Engineered protein constructs targeting siglec-8 and CD117

Abstract

The present disclosure provides for engineered protein constructs, compositions, and uses thereof for treating diseases. The engineered protein constructs and compositions comprising a first antigen-binding moiety that binds Sialic acid–binding immunoglobulin-like lectin 8 (SIGLEC-8) and a second antigen-binding moiety that binds CD117.

Description

Docket No. 225365-702601ENGINEERED PROTEIN CONSTRUCTS TARGETING SIGLEC-8 AND CD117CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 688,131 filed on August 28, 2024, which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted electronically in ST.26 xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on August 27, 2025, is named 225365-702601_SL.xml and is 511,482 bytes in size.BACKGROUND

[0003] Antibody engineering is unlocking tremendous opportunities for auto-immune and inflammatory therapies by enabling the design of innovative therapeutic agents. Today, therapeutic antibodies can be tailored for better efficiency or other advantageous clinical properties. Monoclonal antibodies (mAbs) with enhanced antibody-effector functions and bispecific antibodies (BsAb) have been approved for several inflammatory, auto-immune indications besides their primary oncology indications. Still significant unmet needs remain, as a large proportion of patients either fail to achieve remission or fail to respond altogether.BRIEF SUMMARY

[0004] Provided herein are methods of treating an inflammatory disease or disorder in a subject in need thereof, wherein the methods comprise administering an effective amount of a pharmaceutical composition comprising an engineered protein construct to the subject, wherein the engineered protein construct comprises: a first antigen-binding moiety that binds Sialic acidbinding immunoglobulin-like lectin 8 (SIGLEC-8); and a second antigen-binding moiety that binds cluster of differentiation 117 (CD117), wherein the effective amount is sufficient to reduce an inflammation caused by mast cells in the subject as compared to prior to the administration of the engineered protein construct. Further provided herein are methods, wherein the engineered protein construct comprises a heterodimeric human IgG Fc region that comprises a first constant region and a second constant region. Further provided herein are methods, wherein the heterodimeric Fc region is operably linked to the SIGLEC-8 binding heavy chain variable (VH) domain and the CD117 binding heavy chain variable (VH) domain. Further provided herein are methods, wherein at least one of the first constant region and the second constant region comprises mutations M252Y, S254T, and T256E according to EU numbering. Further provided herein areDocket No. 225365-702601 methods, wherein at least one of the first constant region and the second constant region comprises at least one substitution at position L234, L235, P329, or a combination thereof, according to according to EU numbering. Further provided herein are methods, wherein the at least one substitution comprises L234A, L235A, and P329G, according to according to EU numbering. Further provided herein are methods, wherein at least one of the first constant region and the second constant region comprises at least one substitution at position L234, L235, K322, or a combination thereof, according to according to EU numbering. Further provided herein are methods, wherein the at least one of the two constant regions comprises mutations L234A, L235Q, and K322Q according to EU numbering. Further provided herein are methods, wherein the antigen-binding moiety that binds SIGLEC-8 has a lower affinity for SIGLEC-8, as compared to an affinity of the other antigen-binding moiety to CD 117. Further provided herein are methods, wherein the antigen-binding moiety that binds SIGLEC-8 has a higher affinity for SIGLEC-8, as compared to an affinity of the other antigen-binding moiety to CD117. Further provided herein are methods, wherein the engineered protein construct is an antibody, a variant or a functional fragment thereof. Further provided herein are methods, wherein the engineered protein construct comprises an antibody fragment, wherein the antibody fragment is one or more of Fab, Fv, Fab', (Fab')2, scFv, bis-scFv, Fab2, and Fab3. Further provided herein are methods, wherein the first antigen-binding moiety comprises a first Fab fragment and the second antigen binding moiety comprises a second Fab fragment. Further provided herein are methods, wherein the first antigenbinding moiety comprises a Fab fragment and the second antigen binding moiety comprises an ScFv fragment. Further provided herein are methods, wherein the engineered protein construct is a bivalent, trivalent, or tetravalent molecule. Further provided herein are methods, wherein the engineered protein construct is a bivalent molecule. Further provided herein are methods, wherein the engineered protein construct is a tetravalent molecule. Further provided herein are methods, wherein the engineered protein comprises four antigen-binding sites, wherein two of the four antigen-binding sites bind SIGLEC-8, each comprising a Fab fragment, and wherein two of the four antigen-binding sites bind CD117. Further provided herein are methods, wherein the disease or disorder is mast cell disease. Further provided herein are methods, wherein the mast cell disease is systemic mastocytosis, mast cell activation syndrome, or hereditary alpha-tryptasemia.

[0005] Provided herein are engineered protein constructs comprising a first antigen-binding moiety that binds Sialic acid-binding immunoglobulin-like lectin 8 (SIGLEC-8) and a second antigen-binding moiety that binds CD117, wherein the first antigen-binding moiety comprises: a heavy chain variable (VH) region comprising: a heavy chain complementarity-determining region 1 (HCDR1) having an amino acid sequence selected from Table 1, a heavy chain complementaritydetermining region 2 (HCDR2) having an amino acid sequence selected from Table 2, and a heavyDocket No. 225365-702601 chain complementarity-determining region 3 (HCDR3) having an amino acid sequence selected from Table 3; and a light chain variable (VL) region comprising: a light chain complementaritydetermining region 1 (LCDR1) having an amino acid sequence selected from Table 5, a light chain complementarity-determining region 2 (LCDR2) having an amino acid sequence selected from Table 6, and a light chain complementarity-determining region 3 (LCDR3) having an amino acid sequence selected from Table 7. Further provided herein are engineered protein constructs, wherein the first antigen-binding moiety comprises: a heavy chain variable domain (VH) comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from Table 4; and a light chain variable domain (VL) comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from Table 8. Further provided herein are engineered protein constructs, wherein the second antigen-binding moiety comprises: a heavy chain variable (VH) region comprising: a heavy chain complementaritydetermining region 1 (HCDR1) having an amino acid sequence selected from Table 9, a heavy chain complementarity-determining region 2 (HCDR2) having an amino acid sequence selected from Table 10, and a heavy chain complementarity-determining region 3 (HCDR3) having an amino acid sequence selected from Table 11; and a light chain variable (VL) region comprising: a light chain complementarity-determining region 1 (LCDR1) having an amino acid sequence selected from Table 13, a light chain complementarity-determining region 2 (LCDR2) having an amino acid sequence selected from Table 14, and a light chain complementarity-determining region 3 (LCDR3) having an amino acid sequence selected from Table 15. Further provided herein are engineered protein constructs, wherein the first antigen-binding moiety comprises: a heavy chain variable domain (VH) comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from Table 12; and a light chain variable domain (VL) comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical, or 100% identical to an amino acid sequence selected from Table 16. Further provided herein are engineered protein constructs, wherein the SIGLEC-8 is human SIGLEC-8. Further provided herein are engineered protein constructs, wherein the CD 117 is human CD 117. Further provided herein are engineered protein constructs, wherein the engineered protein construct is an antibody, a variant or a functional fragment thereof. Further provided herein are engineered protein constructs, wherein the engineered protein construct comprises an antibody fragment, wherein the antibody fragment is one or more of Fab, Fv, Fab', (Fab')2, scFv, bis-scFv, minibody, Fab2, and Fab3. Further provided herein are engineered protein constructs, wherein the engineered protein construct comprises a heterodimeric antibody or a functional fragment thereof. Further provided herein are engineeredDocket No. 225365-702601 protein constructs, wherein the engineered protein construct comprises a constant region. Further provided herein are engineered protein constructs, wherein a binding affinity of the first antigenbinding moiety for SIGLEC-8 is higher than a binding affinity of the second antigen-binding moiety for CD117. Further provided herein are engineered protein constructs, wherein the binding affinity of the first antigen-binding moiety for SIGLEC-8 is at least two times the binding affinity of the second antigen-binding moiety for CD117. Further provided herein are engineered protein constructs, wherein a binding affinity of the first antigen-binding moiety for SIGLEC-8 is lower than a binding affinity of the second antigen-binding moiety for CD117. Further provided herein are engineered protein constructs, wherein the binding affinity of the second antigen-binding moiety for CD117 is at least two times the binding affinity of the first antigen-binding moiety for SIGLEC-8. Further provided herein are engineered protein constructs, wherein the engineered protein construct comprises at least one of a Fc region and / or a Fab region. Further provided herein are engineered protein constructs, wherein the Fc region comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of amino acid sequences of SEQ ID NOs: 601-603. Further provided herein are engineered protein constructs, wherein at least one of the first antigen-binding moiety and the second antigen-binding moiety comprises a human IgG constant region, wherein the constant region comprises a light chain constant domain and / or a heavy chain constant domain. Further provided herein are engineered protein constructs, wherein the human IgG constant region comprises mutations M252Y, S254T, and T256E according to EU numbering. Further provided herein are engineered protein constructs, wherein the human IgG constant region comprises at least one substitution at position L234, L235, P329, or a combination thereof, according to according to EU numbering. Further provided herein are engineered protein constructs, wherein the human IgG constant region comprises mutations L234A, L235A, and P329G according to according to EU numbering. Further provided herein are engineered protein constructs, wherein the human IgG constant region comprises at least one substitution at position L234, L235, K322, or a combination thereof, according to according to EU numbering. Further provided herein are engineered protein constructs, wherein the human IgG constant region comprises mutations L234A, L235Q, and K322Q according to EU numbering. Further provided herein are engineered protein constructs, wherein the engineered protein construct comprises anti-inflammatory activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100% more than a combined anti-inflammatory activity of a monospecific antibody that binds SIGLEC-8 and a monospecific antibody that binds CD117. Further provided herein are engineered protein constructs, wherein the engineered protein construct comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at leastDocket No. 225365-70260198%, at least 99%, or 100% identical to any one of amino acid sequences of SEQ ID NOs: 604- 615.

[0006] Provided herein are pharmaceutical compositions comprising the engineered protein constructs provided herein, and a pharmaceutically acceptable carrier.

[0007] Provided herein are compositions for use in treating an inflammatory disease or disorder, wherein the composition comprises a first antigen-binding moiety that binds SIGLEC-8 and a second antigen-binding moiety that binds CD 117, wherein administration of an effective amount of the composition in need thereof results in the treatment of inflammatory disease or condition. Further provided herein are compositions, wherein the inflammatory disease is an autoimmune disease. Further provided herein are compositions for use in treating an inflammatory disease or disorder, wherein the autoimmune disease is multiple sclerosis, systemic lupus erythematosus, or rheumatoid arthritis. Further provided herein are compositions for use in treating an inflammatory disease or disorder, wherein the inflammatory disease is a gastrointestinal disorder. Further provided herein are compositions for use in treating an inflammatory disease or disorder, wherein the gastrointestinal disorder is inflammatory bowel disease (IBD), Crohn’s disease, or ulcerative colitis.

[0008] Provided herein are isolated nucleic acids that encode the engineered protein constructs provided herein.

[0009] Provided herein are expression vectors comprising the isolated nucleic acids provided herein.

[0010] Provided herein are isolated host cells comprising the isolated nucleic acids provided herein.

[0011] Provided herein are isolated host cells that express the engineered protein constructs Provided herein.

[0012] Provided herein are methods of producing an engineered protein construct that binds SIGLEC-8 and CD117 comprising incubating the isolated host cells provided herein under conditions suitable for producing the engineered protein constructs provided herein. Further provided herein are methods, wherein the methods further comprise isolating the antibody or antigen-binding fragment thereof.

[0013] Provided herein are methods of reducing the level of SIGLEC-8 and CD117 in mast cells, the methods comprising treating the mast cells with an effective amount of the engineered protein construct of any one of claims 26 to 49 or the pharmaceutical composition of claim 50, wherein the effective amount is sufficient to reduce the level of SIGLEC-8 and CD117 in the mast cells.Docket No. 225365-702601

[0014] Provided herein are methods of treating a disease or disorder in a subject in need thereof, the methods comprising administering to the subject an effective amount of an engineered protein construct provided herein or a pharmaceutical composition provided herein, wherein the effective amount is sufficient to treat the disease or disorder. Further provided herein are methods, wherein the disease or disorder is a mast cell disease. Further provided herein are methods, wherein the mast cell disease is systemic mastocytosis, mast cell activation syndrome, or hereditary alpha- tryptasemia. Further provided herein are methods, wherein the disease or disorder is an autoimmune disease. Further provided herein are methods, wherein the disease or disorder is asthma, an allergy, chronic urticaria, atopic dermatitis, or asthma. Further provided herein are methods, wherein the disease or disorder is eosinophilic esophagitis, eosinophilic gastroenteritis, or duodenitis. Further provided herein are methods, wherein the disease or disorder is a cancer. Further provided herein are methods, wherein the cancer is chronic eosinophilic leukemia, hypereosinophilic syndrome, or chronic myeloid leukemia. Further provided herein are methods, wherein the cancer is an ovarian carcinoma, a seminoma, a glioma, a glioblastoma, a mast cell tumor, a melanoma, or a gastrointestinal tumor. Further provided herein are methods, wherein the disease or disorder is prurigo nodularis, severe combined immunodeficiency, or a retinopathy. Further provided herein are methods, wherein the retinopathy is diabetic retinopathy, retinopathy of prematurity, or wet macular degeneration. Further provided herein are methods, wherein the disease or disorder is central nervous system (CNS) disorders, multiple sclerosis (MS) or artherosclerosis. Further provided herein are methods, wherein the disease or disorder is Hirschsprung's disease. Further provided herein are methods, wherein the disease or disorder is Crohn's disease.BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0016] FIGURE 1 depicts a bispecific antibody comprising a CD117 binding moiety and a SIGLEC-8 binding moiety.

[0017] FIGURES 2A-2D show a bispecific antibody in different format. FIG. 2A shows a bispecific antibody with two Fab fragments. FIG. 2B shows an IgG Fab / ScFv bispecific antibody with AQQ and YTE mutations in IgG. FIG. 2C shows a tetravalent bispecific antibody with two fragments targeting SIGLEC-8 and two fragments targeting CD 117. FIG. 2D shows a bispecific antibody containing a VH-Fc (knob) component and a Fab-Fc (hole) component.Docket No. 225365-702601

[0018] FIGURE 3 is a plot showing binding of anti-SIGLEC-8 antibodies to SIGLEC-8 in SIGLEC-8 overexpressing HEK293 cells. Y axis represents the mean fluorescence intensity (MFI). X axis represents increasing concentrations of antibodies.

[0019] FIGURE 4 is a bar plot showing IL-6 levels in mast cells in presence of SIGLEC-8 antibodies. Y represents

[0020] FIGURE 5 is a plot showing binding of anti-CD117 antibodies to CD117 in Mo7E cells. Y axis represents the mean fluorescence intensity (MFI). X axis represents increasing concentrations of antibodies.

[0021] FIGURE 6 is a plot showing the inhibition of SCF-induced Mo7E cell proliferation by anti-CD117 antibodies. Y axis represents luminescence. X axis represents increasing concentrations of antibodies.

[0022] FIGURE 7 is a plot showing binding of Barzolvolimab or bispecific antibodies to CD117 in Mo7E cells. Y axis represents the mean fluorescence intensity (MFI). X axis represents increasing concentrations of antibodies.DETAILED DESCRIPTION OF THE INVENTION

[0023] Provided herein are engineered protein constructs and compositions targeting CD117 and / or SIGLEC-8 and uses thereof for treating diseases. Also provided herein are engineered bispecific antibodies (BsAbs) comprising two distinct binding specificities within a single biologic which allow the simultaneous targeting of multiple disease-causing cytokines or pathways. Further provided herein are methods of treating autoimmune and inflammatory diseases.Definitions

[0024] Throughout this disclosure, various embodiments are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiments. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range to the tenth of the unit of the lower limit unless the context clearly dictates otherwise. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual values within that range, for example, 1.1, 2, 2.3, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, rangesDocket No. 225365-702601 excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

[0025] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of any embodiment. As used herein, the singular forms “a,” “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and / or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof. As used herein, the term “and / or” includes any and all combinations of one or more of the associated listed items.

[0026] Unless specifically stated or obvious from context, as used herein, the term “about” in reference to a number or range of numbers is understood to mean the stated number and numbers + / - 10% thereof, or 10% below the lower listed limit and 10% above the higher listed limit for the values listed for a range.

[0027] Unless specifically stated, as used herein, the term “nucleic acid” encompasses double- or triple-stranded nucleic acids, as well as single-stranded molecules. In double- or triple-stranded nucleic acids, the nucleic acid strands need not be coextensive (i.e., a double-stranded nucleic acid need not be double-stranded along the entire length of both strands). Nucleic acid sequences, when provided, are listed in the 5’ to 3’ direction, unless stated otherwise. Methods described herein provide for the generation of isolated nucleic acids. Methods described herein additionally provide for the generation of isolated and purified nucleic acids. A “nucleic acid” as referred to herein can comprise at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, or more bases in length. Moreover, provided herein are methods for the synthesis of any number of polypeptide-segments encoding nucleotide sequences, including sequences encoding non-ribosomal peptides (NRPs), sequences encoding non-ribosomal peptidesynthetase (NRPS) modules and synthetic variants, polypeptide segments of other modular proteins, such as antibodies, polypeptide segments from other protein families, including noncoding DNA or RNA, such as regulatory sequences e.g. promoters, transcription factors, enhancers, siRNA, shRNA, RNAi, miRNA, small nucleolar RNA derived from microRNA, or any functional or structural DNA or RNA unit of interest. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, intergenic DNA, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), small nucleolar RNA, ribozymes, complementary DNA (cDNA), which is a DNADocket No. 225365-702601 representation of mRNA, usually obtained by reverse transcription of messenger RNA (mRNA) or by amplification; DNA molecules produced synthetically or by amplification, genomic DNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. cDNA encoding for a gene or gene fragment referred herein may comprise at least one region encoding for exon sequences without an intervening intron sequence in the genomic equivalent sequence.

[0028] As used herein, the term “Fv” is the minimum antibody fragment that contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (three loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.

[0029] As used herein, the term, “heavy chain variable region” or “VH,” when used in reference to an antibody, refers to the fragment of the heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0030] As used herein, the term, “light chain variable region” or “ VL,” when used in reference to an antibody, refers to the fragment of the light heavy chain that contains three CDRs interposed between flanking stretches known as framework regions, these framework regions are generally more highly conserved than the CDRs and form a scaffold to support the CDRs.

[0031] As used herein, the term “antibody” is understood to include proteins having the characteristic two-armed, Y-shape of a typical antibody molecule as well as one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Exemplary antibodies include, but are not limited to, a monoclonal antibody, a polyclonal antibody, a bi-specific antibody, a multispecific antibody, a grafted antibody, a human antibody, a humanized antibody, a synthetic antibody, a chimeric antibody, a camelized antibody, a single-chain Fvs (scFv) (including fragments in which the VL and VH are joined using recombinant methods by a synthetic or natural linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules, including single chain Fab and scFab), a single chain antibody, a Fab fragment (including monovalent fragments comprising the VL, VH, CL, and CHI domains), a F(ab')2 fragment (including bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region), a Fd fragment (including fragments comprising the VH and CHI fragment), a Fv fragment (including fragments comprising the VL and VH domains of aDocket No. 225365-702601 single arm of an antibody), a single-domain antibody (dAb or sdAb) (including fragments comprising a VH domain), an isolated complementarity determining region (CDR), a diabody (including fragments comprising bivalent dimers such as two VL and VH domains bound to each other and recognizing two different antigens), a fragment comprised of only a single monomeric variable domain, disulfide-linked Fvs (sdFv), an intrabody, an anti -idiotypic (anti-Id) antibody, or ab antigen-binding fragments thereof. In some instances, the libraries disclosed herein comprise nucleic acids encoding for an antibody, wherein the antibody is a Fv antibody, including Fv antibodies comprised of the minimum antibody fragment which contains a complete antigenrecognition and antigen-binding site. In some embodiments, the Fv antibody consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association, and the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. In some embodiments, the six hypervariable regions confer antigen-binding specificity to the antibody. In some embodiments, a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen, including single domain antibodies isolated from camelid animals comprising one heavy chain variable domain such as VHH antibodies or nanobodies) has the ability to recognize and bind antigen. In some instances, the libraries disclosed herein comprise nucleic acids encoding for an antibody, wherein the antibody is a single-chain Fv or scFv, including antibody fragments comprising a VH, a VL, or both a VH and VL domain, wherein both domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains allowing the scFv to form the desired structure for antigen binding. In some instances, a scFv is linked to the Fc fragment or a VHH is linked to the Fc fragment (including minibodies). In some instances, the antibody comprises immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, e.g., molecules that contain an antigen binding site. Immunoglobulin molecules are of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG 2, IgG 3, IgG 4, IgA 1 and IgA 2) or subclass.

[0032] As used herein, the term “bispecific” refers to a molecule that is able to specifically bind at least two distinct targets. Typically, a bispecific antibody comprises two antigen-binding sites, each of which is specific for a different target. In some embodiments, a bispecific antibody binds two targets simultaneously.

[0033] An “antigen” is a molecule comprising at least one epitope. The antigen may for example be a polypeptide, nucleic acid, polysaccharide, protein, lipoprotein or glycoprotein.Engineered protein constructs

[0034] Provided herein are engineered proteins constructs. In some embodiments, an engineered protein construct described herein comprise a SIGLEC-8 binding moiety and / or aDocket No. 225365-702601CD117 binding moiety. In some embodiments, an engineered protein construct described herein comprises multispecific molecules described herein. Multispecific molecules are antibodies that are capable of binding at least two different targets. In some embodiments, a Multispecific molecule targets at least two epitopes, including SIGLEC-8 and CD117. In some embodiments, a Multispecific molecule is a bispecific, trispecific antibody, or a fused-nanobody.

[0035] SIGLEC-8 is a part of the sialic acid-binding IG-like lectin (SIGLEC) family of transmembrane proteins which are a CD33 -related group of receptors. SIGLEC-8 contained a short cytoplasmic tail devoid of signaling motifs. This so-called short form is the result from a premature stop codon; the more predominant form of Siglec-8 is a full-length mRNA encoded with its membrane-proximal immunoreceptor tyrosine-based inhibitory signaling motifs or switch motifs and a membrane-distal. Its extracellular binding domain recognizes specific complex carbohydrate glycans such as 6’ -sulfated sialyl Lewis X (6’-sulfo-sLex), and its intracellular immunoreceptor tyrosine-based inhibition motifs (ITIMs) facilitates receptor binding into inhibitory signals. SIGLEC-8 has two splice variants and may function as an inhibitory immunoregulatory receptor. SIGLEC-8 has a finely-tuned specificity toward different sialylated and sulfated carbohydrate ligands. SIGLEC-8 is expressed late in development and is expressed by immune effector cells such as eosinophils, mass cells, and basophils. Specifically, SIGLEC-8 is selectively expressed on mature eosinophils and mast cells, and with low expression on basophils. SIGLEC-8 can be involved in asthma and allergies. SIGLEC-8 can be expressed by eosinophils and / or basophils from subjects with certain cancers (e.g., chronic eosinophilic leukemia, hypereosinophilic syndrome, or chronic myeloid leukemia). SIGLEC-8 can also be expressed by bone marrow mast cells from subjects with mastocytosis and / or aplastic anemia. In some embodiments, an engineered protein construct described herein binds to a SIGLEC-8 variant. In some embodiments, a SIGLEC- 8 variant is rs36498, rsl0409962, or rsl 1672925.

[0036] CD117 is a transmembrane tyrosine kinase growth factor receptor that is the product of c-kit gene expression. CD117 is the receptor for stem cell factor (SCF) and can play a role in early hemopoiesis. CD117 is expressed by breast epithelium cells, germ cells, melanocytes, myeloid cells, and mast cells. CD117 can be expressed by certain types of cancer such as but not limited to ovarian carcinoma, seminoma, glioma, glioblastoma, mast cell tumors, melanoma, and gastrointestinal tumors. CD117 can also be related to chronic urticaria (hives), prurigo nodularis, severe combined immunodeficiency, or retinopathies (e.g., diabetic retinopathy, retinopathy of prematurity, or wet macular degeneration).

[0037] Methods for making multispecific antibodies are known in the art. Traditionally, recombinant production of multispecific antibodies is based on co-expression of two immunoglobulin heavy-chain / light-chain pairs, where the two heavy chains have differentDocket No. 225365-702601 specificities. Purification of correct molecule is usually accomplished by affinity chromatography steps.

[0038] In some embodiments, a multispecific antibody comprises variations in at least one region of the antibody. Exemplary regions of the antibody for variation include, but are not limited to, a complementarity-determining region (CDR), a variable domain, or a constant domain. In some instances, the CDR is CDR1, CDR2, or CDR3. In some instances, the CDR is a heavy domain including, but not limited to, CDRH1, CDRH2, and CDRH3. In some instances, the CDR is a light domain including, but not limited to, CDRL1, CDRL2, and CDRL3. In some instances, the variable domain is variable domain, light chain (VL) or variable domain, heavy chain (VH). In some instances, the CDR1, CDR2, or CDR3 is of a variable domain, light chain (VL). CDR1, CDR2, or CDR3 of a variable domain, light chain (VL) can be referred to as CDRL1, CDRL2, or CDRL3, respectively. CDR1, CDR2, or CDR3 of a variable domain, heavy chain (VH) can be referred to as CDRH1, CDRH2, or CDRH3, respectively. In some instances, the VL domain comprises kappa or lambda chains. In some instances, the constant domain is constant domain, light chain (CL) or constant domain, heavy chain (CH).

[0039] In some embodiments, antibody variable domains with desired binding specificities (antibody-antigen combining sites) can be linked to immunoglobulin constant domain sequences to form multispecific antibodies. In some embodiments, a fusion comprising antibody variable domains is preferably linked with an immunoglobulin heavy-chain constant domain, wherein the immunoglobulin heavy-chain constant domain comprises at least part of a hinge, CH2, and CH3 regions. In some embodiments, it is preferred to have a first heavy-chain constant region (CHI) containing a site necessary for light-chain binding present in at least one of fusions. DNAs encoding immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain may be inserted into separate expression vectors and may be co-transfected into a suitable host organism.

[0040] In some embodiments, an interface between a pair of antibody molecules described herein is engineered to maximize percentage of heterodimers that can be recovered from recombinant cell culture. In this method, one or more small amino acid side chains from the interface of a first antibody molecule is replaced with larger side chains to form a protuberance or knob (e.g., tyrosine or tryptophan). Compensatory cavities or holes of identical or similar size to the large side chain(s) are created on the interface of a second antibody molecule by replacing large amino acid side chains with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing yield of the heterodimer over other unwanted end-products such as homodimers.Docket No. 225365-702601

[0041] Techniques for generating bispecific antibodies from functional antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. The bispecific antibodies can be used as agents for the selective immobilization of enzymes.

[0042] Various techniques for making and isolating functional bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers.

[0043] The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. The functional fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) by a linker which is too short to allow pairing between the two domains on the same chain. Accordingly, VH and VL domains of one functional fragment are forced to pair with a complementary VL and VH domains of another functional fragment, thereby forming two antigenbinding sites. In some embodiments, making functional bispecific antibody fragments comprises the use of single-chain Fv (sFv) dimers.

[0044] Antibodies with more than two valences are contemplated. For example, trispecific antibodies can be prepared. Exemplary bispecific antibodies can bind to two different epitopes, at least one of which originates in the protein antigen of the invention. Alternatively, an anti-antigenic arm of an immunoglobulin molecule can be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD 16) so as to focus cellular defense mechanisms to the cell expressing the particular antigen. Bispecific antibodies can also be used to direct cytotoxic agents to cells which express a particular antigen. These antibodies may possess an antigen-binding arm and an arm which binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds the protein antigen described herein and further binds tissue factor (TF).

[0045] Several strategies have been used to generate multispecific molecules described herein (e.g., bispecific molecules, trispecific molecules), such as chemical cross-linking of functional antibody fragments, forced heterodimerization, quadroma technology, fusion of functional antibody fragments via polypeptide linkers, and use of single domain antibodies. Availability of recombinant DNA technologies has led to the generation of a multitude of bispecific antibody formats. Linkers and mutations have frequently been introduced into different regions of theDocket No. 225365-702601 antibody to force heterodimer formation or to connect different binding moieties into a single molecule.

[0046] Antibodies or IgGs generated by methods as described herein comprise improved binding affinity. In some instances, the antibody comprises a binding affinity (e.g., KD) of less than 1 nM, less than 1.2 nM, less than 2 nM, less than 5 nM, less than 10 nM, less than 11 nm, less than 13.5 nM, less than 15 nM, less than 20 nM, less than 25 nM, or less than 30 nM. In some instances, the antibody comprises a KD of less than 400 nM, less than 350 nM, less than 300 nM, less than 250 nM, less than 200 nM, less than 150 nm, less than 100 nM, less than 50 nM, less than 25 nM, less than 15 nM, or less than 10 nM. In some instances, the antibody comprises a KD of less than 1 nM. In some instances, the antibody comprises a KD of less than 1.2 nM. In some instances, the antibody comprises a KD of less than 2 nM. In some instances, the antibody comprises a KD of less than 5 nM. In some instances, the antibody comprises a KD of less than 10 nM. In some instances, the antibody comprises a KD of less than 13.5 nM. In some instances, the antibody comprises a KD of less than 15 nM. In some instances, the antibody comprises a KD of less than 20 nM. In some instances, the antibody comprises a KD of less than 25 nM. In some instances, the antibody comprises a KD of less than 30 nM.

[0047] In some instances, the affinity of antibodies or IgGs generated by methods as described herein is at least or about 1.5x, 2. Ox, 5x, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 200x, or more than 200x improved binding affinity as compared to a comparator antibody. In some instances, the affinity of antibodies or IgGs generated by methods as described herein is at least or about 1.5x, 2. Ox, 5x, lOx, 20x, 30x, 40x, 50x, 60x, 70x, 80x, 90x, lOOx, 200x, or more than 200x improved function as compared to a comparator antibody. In some instances, the comparator antibody is an antibody with similar structure, sequence, or antigen target.

[0048] In some embodiments, the variant antibodies or IgGs generated by methods as described herein result in a decreased ECso in a T-cell cytotoxicity assay as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 5X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 8X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 10X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 20X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies orDocket No. 225365-702601IgGs have an ECso in a T-cell cytotoxicity assay that is at least 25X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 30X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 40X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgG. In some embodiments, the variant antibodies or IgGs have an ECso in a T-cell cytotoxicity assay that is at least 50X decreased as compared to the ECso in a T-cell cytotoxicity assay of a reference antibody or IgGs.

[0049] Methods as described herein, in some instances, result in increased yield of antibodies or IgGs. In some instances, the yield is at least or about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, or more than 80 micrograms (ug). In some instances, the yield is in a range of about 5 to about 80, about 10 to about 75, about 15 to about 60, about 20 to about 50, or about 30 to about 40 micrograms (ug).Bispecific Antibody

[0050] Disclosed herein are bispecific antibodies (BsAb) that bind to SIGLEC-8 and CD117. FIG. 1 depicts a bispecific antibody that binds SIGLEC-8 and CD117. In some embodiments, BsAb comprises a heavy chain region. In some embodiments, the BsAb comprises a light chain region. In some embodiments, the light chain region comprises a kappa light chain constant region. In some embodiments, the BsAb comprises an BsAb light chain variable region. In some embodiments, the BsAb comprises an BsAb heavy chain variable region. In some embodiments, the BsAb comprises an BsAb light chain variable region and an IgG heavy chain variable region. In some embodiments, the BsAb can be a humanized antibody. In some embodiments, the BsAb can be a chimeric antibody. In some embodiments, the BsAb can be a human antibody. In some embodiments, the BsAb comprises a common light chain (L chain). In some embodiments, the L chain acts against a specific antigen. The use of a common light chain can prioritize heterodimerization in the Fc region. In some embodiments, the L chain comprises a knob mutation. In some embodiments, the L chain comprises a hole mutation. In some embodiments, preferential heterodimer formation is preferred upon binding of the BsAb comprising a common L chain. In some embodiments, the formations of heterodimeric pairs are assembled using glutathione disulfide exchange.

[0051] Disclosed herein are engineered protein constructs. In some embodiments, an engineered protein construct described herein comprises a BsAb described herein. In some embodiments, a BsAb described herein is an engineered BsAb that comprises one or more modifications of amino acids that can result in pH-dependent target binding activity. In someDocket No. 225365-702601 embodiments, an engineered BsAb described herein can exhibit pH-dependent target binding activity SIGLEC-8, CD 117, a variant thereof and a functional fragment thereof. In some embodiments, an engineered BsAb as described herein can readily bind to a target peptide at a neutral pH and dissociates from the target peptide at an acidic pH. Accordingly, upon administration to a subject the engineered BsAb can bind the target peptide in plasma on account of its neutral pH, while remaining dissociated from the target peptide in endosomes which have an acidic pH. Dissociation of the engineered BsAb from the target peptide in endosomes can facilitate recycling of the engineered BsAb into plasma through FcRn, whereas the target peptide can be trafficked to lysosome and degraded. Such characteristics of the engineered BsAb can allow sweeping of a target peptide from the plasma. Accordingly, in some embodiments, the engineered BsAb can comprise a target peptide sweeping activity for a target peptide that is selected from SIGLEC-8, CD117, a variant thereof and a functional fragment thereof.

[0052] In some embodiments, a BsAb described herein is an engineered BsAb that comprises one or more modifications of amino acids that can result in increased FcRn binding at neutral pH. In such embodiments, the engineered BsAb can have increased ability to repeatedly bind to FcRn and remove target peptide from plasma. Alternatively, in some embodiments, a BsAb described herein is an engineered BsAb that comprises one or more modifications of amino acids that can result in increased FcRn binding at acidic pH. In such embodiments, the engineered BsAb can have increased recycling efficiency from endosomes to plasma resulting in improving plasma retention of the engineered BsAb. Accordingly, in some embodiments, a constant domain of an engineered BsAb as described herein can be further modified for increasing FcRn binding activity at neutral pH and / or acidic pH.

[0053] In some embodiments, a BsAb described herein is an engineered BsAb that comprises one or more modifications of amino acids that can result in change of isoelectric point of the BsAb. In some embodiments, the one or more modifications of amino acids can result in change of isoelectric point of a VH sequence of the engineered BsAb. In some embodiments, the one or more modifications of amino acids can result in change of isoelectric point of a VL sequence of the engineered BsAb. In some embodiments, the one or more amino acid modifications can increase isoelectric point of the engineered BsAb. In some embodiments, increased isoelectric point can results in increased elimination rate of target peptides from plasma.

[0054] In some embodiments, a BsAb described herein is an engineered BsAb that comprises one or more modifications of amino acids that can result in pH-dependent target binding activity and / or increased FcRn binding activity. In some embodiments, the one or more modifications of amino acids comprise at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen,Docket No. 225365-702601 at least fourteen, or at least fifteen amino acid modifications. In some embodiments, the one or more modifications of amino acids are in at least one of a Fab region, a scFv region, and a Fc region. In some embodiments, one or more modifications of amino acids are in a VL sequence of a BsAb, a VH sequence of a BsAb, or a combination thereof. In some embodiments, a modification of an amino acid is a deletion, a substitution, or an addition of the amino acid.

[0055] In some embodiments, a BsAb can be a monospecific antibody, including but not limited to an antibody wherein both arms target different epitopes of the same antigen. In some embodiments, a BsAb can be a bi-specific antibody. In some embodiments, a BsAb can be a tri- specific antibody. In some embodiments, a BsAb can be a multi-specific antibody.Antigen-binding moieties

[0056] Provided herein are engineered protein constructs comprising at least one antigenbinding moiety. In some embodiments, the at least one antigen-binding moiety binds SIGLEC-8. In some embodiments, the at least one antigen-binding moiety binds CD117. In some embodiments, the at least one antigen-binding moiety binds both SIGLEC-8 and CD117. In some embodiments, an engineered protein construct described herein comprises a first antigen-binding moiety that binds SIGLEC-8 and a second antigen-binding moiety that binds CD117.

[0057] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises a sequence at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Tables 1-8. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises at least or about 95% sequence identity to any sequence selected from Tables 1-8. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises at least or about 97% sequence identity to any sequence selected from Tables 1-8. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises at least or about 99% sequence identity to any sequence selected from Tables 1-8. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises at least or about 100% sequence identity to any sequence selected from Tables 1-8.

[0058] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises complementarity determining regions (CDRs) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequences selected from Tables 1-3 and Tables 5-7. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises complementarity determining regions (CDRs) comprising at least or about 95% homology to any sequences selected from Tables 1-3 and Tables 5-7. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises complementarity determining regions (CDRs) comprising at least or about 97% homology to any sequences selected from Tables 1-3 and Tables 5-7. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprisesDocket No. 225365-702601 complementarity determining regions (CDRs) comprising at least or about 99% homology to any sequences selected from Tables 1-3 and Tables 5-7. In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises complementarity determining regions (CDRs) comprising at least or about 100% homology to any sequences selected from Tables 1-3 and Tables 5-7. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises complementarity determining regions (CDRs) comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 20, 21, 22, or more than 22 amino acids of sequences selected from Tables 1-3 and Tables 5-7.

[0059] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises complementarity determining regions (CDRs) comprising the amino acid sequence of any one from Tables 1-3 and Tables 5-7, or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of any sequences selected from Tables 1-3 and Tables 5-7. A “functional variant” as described herein with respect to a complementarity determining region (CDR) refers to a variant of a CDR having one or more amino acid modification(s) (e.g., amino acid insertions, substitutions, or deletions) with respect to a CDR sequence of a variable domain that binds to a particular antigen, where the one or more amino acid modification(s) to the CDR does not result in ablation of antigen binding of the variable domain to the particular antigen.

[0060] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises a heavy chain CDR1 (HCDR1) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table1. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR1 comprising at least or about 95% homology of any sequence selected from Table 1. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR1 comprising at least or about 97% homology to any sequence selected from Table 1. In some instances, an antigenbinding moiety that binds SIGLEC-8 comprises an HCDR1 comprising at least or about 99% homology to any sequence selected from Table 1. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR1 comprising at least or about 100% homology to any sequence selected from Table 1. In some instances, an antigen-binding moiety that binds SIGLEC- 8 comprises an HCDR1 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, or more than 8 amino acids of any sequence selected from Table 1.

[0061] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises a heavy chain CDR2 (HCDR2) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table2. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR2Docket No. 225365-702601 comprising at least or about 95% homology to any sequence selected from Table 2. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR2 comprising at least or about 97% homology to any sequence selected from Table 2. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR2 comprising at least or about 99% homology to any sequence selected from Table 2. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR2 comprising at least or about 100% homology to any sequence selected from Table 2. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR2 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 amino acids of any sequence selected from Table 2.

[0062] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises a heavy chain CDR3 (HCDR3) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table 3. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR3 comprising at least or about 95% homology to any sequence selected from Table 3. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR3 comprising at least or about 97% homology to any sequence selected from Table 3. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR3 comprising at least or about 99% homology to any sequence selected from Table 3. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR3 comprising at least or about 100% homology to any sequence selected from Table 3. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an HCDR3 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or more than 22 amino acids of any sequence selected from Table 3.

[0063] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises a light chain CDR1 (LCDR1) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any one from Table 5. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an LCDR1 comprising at least or about 95% homology of any sequence selected from Table 5. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an LCDR1 comprising at least or about 97% homology to any sequence selected from Table 5. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an LCDR1 comprising at least or about 99% homology to any one from Table 5. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises an LCDR1 comprising at least or about 100% homology to any sequence selected from Table 5. In some instances, an antigen-binding moiety that binds SIGLEC-8 thereof comprisesDocket No. 225365-702601LCDR1 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, or more than 8 amino acids of any sequence selected from Table 5.

[0064] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises a light chain CDR2 (LCDR2) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table6. In some instances, the antibody or antigen-binding fragment thereof comprises an LCDR2 comprising at least or about 95% homology to any sequence selected from Table 6. In some instances, the antibody or antigen-binding fragment thereof comprises an LCDR2 comprising at least or about 97% homology to any sequence selected from Table 6. In some instances, the antibody or antigen-binding fragment thereof comprises an LCDR2 comprising at least or about 99% homology to any sequence selected from Table 6. In some instances, the antibody or antigenbinding fragment thereof comprises an LCDR2 comprising at least or about 100% homology to any sequence selected from Table 6. In some instances, the antibody or antigen-binding fragment thereof comprises an LCDR2 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 amino acids of any sequence selected from Table 6.

[0065] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises a light chain CDR3 (LCDR3) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table7. In some instances, the antibody or antigen-binding fragment thereof comprises an LCDR3 comprising at least or about 95% homology to any sequence selected from Table 7. In some instances, the antibody or antigen-binding fragment thereof comprises an LCDR3 comprising at least or about 97% homology to any sequence selected from Table 7. In some instances, the antibody or antigen-binding fragment thereof comprises an LCDR3 comprising at least or about 99% homology to any sequence selected from Table 7. In some instances, the antibody or antigenbinding fragment thereof comprises an LCDR3 comprising at least or about 100% homology to any sequence selected from Table 7. In some instances, the antibody or antigen-binding fragment thereof comprises an LCDR3 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or more than 22 amino acids of any sequence selected from Table 7.

[0066] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 1 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 1, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 52 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 52, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 103 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 103, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 205 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 205, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 307.

[0067] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 2 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 2, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 53 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 53, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 104 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 104, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 206 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 206, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 308 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 308.

[0068] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 3 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 3, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 54 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 54, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 105 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 105, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 207 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 207, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 309 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 309.Docket No. 225365-702601

[0069] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 4 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 4, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 55 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 55, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 106 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 106, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 208 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 208, an LCDR2 comprising the amino acid sequence of ATS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of ATS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 310 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 310.

[0070] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 5 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 5, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 56 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 56, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 107 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 107, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 209 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 209, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 311 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 311.

[0071] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 6 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 6, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 57 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 57, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 108 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 108, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 210 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 210, an LCDR2 comprising the amino acid sequence of AVS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AVS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 312 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 312.

[0072] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 7 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 7, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 58 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 58, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 109 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 109, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 211 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 211, an LCDR2 comprising the amino acid sequence of SAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 313 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 313.

[0073] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 8 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 8, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 59 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 59, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 110 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 110, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 212 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 212, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 314 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 314.Docket No. 225365-702601

[0074] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 9 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 9, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 60 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 60, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 111 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 111, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 213 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 213, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 315 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 315.

[0075] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 10 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 10, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 61 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 61, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 112 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 112, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 214 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 214, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 265 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 265, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 316 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 316.

[0076] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 11 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 11, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 62 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 62, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 112 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 112, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 215 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 215, an LCDR2 comprising the amino acid sequence of ATS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of ATS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 317 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 317.

[0077] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 12 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 12, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 63 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 63, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 114 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 114, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 216 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 216, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 318 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 318.

[0078] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 13 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 13, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 64 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 64, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 115 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 115, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 217 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 217, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 319 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 319.Docket No. 225365-702601

[0079] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 14 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 14, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 65 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 65, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 116 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 116, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 218 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 218, an LCDR2 comprising the amino acid sequence of ATS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of ATS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 320 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 320.

[0080] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 15 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 15, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 66 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 66, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 117 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 117, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 219 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 219, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 321 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 321.

[0081] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 16 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 16, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 67 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 67, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 118 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 118, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 220 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 220, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 322 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 322.

[0082] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 17 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 17, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 68 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 68, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 119 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 119, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 221 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 221, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 323 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 323.

[0083] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 18 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 18, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 69 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 69, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 120 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 120, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 222 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 222, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 324 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 324.Docket No. 225365-702601

[0084] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 19 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 19, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 70 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 70, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 121 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 121, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 223 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 223, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 325 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 325.

[0085] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 20 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 20, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 71 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 71, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 120 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 120, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 224 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 224, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 326 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 326.

[0086] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 21 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 21, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 72 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 72, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 123 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 123, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 225 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 225, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 327 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 327.

[0087] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 22 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 22, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 73 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 73, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 124 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 124, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 226 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 226, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 328 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 328.

[0088] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 23 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 23, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 74 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 74, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 125 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 125, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 227 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 227, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 329 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 329.Docket No. 225365-702601

[0089] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 24 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 24, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 75 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 75, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 126 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 126, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 228 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 228, an LCDR2 comprising the amino acid sequence of AES or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AES, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 330 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 330.

[0090] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 25 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 25, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 76 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 76, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 127 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 127, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 229 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 229, an LCDR2 comprising the amino acid sequence of ASS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of ASS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 331 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 331.

[0091] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 26 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 26, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 77 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 77, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 128 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 128, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 230 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 230, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 332 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 332.

[0092] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 27 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 27, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 78 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 78, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 129 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 129, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 231 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 231, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 333 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 333.

[0093] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 28 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 28, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 79 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 79, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 130 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 130, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 232 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 232, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 334 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 385.Docket No. 225365-702601

[0094] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 29 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 29, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 80 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 80, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 131 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 131, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 233 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 233, an LCDR2 comprising the amino acid sequence of ATS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of ATS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 335 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 335.

[0095] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 30 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 30, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 81 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 81, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 132 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 132, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 234 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 234, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 336 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 336.

[0096] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 31 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 31, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 82 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 82, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 133 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 133, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 235 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 231, an LCDR2 comprising the amino acid sequence of GTT or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GTT, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 337 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 337.

[0097] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 32 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 32, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 83 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 83, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 134 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 134, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 236 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 236, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of QH or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of QH.

[0098] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 33 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 33, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 84 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 84, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 135 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 135, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 237 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 237, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 339 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 339.Docket No. 225365-702601

[0099] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 34 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 34, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 85 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 85, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 136 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 136, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 238 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 238, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 340 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 340.

[0100] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 35 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 35, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 86 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 86, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 137 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 137, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 239 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 239, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 341 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 341.

[0101] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 36 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 36, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 87 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 87, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 138 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 138, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 240 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 240, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 342 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 342.

[0102] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 37 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 37, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 88 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 88, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 139 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 139, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 241 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 241, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 343 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 343.

[0103] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 38 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 38, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 89 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 89, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 140 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 140, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 242 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 242, an LCDR2 comprising the amino acid sequence of GTS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GTS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 344 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 344.Docket No. 225365-702601

[0104] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 39 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 39, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 90 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 90, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 141 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 141, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 243 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 243, an LCDR2 comprising the amino acid sequence of SAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 345 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 345.

[0105] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 40 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 40, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 91 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 91, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 142 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 142, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 244 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 244, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 346 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 346.

[0106] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 41 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 41, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 92 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 92, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 143 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 143, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 245, or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 245, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 347 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 347.

[0107] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 42 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 42, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 93 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 93, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 144 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 144, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 246 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 246, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 348 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 348.

[0108] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 43 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 43, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 94 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 94, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 145 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 145, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 247 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 247, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 349 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 349.Docket No. 225365-702601

[0109] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 44 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 44, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 95 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 95, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 146 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 146, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 248 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 248, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 350 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 350.

[0110] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 45 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 45, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 96 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 96, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 147 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 147, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 249 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 249, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 351 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 351.[oni] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 46 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 46, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 97 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 97, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 148 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 148, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 250 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 250, an LCDR2 comprising the amino acid sequence of GSS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GSS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 352 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 352.

[0112] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 47 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 47, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 98 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 98, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 149 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 149, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 251 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 251, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 353 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 353.

[0113] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 48 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 48, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 99 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 99, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 150 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 150, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 252 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 252, an LCDR2 comprising the amino acid sequence of SAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 354 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 354.Docket No. 225365-702601

[0114] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 49 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 49, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 100 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 100, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 151 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 151, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 253 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 253, an LCDR2 comprising the amino acid sequence of ATS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of ATS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:355 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 355.

[0115] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 50 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 50, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 101 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 101, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 152 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 152, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 254 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 254, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:356 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 356.

[0116] In some embodiments, an antigen-binding moiety that binds SIGLEC-8 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 51 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 51, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 102 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 102, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 153 or a functional variant thereof having one or two amino acid substitutions with respect to the aminoDocket No. 225365-702601 acid sequence of SEQ ID NO: 153, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 255 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 255, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 306 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 306, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 357 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 357.

[0117] In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises a heavy chain variable domain comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table 4. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises a heavy chain variable domain comprising at least or about 95% sequence identity to any sequence selected from Table 4. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises a heavy chain variable domain comprising at least or about 97% sequence identity to any sequence selected from Table 4. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises a heavy chain variable domain comprising at least or about 99% sequence identity to any sequence selected from Table 4. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises a heavy chain variable domain comprising at least or about 100% sequence identity to any sequence selected from Table 4. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises a heavy chain variable domain comprising at least a portion having at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or more than 150 amino acids of any sequence selected from Table 4.

[0118] In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises a light chain variable domain comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table 8. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises a light chain variable domain comprising at least or about 95% sequence identity to any sequence selected from Table 8. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises a light chain variable domain comprising at least or about 97% sequence identity to any sequence selected from Table 8. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises a light chain variable domain comprising at least or about 99% sequence identity to any sequence selected from Table 8. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises a light chain variable domain comprising at least or about 100% sequence identity to any sequence selected from Table 8. In some instances, an antigen-binding moiety that binds SIGLEC-8 comprises a light chain variable domain comprising at least a portion having at least orDocket No. 225365-702601 about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or more than 150 amino acids of any sequence selected from Table 8.

[0119] Provided herein are engineered protein constructs comprising an antigen-binding moiety that binds CD 117, also known as KIT proto-oncogene receptor tyrosine kinase (c-kit) or mast / stem cell growth factor receptor (SCFR). CD117 is a cytokine receptor protein which binds to stem cell factor to form a dimer to activate signal transduction molecules that propagates the SCF signal into the cell. Signaling through CD117 plays a role in cell survival, proliferation, and differentiation. The terms “CD117” and “c-kit” are herein used interchangeably.

[0120] In some embodiments, an antigen-binding moiety that binds CD 117, wherein the region that binds CD117 comprises a sequence at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Tables 9-16. In some instances, an antigen-binding moiety that binds CD117 comprises at least or about 95% sequence identity to any sequence selected from Tables 9-16. In some instances, an antigen-binding moiety that binds CD117 comprises at least or about 97% sequence identity to any sequence selected from Tables 9-16. In some instances, an antigen-binding moiety that binds CD117 comprises at least or about 99% sequence identity to any sequence selected from Tables 9-16. In some instances, an antigen-binding moiety that binds CD117 comprises at least or about 100% sequence identity to any sequence selected from Tables 9-16.

[0121] In some embodiments, an antigen-binding moiety that binds CD117 comprises complementarity determining regions (CDRs) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to sequences selected from Tables 9-11 and 13-15. In some instances, an antigen-binding moiety that binds CD117 comprises complementarity determining regions (CDRs) comprising at least or about 95% homology to sequences selected from Tables 9-11 and 13-15. In some instances, an antigenbinding moiety that binds CD117 comprises complementarity determining regions (CDRs) comprising at least or about 97% homology to sequences selected from Tables 9-11 and 13-15. In some instances, the antibody or antigen-binding fragment thereof comprises complementarity determining regions (CDRs) comprising at least or about 99% homology to sequences selected from Tables 9-11 and 13-15. In some instances, an antigen-binding moiety that binds CD117 comprises complementarity determining regions (CDRs) comprising at least or about 100% homology to sequences selected from Tables 9-11 and 13-15. In some instances, an antigenbinding moiety that binds CD117 comprises complementarity determining regions (CDRs) comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 17, 18, 19, 20, 21, 22, or more than 22 amino acids of sequences selected from Tables 9-11 and 13-15.Docket No. 225365-702601

[0122] In some embodiments, an antigen-binding moiety that binds CD117 comprises a heavy chain CDR1 (HCDR1) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table 9. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR1 comprising at least or about 95% homology of any sequence selected from Table 9. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR1 comprising at least or about 97% homology to any sequence selected from Table 9. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR1 comprising at least or about 99% homology to any sequence selected from Table 9. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR1 comprising at least or about 100% homology to any sequence selected from Table 9. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR1 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, or more than 8 amino acids of any sequence selected from Table 9.

[0123] In some embodiments, the CD117-binding region or moiety comprises a heavy chain CDR2 (HCDR2) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table 10. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR2 comprising at least or about 95% homology to any sequence selected from Table 10. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR2 comprising at least or about 97% homology to any sequence selected from SEQ ID NOs: 433-456. In some instances, an antigenbinding moiety that binds CD117 comprises an HCDR2 comprising at least or about 99% homology to any sequence selected from Table 10. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR2 comprising at least or about 100% homology to any sequence selected from Table 10. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR2 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 amino acids of any sequence selected from Table 10.

[0124] In some embodiments, an antigen-binding moiety that binds CD117 comprises a heavy chain CDR3 (HCDR3) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table 11. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR3 comprising at least or about 95% homology to any sequence selected from Table 11. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR3 comprising at least or about 97% homology to any sequence selected from Table 11. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR3 comprising at least or about 99% homology to any sequence selected from Table 11. In some instances, an antigen-binding moiety that binds CD117Docket No. 225365-702601 comprises an HCDR3 comprising at least or about 100% homology to any sequence selected from Table 11. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR3 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or more than 22 amino acids of any sequence selected from Table 11.

[0125] In some embodiments, an antigen-binding moiety that binds CD117 comprises a light chain CDR1 (LCDR1) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table 13. In some instances, an antigen-binding moiety that binds CD117 comprises an LCDR1 comprising at least or about 95% homology of any sequence selected from Table 13. In some instances, an antigen-binding moiety that binds CD117 comprises an LCDR1 comprising at least or about 97% homology to any sequence selected from Table 13. In some instances, an antigen-binding moiety that binds CD117 comprises an HCDR1 comprising at least or about 99% homology to any sequence selected from Table 13. In some instances, an antigen-binding moiety that binds CD117 comprises an LCDR1 comprising at least or about 100% homology to any sequence selected from Table 13. In some instances, an antigen-binding moiety that binds CD117 comprises an LCDR1 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, or more than 8 amino acids of any sequence selected from Table 13.

[0126] In some embodiments, an antigen-binding moiety that binds CD117 comprises a light chain CDR2 (LCDR2) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table 14. In some instances, an antigen-binding moiety that binds CD117 comprises an LCDR2 comprising at least or about 95% homology to any sequence selected from Table 14. In some instances, an antigen-binding moiety that binds CD117 comprises an LCDR2 comprising at least or about 97% homology to any sequence selected from Table 14. In some instances, an antigen-binding moiety that binds CD 117 comprises an LCDR2 comprising at least or about 99% homology to any sequence selected from Table 14. In some instances, an antigen-binding moiety that binds CD117 comprises an LCDR2 comprising at least or about 100% homology to any sequence selected from Table 14. In some instances, an antigen-binding moiety that binds CD117 comprises LCDR2 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more than 12 amino acids of any sequence selected from Table 14.

[0127] In some embodiments, an antigen-binding moiety that binds CD117 comprises a light chain CDR3 (LCDR3) comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table 15. In some instances, an antigen-binding moiety that binds CD117 comprises an LCDR3 comprising at least or about 95% homology to any sequence selected from Table 15. In some instances, anDocket No. 225365-702601 antigen-binding moiety that binds CD117 comprises an LCDR3 comprising at least or about 97% homology to any sequence selected from Table 15. In some instances, an antigen-binding moiety that binds CD 117 comprises an LCDR3 comprising at least or about 99% homology to any sequence selected from Table 15. In some instances, an antigen-binding moiety that binds CD117 comprises an LCDR3 comprising at least or about 100% homology to any sequence selected from Table 15. In some instances, an antigen-binding moiety that binds CD117 comprises an LCDR3 comprising at least a portion having at least or about 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22 or more than 22 amino acids of any sequence selected from Table 15.

[0128] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 409 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 409, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 433 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 433, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 457 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 457, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 505 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 505, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 553 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 553.

[0129] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 410 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 410, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 434 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 434, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 458 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 458, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 506 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 506, an LCDR2 comprising the amino acid sequence of ATS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of ATS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:Docket No. 225365-702601554 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 554.

[0130] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 411 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 411, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 435 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 435, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 459 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 459, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 507 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 507, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:555 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 555.

[0131] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 412 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 412, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 436 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 436, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 460 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 460, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 508 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 508, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:556 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 556.

[0132] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 413 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 413, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 437 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 437, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 461 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 461, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 509 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 509, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 557 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 557.

[0133] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 414 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 414, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 438 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 438, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 462 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 462, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 510 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 510, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 668 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 558.

[0134] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 415 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 415, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 439 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 439, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 463 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 463, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 511 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 511, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:Docket No. 225365-702601559 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 559.

[0135] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 416 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 416, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 440 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 440, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 464 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 464, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 512 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 512, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:560 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 560.

[0136] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 417 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 417, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 441 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 441, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 465 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 465, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 513 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 513, an LCDR2 comprising the amino acid sequence of AAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of AAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:561 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 561.

[0137] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 418 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 418, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 442 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 442, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 466 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 466, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 514 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 514, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:562 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 562.

[0138] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 419 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 461, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 443 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 443, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 467 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 467, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 515 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 515, an LCDR2 comprising the amino acid sequence of ATS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of ATS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:563 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 563.

[0139] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 420 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 420, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 444 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 444, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 468 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 468, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 516 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 516, an LCDR2 comprising the amino acid sequence of KAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of KAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:Docket No. 225365-702601564 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 564.

[0140] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 421 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 421, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 445 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 445, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 469 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 469, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 517 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 517, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:565 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 565.

[0141] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 422 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 422, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 446 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 446, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 470 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 470, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 518 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 518, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:566 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 566.

[0142] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 423 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 423, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 447 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 447, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 471 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 471, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 519 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 519, an LCDR2 comprising the amino acid sequence of SAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 567 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 567.

[0143] In some embodiments, an antigen-binding moiety or a moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 424 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 424, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 448 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 448, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 472 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 472, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 520 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 520, an LCDR2 comprising the amino acid sequence of ATS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of ATS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 568 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 568.

[0144] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 425 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 425, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 449 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 449, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 473 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 473, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 521 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 521, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:Docket No. 225365-702601569 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 569.

[0145] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 426 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 426, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 450 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 450, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 474 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 474, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 522 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 522, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:570 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 570.

[0146] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 427 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 427, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 451 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 451, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 475 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 475, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 523 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 523, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:571 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 571.

[0147] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 428 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 428, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 452 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequenceDocket No. 225365-702601 of SEQ ID NO: 452, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 476 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 476, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 524 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 524, an LCDR2 comprising the amino acid sequence of DAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of DAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:572 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 572.

[0148] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 429 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 429, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 453 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 453, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 477 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 477, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 525 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 525, an LCDR2 comprising the amino acid sequence of GAS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of GAS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO:573 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 573.

[0149] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 430 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 430, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 454 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 454, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 478 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 478, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 526 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 526, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 550 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 550, and an LCDR3 comprising the amino acidDocket No. 225365-702601 sequence of SEQ ID NO: 574 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 574.

[0150] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 431 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 431, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 455 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 455, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 479 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 479, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 527 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 527, an LCDR2 comprising the amino acid sequence of SEQ ID NO: 551 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 551, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 575 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 575.

[0151] In some embodiments, an antigen-binding moiety that binds CD117 comprises: an HCDR1 comprising the amino acid sequence of SEQ ID NO: 432 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 432, an HCDR2 comprising the amino acid sequence of SEQ ID NO: 456 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 456, an HCDR3 comprising the amino acid sequence of SEQ ID NO: 480 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 480, an LCDR1 comprising the amino acid sequence of SEQ ID NO: 528 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 528, an LCDR2 comprising the amino acid sequence of LGS or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of LGS, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 576 or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 576.

[0152] In some instances, an antigen-binding moiety that binds CD117 comprises a heavy chain variable domain comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table 12. In some instances, an antigen-binding moiety that binds CD117 comprises a heavy chain variable domain comprising at least or about 95% sequence identity to any sequence selected from TableDocket No. 225365-70260112. In some instances, an antigen-binding moiety that binds CD117 comprises a heavy chain variable domain comprising at least or about 97% sequence identity to any sequence selected from Table 12. In some instances, an antigen-binding moiety that binds CD117 comprises a heavy chain variable domain comprising at least or about 99% sequence identity to any sequence selected from Table 12. In some instances, an antigen-binding moiety that binds CD117 comprises a heavy chain variable domain comprising at least or about 100% sequence identity to any sequence selected from Table 12. In some instances, an antigen-binding moiety that binds CD117 comprises a heavy chain variable domain comprising at least a portion having at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or more than 150 amino acids of any sequence selected from Table 12.

[0153] In some instances, an antigen-binding moiety that binds CD117 comprises a light chain variable domain comprising at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to any sequence selected from Table 16. In some instances, an antigen-binding moiety that binds CD117 comprises a light chain variable domain comprising at least or about 95% sequence identity to any sequence selected from Table 16. In some instances, an antigen-binding moiety that binds CD117 comprises a light chain variable domain comprising at least or about 97% sequence identity to any sequence selected from Table 16. In some instances, an antigen-binding moiety that binds CD117 comprises a light chain variable domain comprising at least or about 99% sequence identity to any sequence selected from Table 16. In some instances, an antigen-binding moiety that binds CD117 comprises a light chain variable domain comprising at least or about 100% sequence identity to any sequence selected from Table 16. In some instances, an antigen-binding moiety that binds CD117 comprises a light chain variable domain comprising at least a portion having at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or more than 150 amino acids of any sequence selected from Table 16.Table 1. SIGLEC-8 Variable Heavy Chain CDR1 (HCDR1)Docket No. 225365-702601Docket No. 225365-702601Table 2. SIGLEC-8 Variable Heavy Chain CDR2 (HCDR2)Docket No. 225365-702601Docket No. 225365-702601Table 3. SIGLEC-8 Variable Heavy Chain CDR2 (HCDR3)Docket No. 225365-702601Table 4. SIGLEC-8 Variable Heavy ChainDocket No. 225365-702601Docket No. 225365-702601Docket No. 225365-702601Table 5. SIGLEC-8 Variable Light Chain CDR1 (LCDR1)Docket No. 225365-702601Docket No. 225365-702601Table 6. SIGLEC-8 Variable Light Chain CDR2 (LCDR2)Docket No. 225365-702601Table 7. SIGLEC-8 Variable Light Chain CDR3 (LCDR3)Docket No. 225365-702601Docket No. 225365-702601Table 8. SIGLEC-8 Light ChainDocket No. 225365-702601Docket No. 225365-702601Table 9. CD117 Variable Heavy Chain CDR1 (HCDR1) SequencesVariable Chain CDR1 (HCDR1)Docket No. 225365-702601Table 10. CD117 Variable Heavy Chain CDR2 (HCDR2) SequencesDocket No. 225365-702601Table 11. CD117 Variable Heavy Chain CDR3 (HCDR3) SequencesTable 12. CD117 Variable Heavy Chain SequencesDocket No. 225365-702601Table 13. CD117 Variable Light Chain CDR1 (LCDR1) SequencesDocket No. 225365-702601Table 14. CD117 Variable Light Chain CDR2 (LCDR2) SequencesDocket No. 225365-702601Table 15. CD117 Variable Light Chain CDR3 (LCDR3) SequencesTable 16. CD117 Variable Light Chain SequencesDocket No. 225365-702601

[0154] In some embodiments, an engineered protein described herein comprises: (1) an antigen-binding moiety that binds SIGLEC-8 comprising complementarity determining regions (CDRs) comprising the amino acid sequence of any one from Tables 1-3 and Tables 5-7, or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of any one from Tables 1-3 and Tables 5-7; and (2) an antigen-binding moiety that binds CD117 comprising complementarity determining regions (CDRs) comprising the amino acid sequence of any one from Tables 9-11 and Tables 13-15, or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of any one from Tables 9-11 and Tables 13-15.

[0155] In some embodiments, an engineered protein described herein comprises (1) an antigen-binding moiety that binds SIGLEC-8 comprising complementarity determining regions (CDRs) comprising the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 78, SEQ ID NO: 129, SEQ ID NO: 231, AAS, or SEQ ID NO: 333, or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 78, SEQ ID NO: 129, SEQ ID NO: 231, AAS, or SEQ ID NO: 333; and (2) an antigen-Docket No. 225365-702601 binding moiety that binds CD117 comprises complementarity determining regions (CDRs) comprising the amino acid sequence of any one from SEQ ID NO: 415, SEQ ID NO: 439, SEQ ID NO: 463, SEQ ID NO: 511, AAS, SEQ ID NO: 559, or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of any one from SEQ ID NO: 415, SEQ ID NO: 439, SEQ ID NO: 463, SEQ ID NO: 511, AAS, SEQ ID NO: 559.

[0156] In some embodiments, an engineered protein described herein comprises (1) an antigen-binding moiety that binds SIGLEC-8 comprising complementarity determining regions (CDRs) comprising the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 78, SEQ ID NO: 129, SEQ ID NO: 231, AAS, or SEQ ID NO: 333, or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 78, SEQ ID NO: 129, SEQ ID NO: 231, AAS, or SEQ ID NO: 333; and (2) an antigenbinding moiety that binds CD117 comprises complementarity determining regions (CDRs) comprising the amino acid sequence of any one from SEQ ID NO: 422, SEQ ID NO: 446, SEQ ID NO: 470, SEQ ID NO: 518, GAS, SEQ ID NO: 566, or a functional variant thereof having one or two amino acid substitutions with respect to the amino acid sequence of any one from SEQ ID NO: 422, SEQ ID NO: 446, SEQ ID NO: 470, SEQ ID NO: 518, GAS, SEQ ID NO: 566.

[0157] In some embodiments, an engineered protein described herein comprises an amino acid sequence of SEQ ID NO: 604 and an amino acid sequence of SEQ ID NO: 605. In some embodiments, an engineered protein described herein comprises an amino acid sequence of SEQ ID NO: 606 and an amino acid sequence of SEQ ID NO: 607. In some embodiments, an engineered protein described herein comprises an amino acid sequence of SEQ ID NO: 608 and an amino acid sequence of SEQ ID NO: 609. In some embodiments, an engineered protein described herein comprises an amino acid sequence of SEQ ID NO: 610 and an amino acid sequence of SEQ ID NO: 611. In some embodiments, an engineered protein described herein comprises an amino acid sequence of SEQ ID NO: 612 and an amino acid sequence of SEQ ID NO: 613. In some embodiments, an engineered protein described herein comprises an amino acid sequence of SEQ ID NO: 610, an amino acid sequence of SEQ ID NO: 611, an amino acid sequence of SEQ ID NO: 612, and an amino acid sequence of SEQ ID NO: 613.Table 17. Exemplary Heavy Chain and Light Chain SequencesDocket No. 225365-702601Docket No. 225365-702601Docket No. 225365-702601Constant regions

[0158] In some embodiments, multispecific molecules described herein comprise at least one constant region. In some embodiments, bispecific antibodies described herein comprise two constant regions. In some embodiments, the two constant regions are derived from a human IgGl heavy constant chain. In some embodiments, the two constant regions are a first constant region and a second constant region. In some embodiments, the first constant region is engineered to comprise a knob, and the second constant region is engineered to comprise a hole. Accordingly, in some embodiments, the first constant region comprises a mutation at a position T366, per EU numbering, and the second constant region comprises a mutation at a position Y407, per EU numbering. In some embodiments, the first constant region comprises a mutation at a position T366, per EU numbering, and the second constant region comprises mutations at positions T366 and Y407, per EU numbering. In some embodiments, the first constant region comprises a mutation at a position T366, per EU numbering, and the second constant region comprises mutations at positions T366, L368 and Y407, per EU numbering. In some embodiments, the first constant region comprises S354C mutation, T366W mutation, or a combination thereof, per EU numbering, and the second constant region comprises Y349C mutation, T366S mutation, Y407V mutation, or a combination thereof, per EU numbering.

[0159] Alternatively, in some embodiments, bispecific antibodies described herein comprise two constant regions are derived from a human IgGl heavy constant chain, wherein the two constant regions comprise a first constant region and a second constant region, wherein the first constant region comprises at least two mutations and the second constant region comprises at least one mutation. For example, in some embodiments, the first constant region comprises at least two mutations selected from positions L351, F405 and Y407, per EU numbering, and the secondDocket No. 225365-702601 constant region comprises at least one mutation selected from positions T366, K392 and T394, per EU numbering. In some embodiments, the mutation at position L351 comprises L351 Y and L351 A substitutions. In some embodiments, the mutation at position F405 comprises F405A, F405S, F405T and F405V substitutions. In some embodiments, the mutation at position Y407 comprises Y407A, Y407V, Y407S and Y407I substitutions. In some embodiments, the mutation at position T366 comprises T366L, T366M, T366V and T366I substitutions. In some embodiments, the mutation at position K392 comprises K392C, K392M, K392L, K392I, K392E, K392D and K392F substitutions. In some embodiments, the mutation at position T394 comprises T394D, T394W, T394V and T394S substitutions. In some embodiments, the at least two mutations of the first constant region further comprises one or more mutations at positions Q347, Y349, T350, K370, G371, D399 and S400, per EU numbering. In some embodiments, the mutation at position Q347 comprises Q347R, Q347E and Q347K substitutions. In some embodiments, the mutation at position Y349 comprises Y349C substitution. In some embodiments, the mutation at position T350 comprises T350V substitution. In some embodiments, the mutation at position K370 comprises K370T substitution. In some embodiments, the mutation at position G371 comprises G371D and G371S substitutions. In some embodiments, the mutation at position D399 comprises D399C, D399R and D399K substitutions. In some embodiments, the mutation at position S400 comprises S400D, S400K, S400E and S400R substitutions. In some embodiments, the at least two mutations of the second constant region further comprises one or more mutations at positions T350, S354, E357, K360, Q362E, S364, N390, K409 and T411, per EU numbering. In some embodiments, the mutation at position T350 comprises T350V substitution. In some embodiments, the mutation at position S354 comprises S354C substitution. In some embodiments, the mutation at position E357 comprises E357Q substitution. In some embodiments, the mutation at position K360 comprises K360D and K360E substitutions. In some embodiments, the mutation at position Q362 comprises Q362E substitution. In some embodiments, the mutation at position S364 comprises S364R substitution. In some embodiments, the mutation at position N390 comprises N390K, N390R, N390D and N390E substitutions. In some embodiments, the mutation at position K409 comprises K409L, K409M, K409F and K409W substitutions. In some embodiments, the mutation at position T411 comprises T411R, T411D, T411I, T411K, T411E, T411N, T411S and T411L substitutions.

[0160] Alternatively, in some embodiments, bispecific antibodies described herein comprise two constant regions are derived from a human IgGl heavy constant chain, wherein the two constant regions comprise a first constant region and a second constant region, wherein the first constant region and the second constant region are engineered to electrostatically interact with each other. In some embodiments, the first constant region comprises a substitution at K370, per EU numbering, with a negatively charged amino acid residue (e.g., aspartic acid, glutamic acid),Docket No. 225365-702601 and the second constant region comprises a substitution at E357, per EU numbering, with a positively charged amino acid residue (e.g., arginine, lysine, histidine). The EU numbering system is one of several schemes used to number amino acid residues in the constant region of the heavy chain (CH domains) of IgG antibodies. It was originally derived from the sequence of the human IgGl myeloma protein EU, and is commonly used in structural and functional studies of antibodies. In some embodiments, the first constant region comprises a substitution at K392 or K409, per EU numbering, with a negatively charged amino acid residue (e.g., aspartic acid, glutamic acid), and the second constant region comprises a substitution at D399, per EU numbering, with a positively charged amino acid residue (e.g., arginine, lysine, histidine). In some embodiments, the first constant region comprises a substitution at K439, per EU numbering, with a negatively charged amino acid residue (e.g., aspartic acid, glutamic acid), and the second constant region comprises a substitution at D356, per EU numbering, with a positively charged amino acid residue (e.g., arginine, lysine, histidine).

[0161] In some embodiments, the molecule described herein comprises a bi-specific T-cell engager (BiTE) antibody construct and may be referred to herein as a “BiTE molecule”. A BiTe antibody construct is a type of fusion protein. In some embodiments, the BiTE molecule activates T-cell activity. In some embodiments, the BiTE molecule comprises two functional single-chain variable fragments. In some embodiments, the BiTE molecule comprises two binding domains. In some embodiments, bispecific antibodies described herein comprise a SIGLEC-8 binding domain. In some embodiments, bispecific antibodies described herein further comprise a CD117 binding domain.

[0162] In some embodiments, two antibodies are covalently linked by the use chemical crosslinking reagents. In some embodiments, functional antibody fragments generated from their respective parent antibodies by enzymatic digestion or generated through recombinant technologies may be conjugated using bifunctional reagents.Recombinant multispecific antibodies

[0163] In some embodiments, a multispecific antibody described herein is a multivalent antibody engineered to have multiple bindings sites, thereby allowing it to attach to multiple identical antigens on a target molecule simultaneously. In some embodiments, a multivalent antibody described herein is in a “1 + 1 format.” “1 + 1 format” refers to a bispecific antibody design where each antigen binding moiety has one binding site. In some embodiments, a multivalent antibody described herein is in a “1 + 2 format.” “1 + 2 format” refers to a bispecific antibody design where one antigen binding moiety has one binding site and the other antigen binding moiety has two binding sites. In some embodiments, a multivalent antibody described herein is in a “2 + 2 format.” “2 + 2 format” refers to a bispecific antibody design where eachDocket No. 225365-702601 antigen binding moiety has two binding sites. In some embodiments, multispecific antibody formats are generated by genetic engineering techniques using functional antibody fragment such as scFv or Fab fragments as building blocks connected via polypeptide linkers. Formats based on linked functional antibody fragments include tandem scFv (BiTE), diabodies and tandem- diabodies. These formats include diabody-Fc, tandem diabody-Fc, tandem diabody-CH3, (scFv)4- Fc and DVD-Ig. In some embodiments, the multispecific antibody is a Fab2 antibody, a bis-scFv antibody, a diabody, a DVD-Ig, a TandAb, a tandem scFv-Fc, a one-armed tandem scFv-Fc, a DART, a DART-Fc, or a functional fragment thereof.

[0164] Strategies based on forcing the heterodimerization of two heavy chains have been explored. A first approach coined 'knob into hole' aims at forcing the pairing of two different IgG heavy chains by introducing mutations into the CH3 domains to modify the contact interface. On one chain amino acids with large side chains were introduced, to create a 'knob'. Conversely, bulky amino acids were replaced by amino acids with short side chains to create a 'hole' into the other CH3 domain. By co-expressing these two heavy chains, more than 90% heterodimer formation was observed ('knob- hole') versus homodimers formation ('hole -hole' or 'knob-knob'). A similar concept was developed using strand- exchange engineered domain (SEED) human CH3 domains based on human IgG and human IgA sequences. These engineered domains lead to the formation of heterodimeric molecules that can carry two different specificities.Single domain based antibodies

[0165] In some embodiments, the bispecific antibodies described herein are single domain based antibodies. The immune systems of camelids (lamas and camels) and cartilaginous fish (nurse sharks) use single V-domains fused to a Fc demonstrating that a single domain can confer high affinity binding to an antigen. Camelid, shark and even human V domains represent alternatives to antibodies, but they also be used for BsAbs generation. They can be reformatted into a classical IgG in which each arm has the potential to bind two targets either via its VH or VL domain. Bispecific antibodies of the present disclosure can be made by any process disclosed in the application or otherwise known in the art.Dual variable domain Immunoglobulin

[0166] In some embodiments, bispecific antibodies described herein comprise individually encoded peptides or “segments” which, in a single continuous chain, would comprise a compact tertiary structure. The component peptides are chosen so as to be asymmetric in their assumed structure, so as not to self-associate to form homo- multimers, but rather to associate in a complementary fashion, adopting a stable complex which resembles the parent tertiary structure. On the genetic level, these segments may be encoded by interchangeable cassettes with suitable restriction sites. These standardized cassettes may be fused C- or N-terminally to differentDocket No. 225365-702601 recombinant proteins via a linker or hinge in a suitable expression vector system. Polypeptide segments which do not have the ability to assemble as homodimers are derived by cutting a parental polypeptide which has a compact tertiary structure. These polypeptide segments can then be fused to one or more different functional domains at the genetic level. These distinct polypeptide segments which are now fused to one or more functional domains can be, for example, co-expressed resulting in the formation of a native like parental structure attached to functional domains. This parental structure is formed by the dimerization of the polypeptide segments which were derived from the original parental polypeptide. The resulting multifunctional construct would appear as a compact tertiary structure attached to the one or more functional domains. Once structural sub-domains are identified, the protein is dissected in such a way these sub-domains remain intact. As part of this disclosure, DNA sequences, vectors, preferably bicistronic vectors, vector cassettes, can be made and characterized in that they comprise a DNA sequence encoding an amino acid sequence and optionally at least one further (poly)peptide comprised in the multifunctional polypeptide of the invention, and additionally at least one, preferably singular cloning sites for inserting the DNA encoding at least one further functional domain or that they comprise DNA sequences encoding the amino acid sequences, and optionally the further (poly)peptide(s) comprised in the multifunctional polypeptide of the invention and suitable restriction sites for the cloning of DNA sequences encoding the functional domains, such that upon expression of the DNA sequences after the insertion of the DNA sequences encoding the functional domains into said restriction sites, in a suitable host the multifunctional polypeptide of the invention may be formed. Said vector cassette is characterized in that it comprises the inserted DNA sequence(s) encoding said functional domain(s) and host cells transformed with at least one vector or vector cassette of the invention which can be used for the preparation of said bispecific or multi-functional polypeptides. The host cell may be a mammalian, preferably human, yeast, insect, plant or bacterial, preferably E. coli cell. Bispecific antibodies can be prepared by a method which comprises culturing at least two host cells of the invention in a suitable medium, said host cells each producing only one of said first and said second amino acid sequences attached to at least one further functional domain, recovering the amino acid sequences, mixing thereof under mildly denaturing conditions and allowing in vitro folding of the multifunctional polypeptide of the invention from said amino acid sequences. The method may be characterized in that the further amino acid sequences attached to at least one further functional domain are / is produced by at least one further host cell not producing said first or second amino acid sequence. Additionally, the method may be characterized in that at least one further amino acid sequence attached to at least one further functional domain is produced by the host cell of the invention producing said first or second amino acid sequence.Docket No. 225365-702601

[0167] In some embodiments, when either the second or the first portion of an antibody construct described herein comprises two antibody variable domains, these two antibody variable domains can be a VH- and VL-domain which are associated with one another. However, it is also contemplated that the two antibody variable domains comprised in either the second or the first portion may be two VH domains or two VL regions which are associated with one another. In the event that the two antibody variable domains of the first or second portion are covalently associated with one another, the two antibody variable domains may be designed as an scFv fragment, meaning that the two domains are separated from one another by a peptide linker long enough to allow intermolecular association between these two domains. The design of linkers suitable for this purpose is described in the prior art, for example in the granted patents EP 623 679 Bl, U.S. Pat. No. 5,258,498, EP 573 551 Bl and U.S. Pat. No. 5,525,491. In other words, a bispecific antibody may be a construct with a total of three antibody variable domains. One antibody variable domain specifically binds alone, i.e., without being paired with another antibody variable domain (a) either to a human immune effector cell by specifically binding to an effector antigen on the human immune effector cell or to a target cell, while the remaining two antibody variable domains together specifically bind (b) either to the target antigen on the target cell or to a human immune effector cell by specifically binding to an effector antigen on the human immune effector cell, respectively. In this case, the presence of three antibody variable domains in the bispecific antibody entails unique advantages. Often, an scFv exhibiting the desired binding specificity for a target antigen is already known and optimized, and omitting one of its two antibody variable domains would abolish or at least attenuate its binding characteristics. Such an scFv may make up part of an antibody construct described herein. Specifically, such a three-domain antibody may advantageously comprise an entire scFv as either its effector antigen- or target antigen-conferring portion. Effectively, then, this allows a bispecific antibody to be formed starting from a desired scFv by simple incorporation of only one additional antibody variable domain into the same polypeptide chain as the scFv, wherein the one additional antibody variable domain incorporated has an antigen binding specificity different than that of the scFv. The first and second portions of the bispecific antibody may be separated from one another by a synthetic polypeptide spacer moiety, which covalently (i.e., peptidically) links either the C-terminus of the first portion with the N-terminus of the second portion, or the C-terminus of the second portion with the N-terminus of the first portion. As such, the portions of these bispecific antibodies may be arranged, as either N-(first portion)-(second portion)-C or N-(second portion)-(first portion)-C. In some embodiments, binding sites of a second specificity are fused to the N- or C-terminus of the heavy or light chain, e.g., in the form of an scFv fragment or a variable single domain, resulting in bispecific, tetraval ent molecules (e.g., FIG. 2D). Bispecific molecules generated through fusionDocket No. 225365-702601 of an scFv fragment to a mAb offer great flexibility. ScFv molecules can be linked to the N- terminus but also the C-terminus of the heavy or light chain variable domain of a mAb, generally without compromising productivity or antigen-binding activity. This group of bispecific molecules also includes DVD-Igs, where a second VH and VL domain is fused to the heavy and light chain, respectively, of a mAb, two-in-one antibodies, where a second specificity is introduced into the natural binding site of an IgG molecule, and mAb2 molecules, where a second specificity is built into the CH3 domain of the Fc region. A characteristic feature of all these molecules is a symmetry caused by dimeric assembly of two identical heavy chains, an intrinsic property of these chains.

[0168] Heavy chain heterodimerization can be achieved by engineering a charged CH3 interface to introduce an electrostatic steering effect or using the strand-exchange engineered domain technology (SEEDbody) with CH3 sequences composed of alternating segments from human IgA and IgG. In contrast to bispecific IgG-like molecules, these bispecific antibodies are bivalent with a size basically identical to that of IgG. Fc heterodimerization was recently applied to generate a trivalent, bispecific molecule fusing a VH and a VL domain to the C-termini of the engineered heavy chains (HA-TF Fc variant.) Bispecific antibodies with a molecular mass in the range of 50 -100 kDa can be generated by combining the variable domains of two antibodies. For example, two scFv have been connected by a more or less flexible peptide linker in a tandem orientation (tandem scFv, taFv, tascFv), which can be extended further by additional scFv, e.g., generating bispecific or trispecific triple bodies (sctb). Diabodies are heterodimeric molecules composed of the variable domains of two antibodies arranged either in the order VHA- VLB and VHB-VLA (VH-VL orientation) or in the order VLA-VHB and VLB- VHA (VL-VH orientation). The linker connecting the two domains within one chain is approximately 5 residues leading, after co- expression of the two chains within one cell, to a head-to-tail assembly and hence formation of a compact molecule with two functional binding sites. The diabody (Db) format was further stabilized by introducing interchain disulfide bonds (dsDb, DART molecules) or by generating a single-chain derivative (scDb). ScDbs can be converted into tetravalent molecules by reducing the middle linker, resulting in homodimerzation of two chains. Small bispecific molecules have also been produced by fusing a scFv to the heavy or light chain of a Fab fragment. Furthermore, tandem scFv, diabodies and scDb have been fused to the Fc or a CH3 domain to generate tetravalent derivatives. Also, scFv can be combined with Fc or CH3 domains to generate tetravalent molecules, e.g., fusing scFvs to the N- and C-terminus of an Fc fragment, or using the knobs-into- holes approach to generate bivalent scFv-Fc or scFv-CH3 molecules. A different approach for the generation of bispecific antibodies of the present invention is the dock-and-lock method (DNL). Many of the established bispecific antibody formats can also be combined with additional proteins and components, e.g., drugs, toxins, enzymes and cytokines, enabling dual targeting and deliveryDocket No. 225365-702601 of a fusion partner. In addition, fusion to plasma proteins such as serum albumin or albuminbinding moi eties can be applied to extend the plasma half- life of bispecific antibodies.Structure of Bispecific Antibodies

[0169] In one example, a bispecific antibody may be a binding protein comprising a first polypeptide chain, wherein the polypeptide chain comprises VHl-(Xl)n-VH2-C-(X2)n, wherein VH1 is a first heavy chain variable domain, VH2 is a second heavy chain variable domain, C is a constant domain, XI represents a polypeptide linker, X2 represents an Fc region and n is 0 or 1. In some embodiments, the VH1 and VH2 in the binding protein may be heavy chain variable domains selected from the group consisting of a murine heavy chain variable domain, a human heavy chain variable domain, a CDR grafted heavy chain variable domain, and a humanized heavy chain variable domain. VH1 and VH2 may be capable of binding different antigens. C may be a heavy chain constant domain. For example, XI is a linker peptide. For example, XI is a linker listed herein. In an embodiment, X2 is an Fc region. In another embodiment, X2 is a variant Fc region. In some embodiments, VH1 binds a first antigen and VH2 binds a second antigen. In some embodiments, VH1 binds a second antigen and VH2 binds a first antigen.

[0170] In one example, a bispecific antibody is a binding protein comprising a second polypeptide chain, wherein the polypeptide chain comprises VLl-(Xl)n-VL2-C-(X2)n, wherein VL1 is a first light chain variable domain, VL2 is a second light chain variable domain, C is a constant domain, XI represents a polypeptide linker, X2 represents an Fc region and n is 0 or 1. In some embodiments, the VL1 and VL2 in the binding protein may be light chain variable domains selected from the group consisting of a murine light chain variable domain, a human light chain variable domain, a CDR grafted light chain variable domain, and a humanized light chain variable domain. VL1 and VL2 may be capable of binding different antigens. C may be a heavy chain constant domain. For example, XI is a linker peptide. For example, XI is a linker listed herein. In an embodiment, X2 is an Fc region. In another embodiment, X2 is a variant Fc region. In some embodiments, VL1 binds a first antigen and VL2 binds a second antigen. In some embodiments, VL1 binds a first antigen and VL2 binds a second antigen. In some embodiments, a bispecific antibody construct comprises both the first polypeptide chain and the second polypeptide chain. In some embodiments, a bispecific antibody of the present disclosure is a dualvariable domain immunoglobulin (DVD-Ig™) which combines the target binding domains of two monoclonal antibodies via flexible naturally occurring linkers, which yields a tetravalent IgG - like molecule.

[0171] The present disclosure additionally provides a method of making a DVD-Ig binding protein by preselecting the parent antibodies against a first antigen and a second antigen. A method of making a Dual Variable Domain Immunoglobulin that binds two antigens comprises the stepsDocket No. 225365-702601 of a) obtaining a first parent antibody, or antigen binding fragment thereof, that binds a first antigen; b) obtaining a second parent antibody or antigen binding fragment thereof, that binds a second antigen; c) constructing two copies of a first polypeptide chains, each of which comprises VHl-(Xl)n-VH2-C-(X2)n, wherein, VH1 is a first heavy chain variable domain obtained from said first parent antibody, or antigen binding fragment thereof; VH2 is a second heavy chain variable domain obtained from said second parent antibody or antigen binding fragment thereof, which can be the same as or different from the first parent antibody; C is a heavy chain constant domain; (Xl)n is a linker wherein said (Xl)n is either present or absent; and (X2)n is an Fc region, d) constructing two copies of a second polypeptide chains each of which comprises VLl-(Xl)n- VL2-C-(X2)n, wherein, VL1 is a first light chain variable domain obtained from said first parent antibody, or antigen binding fragment thereof; VL2 is a second light chain variable domain obtained from said second parent antibody, or antigen binding thereof, which can be the same as or different from the first parent antibody; C is a light chain constant domain; (Xl)n is a linker, wherein said (Xl)n is either present or absent; and (X2)n does not comprise an Fc region, wherein said (X2)n is either present or absent; and e) expressing two copies of said first and second polypeptide chains; such that a DVD-Ig binds said first antigen and said second antigen is generated.Generation of First Antigen Binding and / or Second Antigen Binding Domains

[0172] Disclosed herein are dual variable domain immunoglobulins. The variable domains of the DVD binding protein can be obtained from parent antibodies, including polyclonal and mAbs that bind antigens of interest. These antibodies may be naturally occurring or may be generated by recombinant technology, or can be designed de novo. MAbs can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. Monoclonal antibodies can be prepared by methods disclosed herein.

[0173] Disclosed herein are dual variable domain immunoglobulins comprising variable domains. The dual variable domain immunoglobulin (DVD-Ig) molecule is designed such that two different light chain variable domains (VL) from the two different parent monoclonal antibodies are linked in tandem directly or via a short linker by recombinant DNA techniques, followed by the light chain constant domain, and optionally, anFc region. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CHI and Fc region. The variable domains can be obtained using recombinant DNA techniques from a parent antibody generated by any one of the methods described herein. The variable domain may be a murine heavy or light chain variable domain, a CDR a human heavy or light chain variable domain. The first and second variable domains may be linked directly to eachDocket No. 225365-702601 other using recombinant DNA techniques, linked via a linker sequence, or the two variable domains are linked. The variable domains may bind the same antigen or may bind different antigens. The constant domain may be linked to the two linked variable domains using recombinant DNA techniques. Sequence comprising linked heavy chain variable domains may be linked to a heavy chain constant domain and sequence comprising linked light chain variable domains is linked to a light chain constant domain. The constant domains may also be human heavy chain constant domain and human light chain constant domain respectively. The DVD heavy chain may be further linked to an Fc region. The Fc region may be a native sequence Fc region, or a variant Fc region, or a human Fc region. Two heavy chain DVD polypeptides and two light chain DVD polypeptides may be combined to form a DVD-Ig molecule.Fc region variants

[0174] In some embodiments, an Fc region described herein is derived from an IgG heavy constant chain or an IgA heavy constant chain. In some embodiments, the IgG heavy constant chain comprises a IgGl heavy chain constant chain. In some embodiments, the IgGl heavy constant chain comprises a human IgGl heavy chain constant chain. An amino acid sequence of wildtype human IgGl heavy chain constant chain is provided in TABLE 18. In some embodiments, the Fc region is engineered to not bind to Fc gamma receptor (FcyR). In some embodiments, the FcyR comprises FcyRI, FcyRII and FcyRIII.TABLE 18. Amino acid sequence of Fc region of human IgGlDocket No. 225365-702601

[0175] In some embodiments, the Fc region comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a corresponding parent sequence of the Fc region of IgGl (SEQ ID NO: 601).

[0176] In some embodiments, the Fc region described herein comprises one or more mutations relative to a corresponding parent sequence of the Fc region of IgGl (SEQ ID NO: 601). In some embodiments, the Fc region of IgGl comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen or at least fifteen mutations relative to a corresponding parent sequence of SEQ ID NO: 601. In some embodiments, the Fc region described herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen or at least fifteen deletions, substitutions, additions or combinations thereof relative to a corresponding parent sequence of SEQ ID NO: 453. In some embodiments, the Fc region described herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen or at least fifteen substitutions relative to a corresponding parent sequence of SEQ ID NO: 601.

[0177] In some embodiments, a Fc region described herein is derived from a human IgGl heavy chain constant chain. In some embodiments, the human IgGl heavy chain constant chain comprises at least one substitution, at least two substitutions, at least three substitutions, at least four substitutions, at least five substitutions, at least six substitutions or at least seven substitutions. In some embodiments, the at least one substitution is selected from the positions N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396, per EU numbering. In some embodiments, the at least two substitutions are selected from the positions N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396, per EU numbering. In some embodiments, the at least three substitutions are selected from the positions N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396, per EU numbering. In someDocket No. 225365-702601 embodiments, the at least four substitutions are selected from the positions N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396, per EU numbering. In some embodiments, the at least five substitutions are selected from the positions N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396, per EU numbering. In some embodiments, the at least six substitutions are selected from the positions N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396, per EU numbering. In some embodiments, the at least seven substitutions are selected from the positions N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396, per EU numbering. In some embodiments, the substitutions at the position N297 comprises N297A and N297Q substitutions. In some embodiments, the substitutions at the position C226 comprises C226S substitution. In some embodiments, the substitutions at the position C229 comprises C229S substitution. In some embodiments, the substitutions at the position E233 comprises E233P and E233D substitutions. In some embodiments, the substitutions at the position L234 comprises L234A, L234D, L234E, L234G, L234H, L234K, L234Q, L234R, L234S, L234T and L234F substitutions. In some embodiments, the substitutions at the position L235 comprises L235A, L235S, L235T, L235H, L235K, L235Q, L235D, L235I, L235V, L235R, L235E and L235G substitutions. In some embodiments, the substitutions at the position G236 comprises G236R substitution. In some embodiments, the substitutions at the position G237 comprises G237A and G237D substitutions. In some embodiments, the substitutions at the position P238 comprises P238A, P238D and P238S substitutions. In some embodiments, the substitutions at the position F243 comprises F243L substitution. In some embodiments, the substitutions at the position M252 comprises M252Y substitution. In some embodiments, the substitutions at the position S254 comprises S254T substitution. In some embodiments, the substitutions at the position T256 comprises T256E substitution. In some embodiments, the substitutions at the position D265 comprises D265A substitution. In some embodiments, the substitutions at the position S267 comprises S267E substitution. In some embodiments, the substitutions at the position H268 comprises H268A and H268D substitutions. In some embodiments, the substitutions at the position D270 comprises D270A substitution. In some embodiments, the substitutions at the position P271 comprises P271 G substitution. In some embodiments, the substitutions at the position R292 comprises R292P substitution. In some embodiments, the substitutions at the position Y300 comprises Y300L substitutions. In some embodiments, the substitutions at the position K322 comprises K322A andDocket No. 225365-702601K322Q substitutions. In some embodiments, the substitutions at the position A327 comprises A327Q and A327G substitutions. In some embodiments, the substitutions at the position L328 comprises L328E and L328F substitutions. In some embodiments, the substitutions at the position P329 comprises P329G and P329A substitutions. In some embodiments, the substitutions at the position A330 comprises A330S, A330R and A330L substitutions. In some embodiments, the substitutions at the position P331 comprises P331S substitution. In some embodiments, the substitutions at the position P396 comprises P396L substitution.

[0178] In some embodiments, a human IgGl heavy chain constant chain described herein comprises two substitutions. In some embodiments, the two substitutions are located at positions L234 and L235, per EU numbering. In some embodiments, the two substitutions are L234A and L235A substitutions.

[0179] In some embodiments, a human IgGl heavy chain constant chain described herein comprises three substitutions. In some embodiments, the three substitutions are located at positions L234, L235 and P329, per EU numbering. In some embodiments, the two substitutions are L234A, L235A and P329A substitutions. In some embodiments, the three substitutions are located at positions M252, S254 and T256, per EU numbering. In some embodiments, the three substitutions are M252T, S254T and T256E substitutions.

[0180] In some embodiments, the Fc region comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a corresponding parent sequence of the Fc region of IgG2 (SEQ ID NO: 602).

[0181] In some embodiments, the Fc region described herein comprises one or more mutations relative to a corresponding parent sequence of the Fc region of IgG2 (SEQ ID NO: 602). In some embodiments, the Fc region of IgG2 comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen or at least fifteen mutations relative to a corresponding parent sequence of SEQ ID NO: 454. In some embodiments, the Fc region described herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen or at least fifteen deletions, substitutions, additions or combinations thereof relative to a corresponding parent sequence of SEQ ID NO: 602. In some embodiments, the Fc region described herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen or at least fifteen substitutions relative to a corresponding parent sequence of SEQ ID NO: 602.Docket No. 225365-702601

[0182] In some embodiments, a Fc region described herein is derived from a human IgG2 heavy chain constant chain. In some embodiments, the human IgG2 heavy chain constant chain comprises at least one substitution, at least two substitutions, at least three substitutions, at least four substitutions, at least five substitutions, at least six substitutions or at least seven substitutions. In some embodiments, the at least one substitution is selected from the positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331, per EU numbering. In some embodiments, the at least two substitutions are selected from the positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331, per EU numbering. In some embodiments, the at least three substitutions are selected from the positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331, per EU numbering. In some embodiments, the at least four substitutions are selected from the positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331, per EU numbering. In some embodiments, the at least five substitutions are selected from the positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331, per EU numbering. In some embodiments, the at least six substitutions are selected from the positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331, per EU numbering. In some embodiments, the at least seven substitutions are selected from the positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331, per EU numbering. In some embodiments, the substitutions at the position C232 comprises C232S substitution. In some embodiments, the substitutions at the position C233 comprises C233S substitution. In some embodiments, the substitutions at the position V234 comprises V234A substitution. In some embodiments, the substitutions at the position G237 comprises G237A substitution. In some embodiments, the substitutions at the position P238 comprises P238S substitution. In some embodiments, the substitutions at the position M252 comprises M252Y substitution. In some embodiments, the substitutions at the position S254 comprises S254T substitution. In some embodiments, the substitutions at the position T256 comprises T256E substitution. In some embodiments, the substitutions at the position H268 comprises H268A, H268E and H268Q substitutions. In some embodiments, the substitutions at the position N297 comprises N297A and N297Q substitutions. In some embodiments, the substitutions at the position V309 comprises V309L substitution. In some embodiments, the substitutions at the position A330 comprises A330S substitution. In some embodiments, the substitutions at the position P331 comprises P331 S substitution.

[0183] In some embodiments, an Fc region comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to a corresponding parent sequence of the Fc region of IgG4. In someDocket No. 225365-702601 embodiments, an Fc region comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to SEQ ID NO: 603.

[0184] In some embodiments, an Fc region described herein comprises one or more mutations relative to a corresponding parent sequence of the Fc region of IgG4. In some embodiments, an Fc region described herein comprises one or more mutations relative to SEQ ID NO: 603. In some embodiments, the Fc region of IgG4 comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen or at least fifteen mutations relative to a corresponding parent sequence of SEQ ID NO: 603. In some embodiments, the Fc region described herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen or at least fifteen deletions, substitutions, additions or combinations thereof relative to a corresponding parent sequence of SEQ ID NO: 603. In some embodiments, the Fc region described herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen or at least fifteen substitutions relative to a corresponding parent sequence of SEQ ID NO: 603.

[0185] In some embodiments, an Fc region described herein is derived from a human IgG4 heavy chain constant chain. In some embodiments, the human IgG4 heavy chain constant chain comprises at least one substitution, at least two substitutions, at least three substitutions, at least four substitutions, at least five substitutions, at least six substitutions or at least seven substitutions. In some embodiments, the at least one substitution is selected from the positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394, per EU numbering. In some embodiments, the at least two substitutions are selected from the positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394, per EU numbering. In some embodiments, the at least three substitutions are selected from the positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394, per EU numbering. In some embodiments, the at least four substitutions are selected from the positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394, per EU numbering. In some embodiments, the at least five substitutions are selected from the positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394, per EU numbering. In some embodiments, the at least six substitutions are selected from the positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394, per EU numbering. In some embodiments, the at least seven substitutions are selectedDocket No. 225365-702601 from the positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394, per EU numbering. In some embodiments, the substitutions at the position S228 comprises S228P substitution. In some embodiments, the substitutions at the position E233 comprises E233P substitution. In some embodiments, the substitutions at the position F234 comprises F234V substitution. In some embodiments, the substitutions at the position L235 comprises L235A substitution. In some embodiments, the substitutions at the position G237 comprises G237A substitution. In some embodiments, the substitutions at the position S241 comprises S241P substitution. In some embodiments, the substitutions at the position L248 comprises L248E substitution. In some embodiments, the substitutions at the position M252 comprises M252Y substitution. In some embodiments, the substitutions at the position S254 comprises S254T substitution. In some embodiments, the substitutions at the position T256 comprises T256E substitution. In some embodiments, the substitutions at the position N297 comprises N297A and N297Q substitutions. In some embodiments, the substitutions at the position E318 comprises E318A substitution. In some embodiments, the substitutions at the position T394 comprises T394D substitution.

[0186] The design of the “dual-specific multivalent full length binding proteins” of the present disclosure leads to a dual variable domain light chain and a dual variable domain heavy chain which assemble primarily to the desired “dual-specific multivalent full length binding proteins”. Construction of DVD Molecules

[0187] Provided herein are dual variable domain immunoglobulins. The dual variable domain immunoglobulin (DVD-Ig) molecule is designed such that two different light chain variable domains (VL) from the two parent monoclonal antibodies, which can be the same or different, are linked in tandem directly or via a short linker by recombinant DNA techniques, followed by the light chain constant domain, and optionally, an Fc region. Similarly, the heavy chain comprises two different heavy chain variable domains (VH) linked in tandem, followed by the constant domain CHI and Fc region.

[0188] Provided herein are dual variable domain immunoglobulins comprising variable domains. The variable domains can be obtained using recombinant DNA techniques from a parent antibody generated by any one of the methods described herein. In an embodiment, the variable domain is a murine heavy or light chain variable domain. In another embodiment, the variable domain is a CDR grafted or a humanized variable heavy or light chain domain. In an embodiment, the variable domain is a human heavy or light chain variable domain.

[0189] Disclosed herein are dual variable domain immunoglobulins comprising a first variable domain and a second variable domain. In one embodiment the first and second variable domains are linked directly to each other using recombinant DNA techniques. In another embodiment theDocket No. 225365-702601 variable domains are linked via a linker sequence. In an embodiment, two variable domains are linked. Three or more variable domains may also be linked directly or via a linker sequence. The variable domains may bind the same antigen or may bind different antigens. DVD-Ig molecules of the invention may include one immunoglobulin variable domain and one non-immunoglobulin variable domain, such as ligand binding domain of a receptor, or an active domain of an enzyme. DVD-Ig molecules may also comprise two or more non-Ig domains.

[0190] In an embodiment a constant domain is linked to the two linked variable domains using recombinant DNA techniques. In an embodiment, sequence comprising linked heavy chain variable domains is linked to a heavy chain constant domain and sequence comprising linked light chain variable domains is linked to a light chain constant domain. In an embodiment, the constant domains are human heavy chain constant domain and human light chain constant domain respectively. DVD-Ig molecules described herein may include a DVD heavy chain. In an embodiment, the DVD heavy chain is further linked to an Fc region. The Fc region may be a native sequence Fc region, or a variant Fc region. In another embodiment, the Fc region is a human. In another embodiment two heavy chain DVD polypeptides and two light chain DVD polypeptides are combined to form a DVD-Ig molecule.

[0191] Binding proteins of the present invention may be produced by any of a number of techniques known in the art. For example, expression from host cells, wherein expression vector(s) encoding the DVD heavy and DVD light chains is (are) transfected into a host cell by standard techniques. The various forms of the term “transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE-dextran transfection and the like. Although it is possible to express the DVD proteins of the invention in either prokaryotic or eukaryotic host cells, DVD proteins are expressed in eukaryotic cells, for example, mammalian host cells, because such eukaryotic cells (and in particular mammalian cells) are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active DVD protein.

[0192] Exemplary mammalian host cells for expressing the recombinant antibodies of the invention include Chinese Hamster Ovary (CHO cells), NSO myeloma cells, COS cells, SP2 and PER.C6 cells. When recombinant expression vectors encoding DVD proteins are introduced into mammalian host cells, the DVD proteins are produced by culturing the host cells for a period of time sufficient to allow for expression of the DVD proteins in the host cells or secretion of the DVD proteins into the culture medium in which the host cells are grown. DVD proteins can be recovered from the culture medium using standard protein purification methods.Docket No. 225365-702601

[0193] In an exemplary system for recombinant expression of DVD proteins in constructs described herein, a recombinant expression vector encoding both the DVD heavy chain and the DVD light chain is introduced into dhfr-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the DVD heavy and light chain genes are each operatively linked to CMV enhancer / AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The recombinant expression vector also carries a DHFR gene, which allows for selection of CHO cells that have been transfected with the vector using methotrexate selection / amplification. The selected transformant host cells are cultured to allow for expression of the DVD heavy and light chains and intact DVD protein is recovered from the culture medium. Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the DVD protein from the culture medium. Still further the invention provides a method of synthesizing a DVD protein of the invention by culturing a host cell of the invention in a suitable culture medium until a DVD protein of the invention is synthesized. The method can further comprise isolating the DVD protein from the culture medium.

[0194] An important feature of DVD-Ig is that it can be produced and purified in a similar way as a conventional antibody. The production of DVD-Ig results in a homogeneous, single major product with desired dual-specific activity, without any sequence modification of the constant region or chemical modifications of any kind. Other previously described methods to generate “bispecific”, “multi-specific”, and “multi-specific multivalent” full length binding proteins do not lead to a single primary product but instead lead to the intracellular or secreted production of a mixture of assembled inactive, mono-specific, multi-specific, multivalent, full length binding proteins, and multivalent full length binding proteins with combination of different binding sites. In some embodiments, there are 16 possible combinations of heavy and light chains. Consequently only 6.25% of protein is likely to be in the desired active form, and not as a single major product or single primary product compared to the other 15 possible combinations. Separation of the desired, fully active forms of the protein from inactive and partially active forms of the protein using standard chromatography techniques, typically used in large scale manufacturing, is yet to be demonstrated.

[0195] The design of the dual-specific multivalent full length binding proteins for use in constructs described herein leads to a dual variable domain light chain and a dual variable domain heavy chain which assemble primarily to the desired dual-specific multivalent full length binding proteins.

[0196] In some embodiments, at least 50%, at least 75% and at least 90% of the assembled, and expressed dual variable domain immunoglobulin molecules are the desired dual-specificDocket No. 225365-702601 tetravalent protein. This aspect of the invention particularly enhances the commercial utility of the invention. Therefore, the present invention includes a method to express a dual variable domain light chain and a dual variable domain heavy chain in a single cell leading to a single primary product of a dual-specific tetravalent full length binding protein.

[0197] Provided herein are methods of expressing a dual variable domain light chain and a dual variable domain heavy chain in a single cell leading to a primary product of a dual-specific tetravalent full length binding protein, where the primary product is more than 50% of all assembled protein, comprising a dual variable domain light chain and a dual variable domain heavy chain.

[0198] Provided herein are methods of expressing a dual variable domain light chain and a dual variable domain heavy chain in a single cell leading to a single primary product of a dualspecific tetraval ent full length binding protein, where the primary product is more than 75% of all assembled protein, comprising a dual variable domain light chain and a dual variable domain heavy chain.

[0199] Provided herein are methods of expressing a dual variable domain light chain and a dual variable domain heavy chain in a single cell leading to a single primary product of a dualspecific tetravalent full length binding protein, where the primary product is more than 90% of all assembled protein, comprising a dual variable domain light chain and a dual variable domain heavy chain.Binding Affinity

[0200] Binding affinity is generally represented by the dissociation constant (KD). KD values for antibodies can be determined by any of the methods known in the art. Exemplary methods for determining KD includes by using surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), spectroscopic assays, biolayer interferometry (BLI), and grating-coupled interferometry (GCI).

[0201] In some embodiments, a multispecific antibody described herein comprises a binding affinity in a range of from 1 pM to 1 pM, from 10 pM to 1 pM, from 100 pM to 1 pM, from 1 nM to 1 pM, from 10 nM to 1 pM, from 100 nM to 1 pM, from 500 nM to 1 pM, from 1 pM to 500 nM, from 10 pM to 500 nM, from 100 pM to 500 nM, from 1 nM to 500 nM, from 10 nM to 500 nM, from 100 nM to 500 nM, from 1 pM to 100 nM, from 10 pM to 100 nM, from 100 pM to 100 nM, from 1 nM to 100 nM, from 10 nM to 100 nM, from 1 pM to 10 nM, from 10 pM to 10 nM, from 100 pM to 10 nM, from 1 nM to 10 nM, from 1 pM to 1 nM, from 10 pM to 1 nM, from 100 pM to 1 nM, from 1 pM to 100 pM, or from 10 pM to 100 pM.

[0202] In some embodiments, aBsAb antibody described herein comprises an average binding affinity for target antigen is in a range of from 1 pM to 1 pM, from 10 pM to 1 pM, from 100 pMDocket No. 225365-702601 to 1 pM, from 1 nM to 1 pM, from 10 nM to 1 pM, from 100 nM to 1 pM, from 500 nM to 1 pM, from 1 pM to 500 nM, from 10 pM to 500 nM, from 100 pM to 500 nM, from 1 nM to 500 nM, from 10 nM to 500 nM, from 100 nM to 500 nM, from 1 pM to 100 nM, from 10 pM to 100 nM, from 100 pM to 100 nM, from 1 nM to 100 nM, from 10 nM to 100 nM, from 1 pM to 10 nM, from 10 pM to 10 nM, from 100 pM to 10 nM, from 1 nM to 10 nM, from 1 pM to 1 nM, from 10 pM to 1 nM, from 100 pM to 1 nM, from 1 pM to 100 pM, or from 10 pM to 100 pM.

[0203] In some embodiments, a multispecific antibody described herein comprises at least two antigen-binding moieties. In some embodiments, the at least two antigen-binding moieties comprises a first antigen-binding moiety and a second antigen-binding moiety. In some embodiments, the first antigen-binding moiety comprises a SIGLEC-8 binding moiety. In some embodiments, the second antigen-binding moiety comprises a CD117 binding moiety. In some embodiments, the binding affinity of multispecific antibody is measured with only one target molecule.

[0204] In some embodiments, a multispecific antibody described herein comprises a SIGLEC- 8 binding moiety and a CD117 binding moiety. In some embodiments, a binding affinity of the SIGLEC-8 binding moiety for SIGLEC-8 is lower than a binding affinity of CD117 binding moiety for CD117. In some embodiments, the multispecific antibody comprises the binding affinity of the SIGLEC-8 binding moiety for SIGLEC-8 that is at least two times, at least three time, at least four times, at least five times, at least ten times, at least fifteen times, at least twenty times, at least forty times, at least sixty times, at least eight times, or at least hundred times higher than the binding affinity of the CD117 binding moiety for CD117. Alternatively, in some embodiments, a binding affinity of CD117 binding moiety for CD117 is lower than a binding affinity of the SIGLEC-8 binding moiety for SIGLEC-8. Accordingly, in some embodiments, the multispecific antibody comprises a binding affinity of the CD117 binding moiety for CD117 that is at least two times, at least three time, at least four times, at least five times, at least ten times, at least fifteen times, at least twenty times, at least forty times, at least sixty times, at least eight times, or at least hundred times higher than the binding affinity of the SIGLEC-8 binding moiety for SIGLEC-8.

[0205] In some embodiments, a multispecific antibody described herein comprises a SIGLEC- 8 binding moiety and a CD117 binding moiety that binds CD117. In some embodiments, a binding affinity of the SIGLEC-8 binding moiety for SIGLEC-8 is lower than a binding affinity of the CD117 binding moiety for CD117. In some embodiments, a binding affinity of the SIGLEC-8 binding moiety for SIGLEC-8 is at least two times, at least three time, at least four times, at least five times, at least ten times, at least fifteen times, at least twenty times, at least forty times, at least sixty times, at least eight times, or at least hundred times higher than a binding affinity of the CD117 binding moiety for CD117. Alternatively, in some embodiments, a binding affinity of theDocket No. 225365-702601CD117 binding moiety for an CD117 is lower than a binding affinity of the SIGLEC-8 binding moiety for SIGLEC-8. Accordingly, in some embodiments, a binding affinity of the CD117 binding moiety for CD117 is at least two times, at least three time, at least four times, at least five times, at least ten times, at least fifteen times, at least twenty times, at least forty times, at least sixty times, at least eight times, or at least hundred times higher than a binding affinity of the SIGLEC-8 binding moiety for SIGLEC-8.

[0206] Alternatively, in some embodiments, multispecific antibodies described herein undergo cooperative binding event. For example, in some embodiments, a multispecific antibody undergoes a positive cooperative binding event, wherein binding of the multispecific antibody to a first target molecule results in increase in binding affinity for a second target molecule. For example, in some embodiments, a binding affinity of a SIGLEC-8 bound multispecific antibody for CD117 is lower than a binding affinity of a SIGLEC-8 unbound multispecific antibody for CD117. In some embodiments, a binding affinity of CD117 bound multispecific antibody for SIGLEC-8 is lower than a binding affinity of CD 117 unbound multispecific antibody for SIGLEC- 8. Alternatively, in some embodiments, a multispecific antibody undergoes a negative cooperative binding event, wherein binding of the multispecific antibody to a first target molecule results in decrease in binding affinity for a second target molecule. For example, in some embodiments, a binding affinity of a SIGLEC-8 bound multispecific antibody for CD117 is higher than a binding affinity of a SIGLEC-8 unbound multispecific antibody for CD117. In some embodiments, a binding affinity of CD117 bound multispecific antibody for SIGLEC-8 is higher than a binding affinity of CD117 unbound multispecific antibody for SIGLEC-8. In some embodiments, a binding affinity of SIGLEC-8 bound multispecific antibody for CD117 is lower than a binding affinity of a SIGLEC-8 unbound multispecific antibody for CD117. In some embodiments, a binding affinity of CD117 bound multispecific antibody for SIGLEC-8 is lower than a binding affinity of CD117 unbound multispecific antibody for SIGLEC-8. Alternatively, in some embodiments, a multispecific antibody undergoes a negative cooperative binding event, wherein binding of the multispecific antibody to a first target molecule results in decrease in binding affinity for a second target molecule. For example, in some embodiments, a binding affinity of a SIGLEC-8 bound multispecific antibody for CD117 is higher than a binding affinity of a SIGLEC-8 unbound multispecific antibody for CD117. In some embodiments, a binding affinity of CD117 bound multispecific antibody for SIGLEC-8 is higher than a binding affinity of CD 117 unbound multispecific antibody for SIGLEC-8.Kappa-lambda antibodies

[0207] Provided herein are multispecific (e.g., bispecific, trispecific) antibodies in the kappa - lambda antibody format. Bispecific antibodies provided herein have a common heavy chain, twoDocket No. 225365-702601 light chains - one Kappa (K), one Lambda (X) - that each has a different specificity (i.e., two light chains, two specificities). The methods provided herein produce molecules having specific binding where diversity is restricted to the VL region. These methods produce bispecific antibodies through controlled co-expression of the three chains (one VH chains, two VL chains), and purification.

[0208] This type of molecule is composed of two copies of a unique heavy chain polypeptide, a first light chain variable region fused to a constant Kappa domain and a second light chain variable region fused to a constant Lambda domain. Each combining site displays a different antigen specificity to which both the heavy and light chain contribute. The light chain variable regions can be of the Lambda or Kappa family and are preferably fused to a Lambda and Kappa constant domains, respectively. This is preferred in order to avoid the generation of non-natural polypeptide junctions. However, it is also possible to obtain bispecific antibodies of the invention by fusing a Kappa light chain variable domain to a constant Lambda domain for a first specificity and fusing a Lambda light chain variable domain to a constant Kappa domain for the second specificity.

[0209] An essential step of exemplary methods is the identification of two antibody Fv regions (each composed by a variable light chain and variable heavy chain domain) having different antigen specificities that share the same heavy chain variable domain. Numerous methods have been described for the generation of monoclonal antibodies and functional fragments thereof. Fully human antibodies are antibody molecules in which the sequence of both the light chain and the heavy chain, including the CDRs 1 and 2, arise from human genes. The CDR3 region can be of human origin or designed by synthetic means. Such antibodies are termed “human antibodies,” or “fully human antibodies” herein. Human monoclonal antibodies can be prepared by using the trioma technique; the human B-cell hybridoma technique; and the EBV hybridoma technique to produce human monoclonal antibodies. Human monoclonal antibodies may be utilized and may be produced by using human hybridomas or by transforming human B-cells with Epstein Barr Virus in vitro.

[0210] Monoclonal antibodies may be generated, e.g., by immunizing an animal with a target antigen or an immunogenic fragment, derivative or variant thereof. Alternatively, the animal is immunized with cells transfected with a vector containing a nucleic acid molecule encoding the target antigen, such that the target antigen is expressed and associated with the surface of the transfected cells. A variety of techniques are well-known in the art for producing xenogenic nonhuman animals.

[0211] Alternatively, antibodies may be obtained by screening a library that contains antibody or antigen binding domain sequences for binding to the target antigen. This library may beDocket No. 225365-702601 prepared, e.g., in bacteriophage as protein or peptide fusions to a bacteriophage coat protein that is expressed on the surface of assembled phage particles and the encoding DNA sequences contained within the phage particles (i.e., “phage displayed library”).

[0212] Hybridomas resulting from myeloma / B cell fusions are then screened for reactivity to the target antigen. Monoclonal antibodies may be prepared, for example, using hybridoma methods. In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes can be immunized in vitro.

[0213] Kappa-lambda antibodies having the same heavy chain variable domain can be generated by the use of antibody libraries in which the heavy chain variable domain is the same for all the library members and thus the diversity is confined to the light chain variable domain. However, as the light chain variable domain is expressed in conjunction with the heavy variable domain, both domains can contribute to antigen binding. To further facilitate the process, antibody libraries containing the same heavy chain variable domain and either a diversity of Lambda variable light chains or Kappa variable light chains can be used in parallel for in vitro selection of antibodies against different antigens. This approach enables the identification of two antibodies having a common heavy chain but one carrying a Lambda light chain variable domain and the other a Kappa light chain variable domain that can be used as building blocks for the generation of a bispecific antibody in the full immunoglobulin format of the invention. Numerous methods for the modification of the Fc portion have been described and are applicable to antibodies of the invention, (see for example Strohl, WR Curr Opin Biotechnol 2009 (6): 685- 91).

[0214] Another step of exemplary embodiments is the optimization of co-expression of the common heavy chain and two different light chains into a single cell to allow for the assembly of a bispecific antibody of the invention. If all the polypeptides get expressed at the same level and get assembled equally well to form an immunoglobulin molecule then the ratio of monospecific (same light chains) and bispecific (two different light chains) should be 50%.

[0215] The co-expression of the heavy chain and two light chains generates a mixture of three different antibodies into the cell culture supernatant: two monospecific bivalent antibodies and one bispecific bivalent antibody. The latter has to be purified from the mixture to obtain the molecule of interest. The method described herein greatly facilitates this purification procedure by the use of affinity chromatography media that specifically interact with the Kappa or Lambda light chain constant domains such as the CaptureSelect Fab Kappa and CaptureSelect Fab Lambda affinity matrices (BAC BV, Holland). This multi-step affinity chromatography purification approach is efficient and generally applicable to antibodies of the invention. This is in sharp contrast to specificDocket No. 225365-702601 purification methods that have to be developed and optimized for each bispecific antibodies derived from quadromas or other cell lines expressing antibody mixtures. Indeed, if the biochemical characteristics of the different antibodies in the mixtures are similar, their separation using standard chromatography technique such as ion exchange chromatography can be challenging or not possible at all.

[0216] In some embodiments, purified bispecific antibodies described herein are characterized as follows. The flow- through and elution from each affinity purification step can be analyzed by SDS-PAGE. The specificity and affinity of KX- bodies can be determined by ELISA and surface plasmon resonance. The methods of the invention can allow for the identification of antibodies with affinities in the sub-nanomolar to nanomolar range without optimization. This is not obvious as the diversity in antibody libraries described herein is restricted to the light chain which contributes less to the binding energy in standard antibodies.

[0217] To avoid the requirement of having access to two antibodies having light chain variable domains of the Kappa and Lambda type being perceived as a limitation to the instant invention, the methods described herein allow for the generation of hybrid light chain in which a Lambda variable domain can be fused to a Kappa constant domain and conversely a Kappa variable domain can be fused to a Lambda constant domain. In some embodiments, the methods of generating bispecific and / or multi-specific antibodies use a complete serum- free chemically defined process. These methods incorporate the most widely used mammalian cell line in pharmaceutical industry, the Chinese Hamster Ovary (CHO) cell line. The methods described therein are used to generate both semi-stable and stable cell lines. The methods can be used to manufacture bispecific and / or multi-specific antibodies of the invention at small scale (e.g., in an Erlenmeyer flask) and at midscale (e.g., in 25L Wave bag). The methods are also readily adaptable for larger scale production of bispecific and / or multi-specific antibodies, as well as antibody mixtures of the invention.Nucleotides and Vectors

[0218] Provided herein is a polynucleotide encoding one or more of the engineered proteins provided herein. Further provided herein is a vector comprising: a polynucleotide encoding a multimeric protein complex provided herein. In some embodiments, the nucleic acid or polynucleotide encoding an engineered protein provided herein is DNA. In some embodiments, the nucleic acid or polynucleotide encoding an engineered protein provided herein is RNA. In some embodiments, the polynucleotide is chemically modified for stability.

[0219] In some embodiments, vectors provided herein are a viral vector. Exemplary viral vectors include, but are not limited to, lentiviral vectors, retroviral vectors, adeno-associated viral vectors (AAV), adenoviral vectors, herpes simplex viral vectors, alphaviral vectors, flaviviral vectors, rhabdoviral vectors, measles viral vectors, Newcastle disease viral vectors, poxviralDocket No. 225365-702601 vectors, and picomaviral vectors. In some embodiments, polynucleotides encoding an engineered protein provided herein are contained in an AAV viral vector. In some embodiments, vectors provided herein are an oncolytic viral vector.Cells

[0220] Provided herein are cells expressing an engineered protein provided herein. In some embodiments, the cell is contacted in vitro or ex vivo with a polynucleotide encoding for an engineered protein provided herein. In some embodiments, the cell is contacted in vitro or ex vivo with a vector encoding for an engineered protein provided herein. In some embodiments, the cell is contacted in vitro or ex vivo with an oncolytic virus encoding for an engineered protein provided herein.

[0221] In some embodiments, the cell is a lymphocyte, a leukocyte, a myeloid cell, a T cell, a natural killer cell, a macrophage, a dendritic cell, a stem cell, an induced pluripotent stem-cell derived myeloid cell, an induced pluripotent stem-cell derived leukocyte, an induced pluripotent stem-cell derived lymphocyte. In some embodiments, the cell is a genetically modified cell. In some embodiments, the genetically modified cell is a chimeric antigen receptor (CAR) T cell, an engineered T cell receptor (TCR) T cell, or a tumor-infiltrating lymphocyte.Compositions and Pharmaceutical compositions

[0222] Disclosed herein are compositions comprising an engineered protein construct described herein. A pharmaceutical composition is a composition comprising one or more substances that have medicinal properties, together with a pharmaceutical acceptable carrier. In some embodiments, a composition described herein can be formulated as pharmaceutical compositions and administered to a subject in need thereof in a variety of forms adapted to the chosen route of administration. In some embodiments, a composition described herein comprises one or more salts in an amount required to bring osmolality of the composition into an acceptable range. Such salts include, but are not limited to, those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions; suitable salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite and ammonium sulfate.

[0223] In some embodiments, a pharmaceutical composition described herein is administered by any suitable administration route, including but not limited to, oral, parenteral (e.g., intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intra-articular, intraperitoneal, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, subcapsular, subarachnoid, intraspinal, epidural, intrastemal, or intracranial), intranasal, buccal, sublingual, spinal or rectal administration routes. In some embodiments, a pharmaceutical composition is formulated for parenteral (e.g.,Docket No. 225365-702601 intravenous, subcutaneous, intramuscular, intracerebral, intracerebroventricular, intra-articular, intraperitoneal, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcuticular, subcapsular, subarachnoid, intraspinal, epidural, intrastemal, or intracranial) administration. A pharmaceutical compositions described herein can be formulated into any suitable dosage form, including but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions and the like, for oral ingestion by an individual to be treated, solid oral dosage forms, aerosols, controlled release formulations, fast melt formulations, effervescent formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations, and mixed immediate release and controlled release formulations.

[0224] In some embodiments, a pharmaceutical composition described herein comprise one or more stabilizers, filling agents, surfactants, or combinations thereof. In some embodiments, the stabilizer comprises amino acids (e.g., glycine, alanine, lysine, arginine, and threonine), carbohydrates (e.g., glucose, sucrose, trehalose, raffinose, and maltose), polyols (e.g., glycerol, mannitol, sorbitol, cyclodextrins or dextran of any kind and molecular weight, and polyethylene glycol (PEG)). In some embodiments, the filling agent comprises lactose, sucrose, glucose, mannitol, sorbitol, calcium carbonate, and magnesium stearate. In some embodiments, the surfactant comprises anionic surfactant, cationic surfactant, zwitterionic surfactant, or nonionic surfactant. In some embodiments, the surfactant comprises alkyl ethoxylate, nonylphenol ethoxylate, amine ethoxylate, polyethylene oxide, polypropylene oxide, fatty alcohols (e.g., cetyl alcohol or oleyl alcohol), cocamide MEA, cocamide DEA, polysorbates, and dodecyl dimethylamine oxide. In some embodiments, the surfactant is polysorbate 20. In some embodiments, the surfactant is polysorbate 80.

[0225] In some embodiments, a pharmaceutical composition described herein is formulated into a capsule. In some embodiments, a pharmaceutical composition described herein is formulated into a solution (for example, for IV administration). In some embodiments, a pharmaceutical composition described herein is formulated as an infusion. In some embodiments, the pharmaceutical composition is formulated as an injection.

[0226] The pharmaceutical solid dosage forms described herein optionally include a compound described herein and one or more pharmaceutically acceptable additives such as a compatible carrier, binder, filling agent, suspending agent, flavoring agent, sweetening agent, disintegrating agent, dispersing agent, surfactant, lubricant, colorant, diluent, solubilizer, moistening agent, plasticizer, stabilizer, penetration enhancer, wetting agent, anti-foaming agent, antioxidant, preservative, or one or more combination thereof.Docket No. 225365-702601Methods of Treatment

[0227] Disclosed herein, in some embodiments, are methods of treating a subject in need thereof, comprising administering to the subject, an effective amount of one or more engineered proteins, compositions, or pharmaceutical compositions described herein. In some embodiments, an engineered protein comprises a SIGLEC-8 binding moiety described herein. In some embodiments, an engineered protein comprises a CD117 binding moiety described herein. In some embodiments, an engineered protein comprises both, a SIGLEC-8 binding moiety described herein, and a CD117 binding moiety described herein.

[0228] In some instances, an engineered protein described herein is used to treat a disease or disorder in a subject. Exemplary diseases include, but are not limited to, cancer, inflammatory diseases or disorders, a metabolic disease or disorder, a cardiovascular disease or disorder, an immunodeficiency disease or disorder, a respiratory disease or disorder, pain, a digestive disease or disorder, a reproductive disease or disorder, an endocrine disease or disorder, an immune disease or disorder, an autoimmune disease or disorder, or a neurological disease or disorder. In some instances, the cancer is a solid cancer or a hematologic cancer. In some instances, the disease or disorder is an immunodeficiency disease. In some instances, the disease or disorder is an inflammatory disease. In some instances, the disease or disorder is an immune disorder (e.g., allergies or asthma). In some instances, the disease or disorder is a mast cell disease (e.g., systemic mastocytosis, mast cell activation syndrome, or hereditary alpha-tryptasemia).

[0229] In some instances, the disease or disorder is associated with SIGLEC-8 dysfunction. In some instances, the disease or disorder is associated with aberrant signaling via SIGLEC-8. In some instances, the disease or disorder is a mast cell disease (e.g., systemic mastocytosis, mast cell activation syndrome, or hereditary alpha-tryptasemia). In some instances, the disease or disorder is an eosinophilic reflux disease (e.g., eosinophilic esophagitis or gastroenteritis). In some instances, the disease or disorder is duodenitis. In some instances, the disease or disorder is cancer. In some instances, the disease or disorder is an immune disease. In some instances, the disease or disorder is an allergy (e.g., a nut allergy or allergic conjunctivitis), chronic urticaria (e.g., hives), atopic dermatitis, or asthma (e.g., allergic asthma).

[0230] For example, SIGLEC-8 is a part of the sialic acid-binding IG-like lectin (SIGLEC) family of transmembrane proteins which are a CD33-related group of receptors. SIGLEC-8 has two splice variants and may function as an inhibitory immunoregulatory receptor. SIGLEC-8 has a finely-tuned specificity toward different sialylated and sulfated carbohydrate ligands. SIGLEC- 8 is expressed late in development and is expressed by immune effector cells such as eosinophils, mass cells, and basophils. SIGLEC-8 can be involved in asthma and allergies. SIGLEC-8 can be expressed by eosinophils and / or basophils from subjects with certain cancers (e.g., chronicDocket No. 225365-702601 eosinophilic leukemia, hypereosinophilic syndrome, or chronic myeloid leukemia). SIGLEC-8 can also be expressed by bone marrow mast cells from subjects with mastocytosis and / or aplastic anemia.

[0231] In some instances, the disease or disorder is associated with CD117 dysfunction. For example, CD117 is a transmembrane tyrosine kinase growth factor receptor that is the product of c-kit gene expression. CD117 is the receptor for stem cell factor (SCF) and can play a role in early hemopoiesis. CD117 is expressed by breast epithelium cells, germ cells, melanocytes, myeloid cells, and mast cells. CD117 can be expressed by certain types of cancer such as but not limited to ovarian carcinoma, seminoma, glioma, glioblastoma, mast cell tumors, melanoma, and gastrointestinal tumors. CD117 can also be related to chronic urticaria (hives), prurigo nodularis, severe combined immunodeficiency, or retinopathies (e.g., diabetic retinopathy, retinopathy of prematurity, or wet macular degeneration).

[0232] In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein is used to treat inflammatory disease. Inflammatory diseases include, but are not limited to autoimmune diseases, cardiovascular diseases, gastrointestinal disorders, lung diseases, metabolic disease, and neurodegenerative diseases. In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein is used to treat autoimmune disease, autoimmune disease includes, but are not limited to ulcerative colitis, Crohn’s disease and bowel obstruction diseases, autoimmune hepatitis, systemic lupus erythematosus, and rheumatoid arthritis.

[0233] In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein is used to treat central nervous system (CNS) disorders, multiple sclerosis (MS) or artherosclerosis. In some embodiments, central nervous system (CNS) disorders include, but are not limited to, acute and neurodegenerative diseases. In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein is used to treat inflammatory demyelinating diseases (IDDs) in a subject in need thereof. IDDs include, but are not limited to, multiple sclerosis (MS) and antibody mediated IDDs. In some embodiments, antibody mediated IDDs comprises neuromyelitis optica spectrum disorder (NMOSD) and myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD).

[0234] In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein is used for reducing the level of SIGLEC-8 and / or CD117 in mast cells in a subject in need thereof. In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein is used for inhibiting the activation of mast cells in a subject in need thereof. While dendritic cells (DCs), major antigen-presenting cells, have been proposed to play a central role in the pathogenesis of these demyelinating diseases of the CNS.Docket No. 225365-702601The AXL+SIGLEC6+ DC and cKit+SIGLEC-8+ mast cells have been only recently identified in humans. Without bound by any theory, it is believed that an engineered protein or a composition targeting both SIGLEC-8 and CD117 in master cells inactivate master cells more specifically and efficiently, thereby reducing off-target effect significantly.

[0235] In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein is used to treat multiple sclerosis (MS) and Artherosclerosis in a subject in need thereof, multiple sclerosis (MS) is an autoimmune illness which triggers neurological progressive and persistent functions, associated with an abnormal B-cell response and upregulation of T-cell reactivity against a multitude of antigens. Mast cells are also the forefront of the inflammation and demyelination by presenting myelin antigens to T cells or disrupting the blood-brain barrier and permitting entry of inflammatory cells and cytokines. Alongside, eosinophils infiltration located meningeal and perivascular in spinal cord lesions have been observed in patient with MS and relapsing remitting MS (RRMS). In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein described herein modulates direct cytotoxicity of eosinophils to other cells.

[0236] In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein described herein is used to treat Type 2 inflammation. Type 2 inflammation is driven by both the adaptive and innate arms of the immune system and underpins the pathophysiology of several chronic upper and lower airway diseases, including asthma, pulmonary fibrosis, lung fibrosis and chronic inflammatory airway diseases, asthma, chronic rhinosinusitis, eosinophilic COPD and allergic rhinitis. Therefore, in some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein described herein is used to treat pulmonary fibrosis, lung fibrosis and chronic inflammatory airway diseases, asthma, chronic rhinosinusitis, eosinophilic COPD and allergic rhinitis.

[0237] In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein described herein is used to treat Idiopathic pulmonary fibrosis (IPF). Idiopathic pulmonary fibrosis (IPF) is a progressive, usually fatal lung disease of unknown etiology, characterized by the accumulation of fibroblasts / myofibroblasts in the alveolar interstitium, with aberrant remodeling of the lung parenchyma. A direct implication of the SIGLEC-8-c-Kit axis in the pathogenesis of bleomycin induced lung fibrosis in-vivo model was demonstrated. Activation of the SIGLEC-8-c-Kit pathway results in the recruitment of cells from the bone marrow compartment to sites of lung injury, with consequent paracrine activation of fibroblasts. Elevated c-Kit expression in IPF lung fibroblast cultures suggests potential relevance to human pulmonary fibrosis. In some embodiments, an engineered protein construct, a composition, or a pharmaceutical composition described herein attenuates SIGLEC-8 / cKITDocket No. 225365-702601 expression and function. In some embodiments, an engineered protein described herein inhibits mast cell activation and reduces immune cells recruitment and concurrent airway inflammation.

[0238] In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein is used to treat Hirschsprung’s disease and HD associated enterocolitis, inflammatory bowel disease (IBD), Crohn’s diseases, peritoneal adhesion, and hypereosinophilic syndrome.

[0239] In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein is used to treat chronic diabetic foot ulcers or skin ulceration including atopic dermatitis, HS, PG, chronic venous leg ulcer, vasculitis and melanoma.

[0240] In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein is used for treating osteopenia, osteoporosis, atherosclerosis, myofibroblastic diseases, and myeloproliferative disorders in a subject in need thereof.

[0241] In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein is used for targeting both mast cells and eosinophils in a subject in need thereof. Mast cells and eosinophils are important cells of the immune system with critical roles in allergic and autoimmune disorders, cardiovascular diseases and cancer. Human mast cells derive from CD34+, CD117+ pluripotent hematopoietic stem cells in the bone marrow. In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein promotes immunosuppressive environment through DCs, mast cells and / or eosinophils. In some embodiments, an engineered protein, a composition, or a pharmaceutical composition described herein is used for treating small cell lung cancer (SCLC).

[0242] In some embodiments, the subject is a mammal. In some instances, the subject is a mouse, rabbit, dog, or human. Subjects treated by methods described herein may be infants, adults, or children. Pharmaceutical compositions comprising antibodies or antibody fragments as described herein may be administered intravenously or subcutaneously.EXEMPLARY EMBODIMENTS

[0243] Provided herein are engineered protein constructs comprising a first antigen-binding moiety that binds Sialic acid-binding immunoglobulin-like lectin 8 (“SIGLEC-8”) and a second antigen-binding moiety that binds Tyrosine-protein kinase KIT, CD117 (“CD117”). Further provided herein are engineered protein constructs, wherein the engineered protein construct is an antibody, a variant or a functional fragment thereof. Further provided herein are engineered protein constructs, wherein the engineered protein construct is a Fab2 antibody, a bis-scFv antibody, a diabody, a DVD-Ig, a TandAb, a tandem scFv-Fc, a one-armed tandem scFv-Fc, a DART, a DART-Fc, or a functional fragment thereof. Further provided herein are engineered proteinDocket No. 225365-702601 constructs, wherein the engineered protein construct comprises a heterodimeric antibody or a functional fragment thereof. Further provided herein are engineered protein constructs, wherein the engineered protein construct comprises a constant region. Further provided herein are engineered protein constructs, wherein a binding affinity of the first antigen-binding moiety for SIGLEC-8 is higher than a binding affinity of the second antigen-binding moiety for CD117. Further provided herein are engineered protein constructs, wherein the binding affinity of the first antigen-binding moiety for SIGLEC-8 is at least two times the binding affinity of the second antigen-binding moiety for CD 117. Further provided herein are engineered protein constructs, wherein a binding affinity of the first antigen-binding moiety for SIGLEC-8 is lower than a binding affinity of the second antigen-binding moiety for CD117. Further provided herein are engineered protein constructs, wherein the binding affinity of the second antigen-binding moiety for CD117 is at least two times the binding affinity of the first antigen-binding moiety for SIGLEC-8. Further provided herein are engineered protein constructs, wherein the engineered protein construct comprises at least one of a Fc region and / or a Fab region. Further provided herein are engineered protein constructs, wherein the Fc region comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or 100% identical to any one of amino acid sequences of SEQ ID NOs: 601-603. Further provided herein are engineered protein constructs, wherein at least one of the first antigen-binding moiety and the second antigen-binding moiety comprises a light chain constant domain and / or a heavy chain constant domain. Further provided herein are engineered protein constructs, wherein the heavy chain constant domain of the first antigen-binding moiety comprises the Fc region having S354C mutation and T366W mutation, per EU numbering, and the heavy chain constant domain of the second antigen-binding moiety comprises the Fc region having a Y349C mutation, a T366S mutation and a Y407V mutation, per EU numbering. Further provided herein are engineered protein constructs, wherein the heavy chain constant domain of the first antigen-binding moiety comprises the Fc region having a S354C mutation and a T366W mutation, per EU numbering, and the heavy chain constant domain of the second antigen-binding moiety comprises the Fc region having a Y349C mutation, a T366S mutation and a Y407V mutation, per EU numbering. Further provided herein are engineered protein constructs, wherein the heavy chain constant domain of the second antigen-binding moiety comprises the Fc region having a S354C mutation and a T366W mutation, per EU numbering, and the heavy chain constant domain of the first antigen-binding moiety comprises the Fc region having a Y349C mutation, a T366S mutation and Y407V mutation, per EU numbering. Further provided herein are engineered protein constructs, wherein the heavy chain constant domain of the first antigen-binding moiety or the second antigen-binding moiety comprises an Fc region comprising a human IgGl heavy chain constant chain having at least oneDocket No. 225365-702601 substitution is selected from positions N297, C226, C229, E233, L234, L235, G236, G237, P238, F243, M252, S254, T256, D265, S267, H268, D270, P271, R292, Y300, K322, A327, L328, P329, A330, P331, and P396, per EU numbering. Further provided herein are engineered protein constructs, wherein the heavy chain constant domain of the first antigen-binding moiety or the second antigen-binding moiety comprises an Fc region comprising a human IgG2 heavy chain constant chain having at least one substitution is selected from positions C232, C233, V234, G237, P238, M252, S254, T256, H268, N297, V309, A330, and P331, per EU numbering. Further provided herein are engineered protein constructs, wherein the heavy chain constant domain of the first antigen-binding moiety or the second antigen-binding moiety comprises an Fc region comprising a human IgG4 heavy chain constant chain having at least one substitution is selected from positions S228, E233, F234, L235, L236, G237, S241, L248, M252, S254, T256, N297, E318, and T394, per EU numbering. Further provided herein are engineered protein constructs, wherein the engineered protein construct comprises anti-inflammatory activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, or more than a combined anti-inflammatory activity of a monospecific antibody that binds SIGLEC-8 and a monospecific antibody that binds CD117. Further provided herein are engineered protein constructs, wherein the first antigen-binding moiety comprises: a heavy chain variable (VH) region comprising: a heavy chain complementarity-determining region 1 (HCDR1) having an amino acid sequence selected from Table 1, a heavy chain complementaritydetermining region 2 (HCDR2) having an amino acid sequence selected from Table 2, and a heavy chain complementarity-determining region 3 (HCDR3) having the amino acid sequence of any one from Table 3; and a light chain variable (VL) region comprising: a light chain complementarity-determining region 1 (LCDR1) having an amino acid sequence selected from Table 5, a light chain complementarity-determining region 2 (LCDR2) having an amino acid sequence selected from Table 6, and a light chain complementarity-determining region 3 (LCDR3) having an amino acid sequence selected from Table 7. Further provided herein are engineered protein constructs, wherein the first antigen-binding moiety comprises: a heavy chain variable domain (VH) comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from Table 4; and a light chain variable domain (VL) comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of any one from Table 8. Further provided herein are engineered protein constructs, wherein the second antigen-binding moiety comprises: a heavy chain variable (VH) region comprising: a heavy chain complementaritydetermining region 1 (HCDR1) having an amino acid sequence selected from Table 9, a heavy chain complementarity-determining region 2 (HCDR2) having an amino acid sequence selectedDocket No. 225365-702601 from Table 10, and a heavy chain complementarity-determining region 3 (HCDR3) having an amino acid sequence selected from Table 11; and a light chain variable (VL) region comprising: a light chain complementarity-determining region 1 (LCDR1) having an amino acid sequence selected from Table 13, a light chain complementarity-determining region 2 (LCDR2) having an amino acid sequence selected from Table 14, and a light chain complementarity-determining region 3 (LCDR3) having an amino acid sequence selected from Table 15. Further provided herein are engineered protein constructs, wherein the first antigen-binding moiety comprises: a heavy chain variable domain (VH) comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from Table 12; and a light chain variable domain (VL) comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical to an amino acid sequence selected from Table 16. Further provided herein are engineered protein constructs, wherein the SIGLEC-8 is human SIGLEC-8. Further provided herein are engineered protein constructs, wherein the CD117 is human CD 117. Further provided herein are engineered protein constructs, wherein the first antigen-binding moiety that binds SIGLEC-8 binds to SIGLEC-8 with a KD of less than 75 nM, less than 50 nM, less than 25 nM, or less than 10 nM. Further provided herein are engineered protein constructs, wherein the second antigen-binding moiety that binds CD117 binds to CD117 with a KD of less than 75 nM, less than 50 nM, less than 25 nM, or less than 10 nM.

[0244] Provided herein are pharmaceutical compositions comprising the engineered protein constructs provided herein, and a pharmaceutically acceptable carrier.

[0245] Provided herein are compositions for use in treating an inflammatory disease or disorder, wherein the composition comprises a first antigen-binding moiety that binds SIGLEC-8 and a second antigen-binding moiety that binds CD 117, wherein administration of an effective amount of the composition in need thereof results in the treatment of inflammatory disease or condition. Further provided herein are compositions, wherein the inflammatory disease is an autoimmune disease. Further provided herein are compositions, wherein the autoimmune disease is multiple sclerosis, systemic lupus erythematosus, or rheumatoid arthritis. Further provided herein are compositions, wherein the inflammatory disease is a gastrointestinal disorder. Further provided herein are compositions, wherein the gastrointestinal disorder is inflammatory bowel disease (IBD), Crohn’s disease, or ulcerative colitis.

[0246] Provided herein are isolated nucleic acids that encode the engineered protein constructs provided herein.

[0247] Provided herein are expression vectors comprising the isolated nucleic acids provided herein.Docket No. 225365-702601

[0248] Provided herein are isolated host cells comprising the isolated nucleic acids or the expression vectors provided herein.

[0249] Provided herein are isolated hosts cell that express the engineered protein constructs Provided herein.

[0250] Provided herein are methods of producing an engineered protein construct that binds SIGLEC-8 and CD117, comprising incubating the isolated host cells provided herein under conditions suitable for producing the engineered protein construct. Provided herein are methods, further comprising isolating the antibody or antigen-binding fragment thereof.

[0251] Provided herein are methods of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the engineered protein construct or the pharmaceutical composition provided herein. Further provided herein are methods, wherein the disease or disorder is a mast cell disease. Further provided herein are methods, wherein the mast cell disease is systemic mastocytosis, mast cell activation syndrome, or hereditary alpha-tryptasemia. Further provided herein are methods, wherein the disease or disorder is an autoimmune disease. Further provided herein are methods, wherein the disease or disorder is asthma, an allergy, chronic urticaria, atopic dermatitis, or asthma. Further provided herein are methods, wherein the disease or disorder is eosinophilic esophagitis, eosinophilic gastroenteritis, or duodenitis. Further provided herein are methods, wherein the disease or disorder is a cancer. Further provided herein are methods, wherein the cancer is chronic eosinophilic leukemia, hypereosinophilic syndrome, or chronic myeloid leukemia. Further provided herein are methods, wherein the cancer is an ovarian carcinoma, a seminoma, a glioma, a glioblastoma, a mast cell tumor, a melanoma, or a gastrointestinal tumor. Further provided herein are methods, wherein the disease or disorder is chronic urticaria, prurigo nodularis, severe combined immunodeficiency, or a retinopathy. Further provided herein are methods, wherein the retinopathy is diabetic retinopathy, retinopathy of prematurity, or wet macular degeneration. Further provided herein are methods, wherein the disease or disorder is central nervous system (CNS) disorders, multiple sclerosis (MS) or artherosclerosis. Further provided herein are methods, wherein the disease or disorder is Hirschsprung's disease. Further provided herein are methods, wherein the disease or disorder is Crohn's disease.

[0252] The following examples are set forth to illustrate more clearly the principle and practice of embodiments disclosed herein to those skilled in the art and are not to be construed as limiting the scope of any claimed embodiments. Unless otherwise stated, all parts and percentages are on a weight basis.EXAMPLESDocket No. 225365-702601

[0253] The following examples are given for the purpose of illustrating various embodiments of the disclosure and are not meant to limit the present disclosure in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the disclosure. Changes therein and other uses which are encompassed within the spirit of the disclosure as defined by the scope of the claims will occur to those skilled in the art.Example 1: Bispecific Antibody Constructs

[0254] Bispecific antibody capable of binding two different targets are generated. The two different targets are (a) SIGLEC-8, a variant thereof or a functional fragment thereof, and (b) CD 117, a variant thereof or a functional fragment thereof. The bispecific antibody comprises a first target binding domain and a second target binding domain. The first target binding domain comprises a first VH sequence and a first VL sequence, wherein the first target binding domain binds to SIGLEC-8, a variant thereof or a functional fragment thereof. The second target binding domain comprises a second VH sequence and a second VL sequence, wherein the second target binding domain binds to CD117, a variant thereof or a functional fragment thereof. The first VH sequence comprises any one of the variable heavy chain sequences in Table 1. The first VL sequence comprises any one of variable light chain sequences in Table 1. The second VH sequence comprises any one of the variable heavy chain sequences in Table 2. The second VL sequence comprises any one of variable light chain sequences in Table 2.Example 2. Bispecific antibody administration to subjects

[0255] Bi specific antibody capable of binding two different targets are generated as described in Example 1. Briefly, the two different targets are (a) SIGLEC-8, a variant thereof or a functional fragment thereof, and (b) CD 117, a variant thereof or a functional fragment thereof. The bispecific antibody is administered to a subject in need thereof. The subject is selected according to criteria described in TABLE 19.TABLE 19Docket No. 225365-702601Example 3. Bispecific AntibodyBispecific Generation

[0256] A bispecific antibody is prepared comprising a SIGLEC-8 binding domain and a CD117 binding domain. Briefly, a nucleotide sequences encoding the SIGLEC-8 binding domain and the CD117 binding domain is synthesized. The SIGLEC-8 binding domain comprises a SIGLEC-8 heavy chain sequence and a SIGLEC-8 light chain sequence. The CD117 binding domain comprises a CD117 heavy chain sequence and a CD117 light chain sequence. The nucleotide sequences are, optionally, codon optimized. Combinations of the encoded SIGLEC-8 and CD117 binding domain sequences in the bispecific antibodies are shown in Table 20.Table 20. Combinations of SIGLEC-8 and CD117 binding domains

[0257] Docket No. 225365-702601

[0258] The construct is cloned using Gibson Assembly via the NEBuilder HiFi DNA Assembly Cloning Kit (NEB). After Gibson Assembly the constructs are transformed using DH5a competent cells (NEB) and plated onto LB-CARB trays (Teknova) with growth overnight at 37 degrees Celsius shaking. Single colonies are then picked from each construct on the LB-CARB trays for inoculation into LB-CARB Media (Teknova, prepared fresh) and grown overnight at 37 degrees Celsius in 15 mL falcon tubes, seeded at 5 mL LB-CARB media. The cultured cells tubes are then pelleted, discarding the media and plasmid purification is initiated using the Qiagen plasmid plus midiprep purification kits (for yield), using a vacuum manifold and centrifugation. Plasmid DNA is then confirmed for concentration (on the Nanodrop) and nucleotide sequences are confirmed by Sanger Sequencing.Bispecific Expression & Purification

[0259] Unique heavy and light chain pairs are cloned into vectors designed to express bispecifics and relevant controls in HEK293 cells under the control of a CMV promoter. Antibody expression vectors are complexed with poly ethyl enimine and transfected into HEK293 cultures. Knob and Hole technology is used to enrich for heterodimerization, and disfavor homodimerization, during expression. After 5 days of shaking at 37 degrees Celsius in 293 cell culture media, cultures are harvested and the supernatant containing the secreted antibodies is clarified and filtered. Antibodies are captured out of the supernatant via an agarose-based protein A resin on a FPLC. After several washes with PBS, antibodies are eluted in a phosphoric acid and saline solution (pH 3) and neutralized with a basic phosphate saline solution (pH 11) to about pH 5.5. Analytical SEC and SDS PAGE analysis are used for determining the optimal fractions from each protein A elution to move forward into secondary / polishing purification.

[0260] Automated cation exchange chromatography (CEX), again on the FPLC, using a bind and elute methodology is employed to further enrich the samples for heterodimer bispecifics or controls. After dilution, to lower the salt concentration, a strong cation exchange column (Capto S ImpACT) is used for capturing the material of interest in the protein A elute, followed by washing and elution with increasing levels of sodium chloride at a constant pH of 6.5. The combination of analytic SEC and SDS PAGE is used for identifying fractions from CEX that are suitable for pooling. Following pooling, samples are sterilized, and assayed for purity via analytical SEC and endotoxin by LAL test from Charles River.Example 4. Assays with SIGLEC-8 antibodies

[0261] To select SIGLEC-8 binding arms for SIGLEC-8 / CD117 bispecific antibodies, the following antibodies containing SIGLEC-8 binding arms were tested for binding of SIGLEC-8 inDocket No. 225365-702601SIGLEC-8 overexpressing HEK293 cells: Lirentelimab, SIGLEC-8-04, SIGLEC-8-20, SIGLEC- 8-31, SIGLEC-8-33, SIGLEC-8-61. Briefly, on day 1, HEK293 cells were seeded at a density of 0.5 x io5cells per well in a 48-well plate, with 0.3 mL of complete DMEM per well. On day 2, 0.25 pg of plasmid DNA encoding human Siglec-8 was diluted in 50 pL of serum-free DMEM and added to each well.0.75 pL of 293Tran transfection reagent was added to the diluted DNA, mixed gently, and the mixture was added dropwise onto the cells. After 48 hours post-transfection, at day 4, cells were harvested, transferred to a round-bottom 96-well plate and incubated in presence of a series of anti-Siglec-8 antibody diluted in 100 pL of FACS buffer at the following concentrations: 10, 2, 0.4, 0.08, and 0.016 pg / mL. Cells were then incubated at 4°C for 1 hour, washed with FACS buffer (100 pL *2), resuspended in 100 pL of FACS buffer and stained for 1 hour at 4°C, in presence of anti-human Fc antibody conjugated to Alexa Fluor 647 (AF647 diluted at 1 : 100 in FACS buffer). Prior to be acquired on a BD FACS Canto II flow cytometer, cells were washed (100 pL *2) and resuspended in 150 pL of FACS buffer. Mean Fluorescence intensity (MFI) was measured for each antibody concentration and the results are shown in FIG. 3. The plots of MFI against increasing antibody concentrations in FIG. 3 indicated that each antibody could bind to SIGLEC- 8, suggesting that the SIGLEC-8 binding arm in each antibody can be used for designing a SIGLEC-8 / CD117 bispecific antibody.

[0262] To test inhibition of mast cell activation by anti-Siglec-8 Ab, human mast cells were seeded at a density of 5 x io5cells / mL, with 100 pL per well in a 96-well plate (50 000 cells per well). Antibodies were added at 10 pg / mL and incubated for 2 hours and treated with of IL-33 at 50 ng / mL and no IL-33 for the control well. After 24 hours, supernatants were collected, and IL- 6 levels were quantified by ELISA. As shown in FIG. 4, treatment with IL-33 stimulated mast cells, which in turn triggered secretion of IL-6. Consequently, elevated IL-6 level was detected for all groups treated with IL33 as compared to control. In the absence of SIGLEC-8 antibodies, the level of IL-6 was 270 pg / mL. The level of IL-6 was lower when treated with SIGLEC-8-33 (150 pg / mL) or Lirentelimab (200 pg / mL), suggesting the two antibodies inhibited mast cell activation. A bispecific antibody thus can be designed to include the SIGLEC-8 binding fragment from either of the two antibodies.Example 5. Assays with CD117 antibodies

[0263] To select CD117 binding arms for SIGLEC-8 / CD117 bispecific antibodies, the following antibodies containing CD117 binding arms were tested for binding of CD117 in CD117 overexpressing Mo7e cells: Barzolvolimab, anti-CD117-7 and anti-CDl 17-14. Briefly, 1x105Mo7e cells were seeded in a round-bottom 96-well plate, treated at 4°C for 1 hour with 100 pL ofDocket No. 225365-702601 an 8-point dilution of anti-CDl 17 mAb, washed with FACS buffer (100 pL *2) and resuspended in 100 pL of FACS buffer before being stained with anti-human Fc antibody conjugated to Alexa Fluor 647 (AF647 diluted at 1 : 100 in FACS buffer). After 1 hour at 4°C, cells were washed with FACS buffer (100 pL *2), resuspended in 150 pL of FACS buffer and acquired on a BD FACSCanto II flow cytometer. Mean Fluorescence intensity (MFI) was measured for each antibody concentration and the results are shown in FIG. 5. The plots of MFI against increasing antibody concentrations in FIG. 5 indicated that each antibody could bind to CD 117, suggesting that the CD117 binding arm in each antibody can be used for designing a SIGLEC-8 / CD117 bispecific antibody.

[0264] Assays were also performed in Mo7e cells to further test the function of CD117 antibodies. Briefly, Mo7e cells were resuspended in GM-CSF-free medium at a density of 0.5* 106cells / mL and transferred into a 6-well culture plate to be incubated for 16 hours at 37 °C in a 5% CO2. Mo7e cells were then harvested, centrifuged and resuspended at I MO5cells / mL in RPMI + 10% FBS prior to be dispensed into a 96-well plate, treated with anti-CDl 17 mAb and the isotype control, and incubated for an additional Ih at 37 °C in a 5% CO2. A final volume of SCF at 100 ng / mL was added and cells were incubated for 6 days. At day 8, cells were checked per microscopy, and proliferation was quantified using the CellTiter-Glo® Luminescent Cell Viability Assay from Promega. As shown in FIG. 6, SCF-induced Mo7 cell proliferation was inhibited in the presence of increasing concentration of Barzolvolimab, anti-CDl 17-7, and anti-CDl 17-14. Because SCF binds CD117. The results suggest the three anti-CDl 17 antibodies bind CD117, thereby blocking SCF from inducing cell proliferation.Example 6. Binding Assay with Bispecific Antibodies

[0265] Two bispecific antibodies targeting SIGLEC-8 and CD117 were generated: (1) a Fab / Fab bispecific antibody having a Fab fragment targeting Siglec8 and a Fab fragment targeting CD117; (2) a Fab / ScFv bispecific antibody having a Fab fragment targeting SIGLEC-8 and an ScFv fragment targeting CD117. Briefly, The Fab / Fab bispecific antibody was constructed with (a) a heavy chain (SEQ ID NO: 610) and a light chain (SEQ ID NO: 611) for SIGLEC-8 binding and (b) a heavy chain (SEQ ID NO: 612) and a light chain (SEQ ID NO: 613) for CD117 binding. The Fab / ScFv bispecific antibody was constructed with a heavy chain (SEQ ID NO: 608) and a light chain (SEQ ID NO: 609).

[0266] Assays were performed in Mo7e cells to confirm the binding of the bispecific antibodies to CD117. Briefly, 1 x 105Mo7e cells were seeded in a round-bottom 96-well plate, treated at 4 °C for 1 hour with 100 pL of an 8-point dilution of anti-CDl 17 mAb, washed with FACS buffer (100 pL *2) and resuspended in 100 pL of FACS buffer. Cells were subsequentlyDocket No. 225365-702601 stained with anti-human Fc antibody conjugated to Alexa Fluor 647 (AF647, diluted at 1 : 100 in FACS buffer), incubated for 1 hour at 4°C, washed with FACS buffer (100 pL *2) and resuspended in 150 pL of FACS buffer. Samples were acquired on a BD FACSCanto II flow cytometer. Mean Fluorescence intensity (MFI) was measured for each antibody concentration and the results are shown in FIG. 7. The plots of MFI against increasing antibody concentrations in FIG. 7 indicated that each bispecific antibody could bind CD117.Example 7. Effects of SIGLEC-8 / CD117 Bispecific Antibodies in Mast Cells

[0267] The bispecific antibodies generate in Example 6 are assayed to confirm their inhibitory effect on mast cell activation. Briefly, human mast cells are seeded at a density of 5 * 105cells / mL, with 100 pL per well in a 96-well plate (50 000 cells per well). Bispecific antibodies are added at 10 pg / mL and incubated for 2 hours and treated with of IL-33 at 50 ng / mL and no IL-33 for the control well. After 24 hours, supernatants were collected, and IL-6 levels were quantified by ELISA. For the effect of the bispecific antibodies on IgE degranulation, instead of treating the cells with IL-33, IgE is added for stimulation.

[0268] The effects on mast cells primed with IL33 and stimulated by IgE are also tested with the bispecific antibody. Upon IL33 priming, mast cells are primed to respond more robustly to IgE-mediated activation, while an anti-SIGLEC-8 antibody or SIGLEC-8 / CD117 bispecific antibody can inhibit IL33 priming.

[0269] To test the effects of the bispecific antibodies on mast cell or Mo7e cells survival, cells are resuspended in GM-CSF-free medium at a density of 0.5* 106cells / mL and transferred into a 6-well culture plate to be incubated for 16 hours at 37 °C in a 5% CO2. Mo7e cells are then harvested, centrifuged and resuspended at LIO5cells / mL in RPMI + 10% FBS prior to be dispensed into a 96-well plate, treated with anti-CD117 mAb and the isotype control, and incubated for an additional Ih at 37 °C in a 5% CO2. A final volume of SCF at 100 ng / mL is added and cells are incubated for 6 days. At day 8, cells were checked per microscopy, and cell survival is quantified using the CellTiter-Glo® Luminescent Cell Viability Assay from Promega.

[0270] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

Docket No. 225365-702601CLAIMSWHAT IS CLAIMED IS:

1. A method of treating an inflammatory disease or disorder in a subject in need thereof, wherein the method comprises administering an effective amount of a pharmaceutical composition comprising an engineered protein construct to the subject, wherein the engineered protein construct comprises:(a) a first antigen-binding moiety that binds Sialic acid-binding immunoglobulin-like lectin 8 (SIGLEC-8); and(b) a second antigen-binding moiety that binds cluster of differentiation 117 (CD117), wherein the effective amount is sufficient to reduce an inflammation caused by mast cells in the subject as compared to prior to the administration of the engineered protein construct.

2. The method of claim 1, wherein the engineered protein construct comprises a heterodimeric human IgG Fc region that comprises a first constant region and a second constant region.

3. The method of claim 2, wherein the heterodimeric Fc region is operably linked to the SIGLEC-8 binding heavy chain variable (VH) domain and the CD117 binding heavy chain variable (VH) domain.

4. The method of claim 2, wherein at least one of the first constant region and the second constant region comprises mutations M252Y, S254T, and T256E according to EU numbering.

5. The method of claim 2, wherein at least one of the first constant region and the second constant region comprises at least one substitution at position L234, L235, P329, or a combination thereof, according to according to EU numbering.

6. The method of claim 5, wherein the at least one substitution comprises L234A, L235A, and P329G, according to according to EU numbering.

7. The method of claim 2, wherein at least one of the first constant region and the second constant region comprises at least one substitution at position L234, L235, K322, or a combination thereof, according to according to EU numbering.

8. The method of claim 7, wherein the at least one of the two constant regions comprises mutations L234A, L235Q, and K322Q according to EU numbering.Docket No. 225365-7026019. The method of claim 1, wherein the antigen-binding moiety that binds SIGLEC-8 has a lower affinity for SIGLEC-8, as compared to an affinity of the other antigen-binding moiety to CD117.

10. The method of claim 1, wherein the antigen-binding moiety that binds SIGLEC-8 has a higher affinity for SIGLEC-8, as compared to an affinity of the other antigen-binding moiety to CD117.

11. The method of claim 1, wherein the engineered protein construct is an antibody, a variant or a functional fragment thereof.

12. The method of claim 1, wherein the engineered protein construct comprises an antibody fragment, wherein the antibody fragment is one or more of Fab, Fv, Fab', (Fab')2, scFv, bis-scFv, Fab2, and Fab3.

13. The method of claim 11, wherein the first antigen-binding moiety comprises a first Fab fragment and the second antigen binding moiety comprises a second Fab fragment.

14. The method of claim 11, wherein the first antigen-binding moiety comprises a Fab fragment and the second antigen binding moiety comprises an ScFv fragment.

15. The engineered protein or the antigen binding fragment of claim 14, wherein each antigen-binding moiety comprises an Fc region in a heavy chain, wherein at least one antigenbinding moiety comprises a modification in an Fc region such that the two heavy chains favor heterodimerization over homodimerization.

16. The engineered protein or the antigen binding fragment of claim 15, wherein the modification results in a heterodimerization in which the Fc regions of the two heterologous heavy chains are operably linked.

17. The engineered protein or the antigen binding fragment of claim 15, wherein the two heterologous heavy chains are connected by a Knob-in-Hole (KiH) structure, wherein one heterologous heavy chain comprises at least one knob mutation and the other heterologous heavy chain comprises at least one hole mutation.

18. The engineered protein or the antigen binding fragment of claim 17, wherein the Fc regions of the heterologous heavy chain comprising at least one knob mutation comprises a mutation at position 354, 366 according to EU numbering.Docket No. 225365-70260119. The engineered protein or the antigen binding fragment of claim 17, wherein the Fc regions of the heterologous heavy chain comprising at least one hole mutation comprises a mutation at position 349, 366, 368, 407, or a combination thereof, according to EU numbering.

20. The engineered protein or the antigen binding fragment of claim 19, wherein the Fc regions of the heterologous heavy chain comprising at least one knob mutation comprises a mutation T336W according to EU numbering and the Fc region of the heterologous heavy chain comprising at least one hole mutation comprises mutations T366S, L368A, and Y407V according to EU numbering.

21. The method of claim 11, wherein the engineered protein construct is a bivalent, trivalent, or tetravalent molecule.

22. The method of claim 21, wherein the engineered protein construct is a bivalent molecule.

23. The method of claim 21, wherein the engineered protein construct is a tetraval ent molecule.

24. The method of claim 23, wherein the engineered protein comprises four antigenbinding sites, wherein two of the four antigen-binding sites bind SIGLEC-8, each comprising a Fab fragment, and wherein two of the four antigen-binding sites bind CD117.

25. The method of claim 1, wherein the disease or disorder is mast cell disease.

26. The method of claim 25, wherein the mast cell disease is systemic mastocytosis, mast cell activation syndrome, or hereditary alpha-tryptasemia.

27. An engineered protein construct comprising a first antigen-binding moiety that binds Sialic acid-binding immunoglobulin-like lectin 8 (SIGLEC-8) and a second antigenbinding moiety that binds CD117, wherein the first antigen-binding moiety comprises:(a) a heavy chain variable (VH) region comprising: a heavy chain complementarity-determining region 1 (HCDR1) having an amino acid sequence selected from Table 1, a heavy chain complementarity-determining region 2 (HCDR2) having an amino acid sequence selected from Table 2, and a heavy chain complementarity-determining region 3 (HCDR3) having an amino acid sequence selected from Table 3; andDocket No. 225365-702601(b) a light chain variable (VL) region comprising: a light chain complementarity-determining region 1 (LCDR1) having an amino acid sequence selected from Table 5, a light chain complementarity-determining region 2 (LCDR2) having an amino acid sequence selected from Table 6, and a light chain complementarity-determining region 3 (LCDR3) having an amino acid sequence selected from Table 7.

28. The engineered protein construct of claim 27, wherein the first antigen-binding moiety comprises:(a) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from Table 4; and(b) a light chain variable domain (VL) comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from Table 8.

29. The engineered protein construct of claim 27, wherein the second antigen-binding moiety comprises:(a) a heavy chain variable (VH) region comprising: a heavy chain complementarity-determining region 1 (HCDR1) having an amino acid sequence selected from Table 9, a heavy chain complementarity-determining region 2 (HCDR2) having an amino acid sequence selected from Table 10, and a heavy chain complementarity-determining region 3 (HCDR3) having an amino acid sequence selected from Table 11; and(b) a light chain variable (VL) region comprising: a light chain complementarity-determining region 1 (LCDR1) having an amino acid sequence selected from Table 13, a light chain complementarity-determining region 2 (LCDR2) having an amino acid sequence selected from Table 14, and a light chain complementarity-determining region 3 (LCDR3) having an amino acid sequence selected from Table 15.

30. The engineered protein construct of claim 27, wherein the first antigen-binding moiety comprises:Docket No. 225365-702601(a) a heavy chain variable domain (VH) comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from Table 12; and(b) and a light chain variable domain (VL) comprising an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, at least 99% identical, or 100% identical to an amino acid sequence selected from Table 16.

31. The engineered protein construct of any one of claims 27 to 30, wherein the SIGLEC-8 is human SIGLEC-8.

32. The engineered protein construct of any one of claims 27 to 31, wherein the CD117 is human CD117.

33. The engineered protein construct of claim 27, wherein the engineered protein construct is an antibody, a variant or a functional fragment thereof.

34. The engineered protein construct of claim 27, wherein the engineered protein construct comprises an antibody fragment, wherein the antibody fragment is one or more of Fab, Fv, Fab', (Fab')2, scFv, bis-scFv, minibody, Fab2, and Fab3.

35. The engineered protein construct of claim 27, wherein the engineered protein construct comprises a heterodimeric antibody or a functional fragment thereof.

36. The engineered protein construct of claim 27, wherein the engineered protein construct comprises a constant region.

37. The engineered protein construct of claim 27, wherein a binding affinity of the first antigen-binding moiety for SIGLEC-8 is higher than a binding affinity of the second antigen-binding moiety for CD117.

38. The engineered protein construct of claim 37, wherein the binding affinity of the first antigen-binding moiety for SIGLEC-8 is at least two times the binding affinity of the second antigen-binding moiety for CD117.

39. The engineered protein construct of claim 27, wherein a binding affinity of the first antigen-binding moiety for SIGLEC-8 is lower than a binding affinity of the second antigenbinding moiety for CD117.Docket No. 225365-70260140. The engineered protein construct of claim 27, wherein the binding affinity of the second antigen-binding moiety for CD117 is at least two times the binding affinity of the first antigen-binding moiety for SIGLEC-8.

41. The engineered protein construct of claim 27, wherein the engineered protein construct comprises at least one of a Fc region and / or a Fab region.

42. The engineered protein construct of claim 41, wherein the Fc region comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to any one of amino acid sequences of SEQ ID NOs: 601-603.

43. The engineered protein construct of claim 27, wherein at least one of the first antigen-binding moiety and the second antigen-binding moiety comprises a human IgG constant region, wherein the constant region comprises a light chain constant domain and / or a heavy chain constant domain.

44. The engineered protein construct of claim 43, wherein the human IgG constant region comprises mutations M252Y, S254T, and T256E according to EU numbering.

45. The engineered protein construct of claim 43, wherein the human IgG constant region comprises at least one substitution at position L234, L235, P329, or a combination thereof, according to according to EU numbering.

46. The engineered protein construct of claim 45, wherein the human IgG constant region comprises mutations L234A, L235A, and P329G according to according to EU numbering.

47. The engineered protein construct of claim 43, wherein the human IgG constant region comprises at least one substitution at position L234, L235, K322, or a combination thereof, according to according to EU numbering.

48. The engineered protein construct of claim 43, wherein the human IgG constant region comprises mutations L234A, L235Q, and K322Q according to EU numbering.

49. The engineered protein construct of any one of claims 27 to 48, wherein the engineered protein construct comprises anti-inflammatory activity that is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at leastDocket No. 225365-70260190%, at least 100% more than a combined anti-inflammatory activity of a monospecific antibody that binds SIGLEC-8 and a monospecific antibody that binds CD117.

50. The engineered protein construct of claim 27, wherein the engineered protein construct comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of amino acid sequences of SEQ ID NOs: 604-615.

51. A pharmaceutical composition comprising the engineered protein construct of any one of claims 27 to 50, and a pharmaceutically acceptable carrier.

52. A composition for use in treating an inflammatory disease or disorder, wherein the composition comprises a first antigen-binding moiety that binds SIGLEC-8 and a second antigen-binding moiety that binds CD117, wherein administration of an effective amount of the composition in need thereof results in the treatment of inflammatory disease or condition.

53. The composition for use of claim 52, wherein the inflammatory disease is an autoimmune disease.

54. The composition for use of claim 53, wherein the autoimmune disease is multiple sclerosis, systemic lupus erythematosus, or rheumatoid arthritis.

55. The composition for use of claim 52, wherein the inflammatory disease is a gastrointestinal disorder.

56. The composition for use of claim 55, wherein the gastrointestinal disorder is inflammatory bowel disease (IBD), Crohn’s disease, or ulcerative colitis.

57. An isolated nucleic acid that encodes the engineered protein construct of any one of claims 27 to 50.

58. An expression vector comprising the isolated nucleic acid of claim 57.

59. An isolated host cell comprising the isolated nucleic acid of claim 57 or the expression vector of claim 58.

60. An isolated host cell that expresses the engineered protein construct of any one of claims 27 to 50.Docket No. 225365-70260161. A method of producing an engineered protein construct that binds SIGLEC-8 and CD117 comprising, incubating the isolated host cell of claim 59 or claim 60 under conditions suitable for producing the engineered protein construct.

62. The method of claim 61, further comprising isolating the antibody or antigenbinding fragment thereof.

63. A method reducing the level of SIGLEC-8 and CD117 in mast cells, the method comprising treating the mast cells with an effective amount of the engineered protein construct of any one of claims 27 to 50 or the pharmaceutical composition of claim 51, wherein the effective amount is sufficient to reduce the level of SIGLEC-8 and CD117 in the mast cells.

64. A method of treating a disease or disorder in a subject in need thereof, the method comprising administering to the subject an effective amount of the engineered protein construct of any one of claims 27 to 50 or the pharmaceutical composition of claim 51, wherein the effective amount is sufficient to treat the disease or disorder.

65. The method of claim 64, wherein the disease or disorder is a mast cell disease.

66. The method of claim 65, wherein the mast cell disease is systemic mastocytosis, mast cell activation syndrome, or hereditary alpha-tryptasemia.

67. The method of claim 64, wherein the disease or disorder is an autoimmune disease.

68. The method of claim 64, wherein the disease or disorder is asthma, an allergy, chronic urticaria, atopic dermatitis, or asthma.

69. The method of claim 64, wherein the disease or disorder is eosinophilic esophagitis, eosinophilic gastroenteritis, or duodenitis.

70. The method of claim 64, wherein the disease or disorder is a cancer.

71. The method of claim 70, wherein the cancer is chronic eosinophilic leukemia, hypereosinophilic syndrome, or chronic myeloid leukemia.

72. The method of claim 70, wherein the cancer is an ovarian carcinoma, a seminoma, a glioma, a glioblastoma, a mast cell tumor, a melanoma, or a gastrointestinal tumor.Docket No. 225365-70260173. The method of claim 64, wherein the disease or disorder is prurigo nodularis, severe combined immunodeficiency, or a retinopathy.

74. The method of claim 73, wherein the retinopathy is diabetic retinopathy, retinopathy of prematurity, or wet macular degeneration.

75. The method of claim 64, wherein the disease or disorder is central nervous system (CNS) disorders, multiple sclerosis (MS) or artherosclerosis.

76. The method of claim 64, wherein the disease or disorder is Hirschsprung's disease.

77. The method of claim 64, wherein the disease or disorder is Crohn's disease.

Images