or culture.

[0175] Embodiment 28. A method of treating an inflammatory disease or neoplasm in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific protein of any one of embodiments 1 to 19, an immunoconjugate of embodiment 20 or 21, or a pharmaceutical composition of embodiment 22.

[0176] Embodiment 29. A method for ameliorating symptoms of an inflammatory disease or neoplasia in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a bispecific protein of any one of embodiments 1 to 19, an immunoconjugate of embodiment 20 or 21, or a pharmaceutical composition of embodiment 22.

[0177] Embodiment 30. The method of embodiment 28 or 29, wherein the inflammatory disease is a chronic inflammatory disease.

[0178] Embodiment 31 The method of embodiment 28 or 29, wherein the inflammatory disease is a mast cell-induced disease.

[0179] 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.

[0180] Embodiment 33 The method of embodiment 28 or 29, wherein the neoplasm is a mast cell-induced neoplasm.

[0181] Embodiment 34. The method of embodiment 28 or 29, wherein the neoplasm is systemic mastocytosis or mast cell leukemia.

[0182] Embodiment 35. A bispecific protein according to any one of embodiments 1 to 19, an immunoconjugate according to embodiment 20 or 21, or a pharmaceutical composition according to embodiment 22, for use as a medicament.

[0183] Embodiment 36. A bispecific protein according to any one of embodiments 1 to 19, an immunoconjugate according to embodiment 20 or 21, or a pharmaceutical composition according to embodiment 22, for use in treating an inflammatory disease or neoplasm.

[0184] Embodiment 37. The bispecific protein, immunoconjugate, or pharmaceutical composition for use according to embodiment 36, wherein the inflammatory disease is a chronic inflammatory disease.

[0185] Embodiment 38. The bispecific protein, immunoconjugate, or pharmaceutical composition for use according to embodiment 36, wherein the inflammatory disease is a mast cell-induced disease.

[0186] Embodiment 39. The bispecific protein, immunoconjugate, or pharmaceutical composition for use according to embodiment 36, wherein said inflammatory disease is chronic urticaria, chronic pruritus, atopic dermatitis, allergic asthma, prurigo nodularis, eosinophilic gastritis, eosinophilic duodenitis, or eosinophilic esophagitis.

[0187] Embodiment 40. The bispecific protein, immunoconjugate, or pharmaceutical composition for use according to embodiment 36, wherein said neoplasm is a mast cell-derived neoplasm.

[0188] Embodiment 41. The bispecific protein, immunoconjugate, or pharmaceutical composition for use according to embodiment 36, wherein said neoplasm is systemic mastocytosis or mast cell leukemia.

[0189] The present disclosure will be further clarified by the following examples, which are intended to be purely illustrative of the disclosure and not limiting in any way. [Example]

[0190] Generation and analysis of anti-c-Kit and anti-CD203c bispecific proteins Materials and Methods IgG / bispecific expression and purification 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 effector function-null human IgG1 ("IgG1 null"; a human IgG1 containing L234A, L235A, and G237A mutations in the lower hinge that suppress normal immunoglobulin ADCC, ADCP, and CDC functions) and a human Cκ domain, respectively. The Fc sequences are shown in Table 4. Cotransfection of the heavy and light chain-containing vectors in a CHO mammalian expression system was followed by Protein A-based IgG purification, quantification, and quality control by denaturing and non-denaturing SDS-PAGE and analytical SEC.

[0191] Expression and purification of common light chain bispecific antibodies was performed in a similar manner using heavy chain constructs carrying the effector function null human IgG1 mutations described above, plus the "knob" or "hole" mutations described in Figure 2. After Protein A purification, the bispecific molecules underwent a second step of preparative cation exchange chromatography to achieve a single POI >95%.

[0192] Direct binding ELISA for IgG / bispecific The binding and cross-reactivity of the molecules to human, rhesus, and cynomolgus CD203c were initially assessed by binding ELISA. Human CD203c His-tagged recombinant protein and rhesus CD203c His-tagged recombinant protein were coated onto the surface of a MaxiSorp™ flat-bottom 96-well plate at a concentration of 1 μg / ml. 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 with 3,3',5,5'-tetramethylbenzidine substrate solution (TMB), and absorbance was measured at 450 nm.

[0193] Binding of KU812 cells to cell surface CD203c All treatments were performed on cells seeded at a seeding density of 0.3e6 / ml in T75 flasks. Cells were cultured in the presence of 80ng / ml SCF and treated with 20ng / ml IL3 24 hours prior to antibody staining. After SCF / IL3 treatment, cells were harvested and stained with a live / dead stain (Zombie UV Biolegend 423108). Anti-CD203c antibodies / bispecifics or their respective isotype controls were then titrated in a 5-fold dilution series starting at 50µg / mL, and cells were stained and detected with anti-human Fc AF647 antibody (JIR 109-605-098). Samples were then analyzed on a Yeti analyzer, gating on 10K live cells. Subsequent analysis was performed using FlowJo software, and data were presented as MFI (median fluorescence intensity).

[0194] 1.27 Heavy Chain Optimized Library Design, Cloning, and Selection One precision-defined complex variant library with two domains (HCDR1 and HCDR2) containing single and double mutations, and one precision-defined complex variant library with a single domain (HCDR3) containing single and double mutations, were synthesized using bulk oligo assembly and high-fidelity oligo pool splicing. All amino acids were represented at all positions except cysteine, and insertion of NG and DG developability risk motifs was avoided. These designs are summarized in Figures 4 and 5. These libraries were cloned into a custom pCAT-Fab-MH1 phagemid vector containing the anti-c-Kit-MH1 VL, transformed into E. coli TG-1 cells, and rescued essentially as previously described in detail (Finlay et al., 2011, Methods Mol Biol 681:383-401).

[0195] Phage selection was performed by coating streptavidin magnetic microbeads with biotinylated CD203c protein (either human or rhesus monkey). The beads were washed three times with PBS and resuspended in PBS (pH 7.4) containing 5% skim milk protein. These beads were coated with 100 nM target protein in round 1 of selection, followed by three successive rounds with a 4-5-fold decrease in antigen concentration. In each round, phage were eluted using trypsin and then reinfected into TG1 cells.

[0196] Generation of periplasmic extracts (small scale) Soluble Fabs were generated for individual E. coli clones. Logarithmically growing E. coli TG1 cells 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 at room temperature and resuspended in PBS (pH 7.4). The supernatants containing soluble Fabs were collected after shaking and centrifugation at room temperature. These were tested for binding to human / rhesus CD203c coated at 1 μg / mL in a direct binding ELISA starting with an 85% periprep and diluted 1.5-fold in an 8-point titration. Anti-HA-HRP was used to detect periprep binding.

[0197] c-kit receptor phosphorylation assay KU812 cells were cultured in RPMI + 10% FCS without SCF for 48 hours. The cells were plated in a 24-well plate at 1x10 cells per well in a total volume of 1.5 mL. 6 Cells were seeded with 40 ng / mL of SCF for 1 hour and then treated with isotype control or test agents for 2 hours. Cells were stimulated with 40 ng / mL of SCF for 5 minutes, washed with PBS, and cell lysates were prepared using lysis buffer according to the manufacturer's instructions (Biotech). Total and phospho-c-kit levels were detected using Biotech's ELISA kits using 6.25 μg and 50 μg of total protein, respectively.

[0198] Affinity determination of optimized CD203c binding domains using SPR After affinity optimization, the CD203c VH binding domains F6 and F6.12 were produced as Fabs and compared to the Fab binding of the parental 1.27 to human and cynomolgus CD203c proteins. A biotinylated version of the CD203c protein was captured on a BiotinCap chip, and the antibody was injected in solution at two-fold serial dilutions. SPR affinity determination assays were performed on a BIAcore T20 using the conditions described below. Biacore chip: CAP chip (Biotin CAPture kit, Cytiva, catalog number 28920234) Surface preparation: Biotin CAPture reagent (Biotin CAPture Kit, Cytiva, Cat. No. 28920234) injected at 2 μl / min for 5 min Ligand capture - biotinylated antigen: bio-huCD203c (Acro, Cat. No. H52H4) and bio-cyCD203c (Icosagen) at 20 nM and 10 nM, respectively, in 1x HBS-EP + pH 7.4, flow cell 2, 10 μl / min for 1 minute Analyte binding: Three test antibodies were injected for 1 min at 30 μl / min into flow cells 1 and 2 in an eight-step 2-fold dilution series (1000 nM to 7.81 nM for 1.27-Fab, F6 Fab, and F6.12 Fab). A complete blank assay run without analyte antibody was included as a blank reference. Off-rate measurement: 600 seconds · Regeneration: Baseline levels were restored using the regeneration solution provided in the Biotin CAPture kit. SPR running buffer: 1x HBS-EP + pH 7.4 (Cytiva, catalog number BR100669) Analysis: Data were analyzed using the Biacore Insight Evaluation software with a steady-state affinity model or a 1:1 binding model. Sensorgrams were referenced to flow cell 1 and 0 nM concentration cycles.

[0199] The data for the three Fabs are summarized in Figures 11A-11D.

[0200] pI engineering of bispecific heavy chains to allow for easy purification 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. Several criteria were used to select mutations that would induce a more negative pI in the anti-c-Kit heavy chain and a more positive pI in the anti-CD203c heavy chain. These included: Distal to antigen-interacting CDRs Analysis of the available NGS antibody repertoire in the published literature Alignment to germline homologs for anti-CD203c (VH1-46 for anti-c-Kit / VH5-51) Comparison with known and approved clinical-stage antibody therapeutics Comparison with the deposited structures of the two germlines in the PDB to provide structural context for the selected mutations All candidate variants were modeled for their predicted impact on stability, dipole moment, developability, and immunogenicity

[0201] For each heavy chain, a set of five double mutants was prioritized that altered pI while improving stability or neutralizing the effect. These double mutants were not located within the same 12-mer sequence stretch to reduce immunogenicity risk, and did not create deleterious charge / hydrophobic patches or post-translational modification sites. They maintained developability parameters within or close to those described for clinical-stage therapeutic antibodies. These double mutation combinations are summarized in Figure 13.

[0202] Generation, purification, and characterization of lead pI-engineered bispecific antibodies Five double mutants of the MH1 anti-c-Kit VH domain were combined with each of the five double mutants of the F6.12 CD203c VH domain to generate 25 bispecific antibodies. Each was produced in 50 mL of ExpiCHO cells. The ExpiCHO cells were cultured for 9 days depending on cell viability, after which they were centrifuged to clarify the supernatant and immediately processed. The purified antibodies were captured from the clarified supernatant using a HiTrap MabSelect Sure Protein A 5 mL column (GE Healthcare, catalog no. 11-0034-95) on an AKTA Pure 25 FPLC system. The acidic fraction corresponding to the affinity-purified antibodies was immediately neutralized with 30% 1 M Tris (pH 8.0). 2.5 μL of each sample was analyzed on a LabChip under non-reducing and reducing conditions using a Protein clear HT chip (catalog no. CLS1486695) according to the manufacturer's instructions. Approximately 10 μg of each sample was analyzed by SEC-HPLC on a Thermo Vanquish Flex UHPLC system (Thermo Fisher). Samples were injected onto a Superdex 200 Increase 5 / 150 GL (catalog number 28-9909-45) column, and the percent monomer peak was quantified. Analytical CEX was performed on each sample to perform an initial assessment of peak separation by cation exchange. Approximately 10 μg of each sample was injected onto a TSKgel SP-STAT (7 μm, 4.6 mm ID x 10 cm L) column pre-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).

[0203] For further characterization of the preferred bispecific, the same process was repeated using a scaled-up 200 mL volume of ExpiCHO cells. After Pro-A purification, several different preparative cation exchange methods were compared to evaluate the optimal resolution of the major heterodimeric bispecific peak. First, the desalted sample was injected onto a HiTrap SP HP 5 mL CEX column (Cytiva, catalog number 17-1151-02). A total of two runs were performed to avoid compromising the resolution of the separation. A salt gradient of 1–20% Buffer B (25 mM NaH2PO4 / Na2HPO4, 1 M NaCl (pH 6.3)) over 40 CV, followed by 20–100% Buffer B over 10 CV was tested. However, the column's resolution was suboptimal, and a RESOURCE S 6mL CEX column (Cytiva, catalog no. 17118001) was selected as an alternative, using 5–12% buffer B over 40 CV, followed by 12–100% buffer B over 10 CV. Several column fractions were collected for analytical SDS-PAGE characterization and LC-MS analysis by LabChip as described above. Each sample was deglycosylated using Rapid PNGase F (non-reducing format) (New England Biolabs, catalog no. P0711S) according to the manufacturer's recommendations. The intact, deglycosylated samples were analyzed by reversed-phase liquid chromatography on a BioAccord system using Protein BEH C4 300Å Waters (catalog no. 186004495). Data were processed using MaxEnt1 software.

[0204] Results and Discussion Lead IgG generation and screening Anti-CD203c antibodies bearing the anti-c-kit MH1 light chain (listed 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 endotoxin content of the preparations was <1 EU / mg.

[0205] Purified IgG was first tested for binding to His-tagged recombinant versions of human and rhesus CD203c (Figures 3A and 3B), and all clones that showed measurable binding to both orthologs were subjected to binding analysis using flow cytometry on KU812 cells, which endogenously express human CD203c. Of the clones tested, only 1.27 retained strong binding to cell surface-expressed CD203c (Figure 3C), and this was selected as the parent clone for subsequent heavy chain mutagenesis and variant library generation.

[0206] 1.27 Heavy Chain Library Generation and Screening The anti-CD203c parent clone 1.27 was used as a template to generate two heavy chain-precise variant libraries, which are depicted in Figures 4 and 5. These libraries did not target the light chain; all variants maintained the common anti-c-kit MH1 light chain, as described in Table 2. These libraries were selected over four iterative rounds of phage selection on human and rhesus CD203c and screened by direct binding ELISA for the ability to bind to both orthologs in a periprep Fab format. Subsequent sequence analysis allowed the selection of a lead panel of optimized clones with sequences derived from both the HCDR1 and HCDR2 / HCDR3 libraries.

[0207] The 1.27 output clones summarized in Table 3 were expressed and purified as IgG and retested in purified IgG format for human / rhesus CD203c binding by direct ELISA and for binding to cell surface CD203c on KU812 cells using a flow-based assay. Based on the data summarized in Figure 6, high-performing clones were prioritized for generation of common light chain bispecifics. The clones generated included HCDR1 / HCDR2 variants, HCDR3 variants, and clones combining mutations from both libraries. These are summarized in Table 3.

[0208] Characterization of optimized common light chain bispecifics 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 achieve a >95% monomer fraction. The purified proteins were evaluated for cooperative binding to both targets, CD203c and c-Kit, in KU812 cells. Figure 7 shows that the bispecific molecules 1.27 / MH1 and F6 / MH1 bind to the dual receptor in KU812 cells, demonstrating enhanced binding compared to bivalent CD203c IgG and bivalent and monovalent c-Kit IgG. This enhanced binding translates into stronger inhibition of c-Kit receptor phosphorylation by the F6 / MH1 bispecific molecule compared to monovalent anti-c-Kit (shown in Figure 8). This inhibition demonstrates that targeting monovalent c-Kit can inhibit receptor phosphorylation, and co-engagement with CD203c greatly enhances the efficacy of this inhibition by enhancing receptor occupancy.

[0209] 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 CDR (Table 2, SEQ ID NOS: 58-61) and paired with the CD203c VH domain (Table 3, SEQ ID NOS: 54-57), binding to c-Kit was retained but binding to CD203c was lost. These data are summarized in Figures 9A and 9B and demonstrate that successful bispecific pairs cannot be predicted a priori.

[0210] A comparative study of the binding of the F6 / MH1 bispecific molecule to human and cynomolgus CD203c was performed by ELISA. The data shown in Figure 10 demonstrate comparable binding to both orthologues.

[0211] Affinity optimization of the anti-CD203c 1.27 binding domain SPR was used to characterize variants from the 1.27 affinity-optimized library. Binding of the Fab version of the parental 1.27, F6 containing mutations in H-CDR1 and H-CDR2, and F6.12 containing mutations across all three VH CDRs was measured against chips coated with biotinylated human or cynomolgus CD203c. 1:1 interaction kinetics determinations showed a 10-fold improvement in KD for F6 to 74 nM and a further improvement to 55 nM for F6.12 (Figures 11A-11D). This improved binding was further demonstrated in KU812 cells, where binding of F6 IgG (EC50 1.311 nM) was improved 75-fold compared to the parental 1.27 IgG (EC50 98.23 nM). This is summarized in Figures 12A-12B.

[0212] pI engineering of MH1 / F6.12 bispecific To improve downstream process development and optimize heterodimer formation, we engineered the pI of both bispecific heavy chains. Five double mutants were generated for the anti-c-Kit VH MH1 to induce a more negative pI, and five double mutants were generated for the anti-CD203c VH F6.12 to induce a more positive pI. These mutations are summarized in Figure 13. A matrix of all 25 potential variant bispecifics was generated and compared for productivity, analytical SEC, and pilot heterodimer formation by analytical CEX. Each bispecific variant exhibited a different CEX profile, with some exhibiting multiple peaks that were not easily resolved. An example of such a profile is shown in Figure 14, where three overlapping peaks are present for MH1_A / F6.12_B. However, some variants exhibited clear resolution and preferential heterodimer formation and could be purified with high yields by preparative CEX. This was the case for MH1_C / F6.12_D, shown in Figure 15.

[0213] Comparison of MH1_C / F6.12_D with the parent bispecific MH1 / F6.12 confirmed that dual target interaction was maintained after pI engineering. Both variants exhibited comparable binding to human and cynomolgus c-Kit (Figure 16) and human and cynomolgus CD203c (Figure 17), with kinetic data summarized in Figure 18. This was also true for binding to cell surface targets, which showed comparable binding before and after pI engineering on KU812 cells (Figures 19A-C).

Claims

1. 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) identical third and fourth polypeptide chains, wherein the third and fourth polypeptide chains comprise an immunoglobulin light chain comprising a light chain variable (VL) domain capable of specifically binding to c-Kit and CD203c, respectively; 2. The bispecific protein comprising:

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

8.

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

8.

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

8.

5. 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; 3. The bispecific protein of claim 2, wherein the VL domain comprises the amino acid sequence of SEQ ID NO:

5.

6. 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; 4. The bispecific protein of claim 3, wherein the VL domain comprises the amino acid sequence of SEQ ID NO:

5.

7. 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; 5. The bispecific protein of claim 4, wherein the VL domain comprises the amino acid sequence of SEQ ID NO:

5.

8. 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 to 51; 2. The bispecific protein of claim 1, wherein the VL domain comprises the amino acid sequence of SEQ ID NO:

5.

9. 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; 2. The bispecific protein of claim 1, wherein the VL domain comprises the amino acid sequence of SEQ ID NO:

5.

10. 10. The bispecific protein of any one of claims 1 to 9, comprising an immunoglobulin constant region.

11. 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. 11. 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. 10. The bispecific protein of any one of claims 1 to 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 knobs-in-holes mutations.

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

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

17. (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 15. The bispecific protein of claim 14, wherein (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 to c-Kit and CD203c, wherein said c-Kit and CD203c are present on the surface of the same cell.

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

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

21. 21. The immunoconjugate of claim 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.

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

23. 18. 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. 20. A nucleic acid molecule encoding the bispecific protein of claim 18 or 19.

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

26. 26. A recombinant host cell comprising a nucleic acid molecule according to claim 23 or 24, or an expression vector according to claim 25.

27. 1. A method for producing a bispecific protein, comprising: Culturing the recombinant host cell of claim 26 under conditions to express the nucleic acid molecule, thereby producing the protein; and isolating said protein from said host cell or culture; The method comprising:

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

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

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

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

32. 30. 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. 30. The method of claim 28 or 29, wherein the neoplasm is a mast cell-derived neoplasm.

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

35. A bispecific protein according to any one of claims 1 to 19, an immunoconjugate according to claim 20 or 21, or a pharmaceutical composition according to claim 22, for use as a medicament.

36. 23. A bispecific protein according to any one of claims 1 to 19, an immunoconjugate according to claim 20 or 21, or a pharmaceutical composition according to claim 22, for use in the treatment of an inflammatory disease or a neoplasm.

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

38. 37. The bispecific protein, immunoconjugate or pharmaceutical composition for use according to claim 36, wherein the inflammatory disease is a mast cell-induced disease.

39. 37. The bispecific protein, immunoconjugate or pharmaceutical composition for use according to 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. 37. The bispecific protein, immunoconjugate, or pharmaceutical composition for use according to claim 36, wherein the neoplasm is a mast cell-induced neoplasm.

41. 37. 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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