Anti-Stem Cell Factor Antibody and Method of Using the Same

Abstract

The present disclosure relates to antibodies and antigen-binding fragments thereof that bind to stem cell factor (SCF). The antibodies and antigen-binding fragments thereof specifically bind to SCF248. The disclosure further relates to methods for producing the antibodies and methods of using the antibodies, including methods for treating inflammatory and / or fibrotic diseases and disorders.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit of priority of U.S. Provisional Application No. 62 / 900,927, filed on September 16, 2019, the entire content of which is incorporated herein by reference.

[0002] Field The present invention relates to stem cell factor (SCF), antibodies that bind to specific sites thereof, and antigen - binding fragments thereof, and methods of using such antibodies and antigen - binding fragments.

[0003] Description of Text File Submitted Electronically The content of the text file submitted electronically together with this specification, namely, the computer - readable format copy of the sequence listing (file name: OPSL_001_01WO_SeqList_ST25; recording date: September 16, 2020; file size: 58 kb) is incorporated herein by reference in its entirety.

Background Art

[0004] Inflammatory diseases are a major cause of morbidity and mortality worldwide. Some types of chronic inflammation can lead to fibrosis. Fibrosis is the formation or development of excessive fibrous connective tissue in an organ or tissue as a reparative or reactive process, which is different from the formation of fibrous tissue as a normal component of an organ or tissue. Chronic inflammation and fibrosis affect almost all tissues and organ systems, and fibrous tissue remodeling can affect cancer metastasis and accelerate chronic graft rejection in transplant patients.

[0005] Stem cell factor (SCF) and its receptor c-Kit are important factors in the persistence of chronic inflammation and fibrotic diseases (El-Koraie, et al., Kidney Int. 60:167 (2001); Powell, et al., Am. J. Physiol. 289:G2 (2005); El Kossi, et al., Am. J. Kidney Dis. 41:785 (2003); Powell, et al., Am. J. Physiol. 277:C183 (1999); Ding et al J Pathol. 2013 Jun;230(2):205-14.; Berlin et al Lab Invest. 2006 Jun;86(6):557-65; Rasky et al Am J Physiol Lung Cell Mol Physiol. 2020 Jan 1;318(1):L200-L211). c-Kit is a type III receptor-tyrosine kinase present in many cell types (Orr-Urtreger et al., Development 109:911 (1990)). Immune cells, such as mast cells, eosinophils, and innate lymphoid cells 2 and 3 (ILC2 and ILC3), are all c-Kit+ cells and may drive the chronic inflammatory process depending on the disease and organ involved. When the inflammatory response is initiated, various mediators, including SCF, activate c-Kit+ immune cells, resulting in the production of cytokines that transform fibroblasts into activated myofibroblasts. Myofibroblasts secrete extracellular matrix proteins, collagen, and fibronectin, leading to tissue fibrosis. Activated myofibroblasts, activated epithelium, endothelium, macrophages, eosinophils, mast cells, monocytes, and other cells also express SCF on the cell surface, further activating c-Kit+ immune cells, resulting in increased cytokine release and persistent inflammation. In the art, there is a need for more efficient and specific treatments for inflammatory diseases. The present disclosure addresses such needs and other needs.

PRIOR ART DOCUMENTS

NON-PATENT DOCUMENTS

[0006]

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Summary of the Invention

Means for Solving the Problems

[0007] In one aspect, the present disclosure provides an antibody and fragments thereof that specifically bind to stem cell factor (SCF). In some embodiments, the antibody and fragments thereof specifically bind to the SCF isoform SCF248. In some embodiments, the antibody and fragments thereof comprise heavy chain complementarity determining regions (CDRs), and heavy chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the antibody and fragments thereof comprise light chain CDRs, and light chain CDR1, CDR2, and CDR3 comprise SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the antibody and fragments thereof comprise heavy chain CDR1, CDR2, and CDR3 that comprise SEQ ID NOs: 1, 37, and 3, respectively. In some embodiments, the antibody and fragments thereof comprise a heavy chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12. In some embodiments, the antibody and fragments thereof comprise a light chain variable region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to a sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, and 17. In some embodiments, the antibody and fragments thereof comprise a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12 and a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, and 17.

[0008] In some embodiments, the antibody or fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 7 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody or fragment thereof according to claim 1, comprising the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 8 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody or fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 9 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody or fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody or fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 11 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody or fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 12 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 16.

[0009] In some embodiments, the antibody or fragment thereof is humanized. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody comprises a human IgG1 domain or a human IgG4 domain. In some embodiments, the antibody is an antigen-binding fragment, and the fragment is selected from Fab, F(ab’)2, Fab’, scFv, and single domain antibody (sdAb).

[0010] In some embodiments, the antibody or fragment thereof blocks the interaction between SCF (e.g., SCF248) and c-Kit. In some embodiments, the antibody specifically binds to SCF248. In some embodiments, the antibody does not bind to SCF220. In some embodiments, the antibody prevents the interaction between SCF248 and c-kit by causing internalization of SCF, so that it is not available on the cell surface.

[0011] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antibody or a fragment thereof provided herein. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0012] In some embodiments, the present disclosure provides an isolated nucleic acid molecule encoding an antibody or a fragment thereof provided herein. In some embodiments, the present disclosure provides an expression vector comprising a nucleic acid encoding an antibody or a fragment thereof. In further specific embodiments, the present disclosure provides a recombinant host cell comprising the expression vector.

[0013] In one aspect, the present disclosure provides a method for generating an antibody that specifically binds to stem cell factor isoform 248 (SCF248), the method comprising immunizing a host animal with a peptide comprising SEQ ID NO: 30 (ASSLRNDSSSSNRKAKNPPGD) or a fragment thereof, and obtaining an antibody from the immunized host animal. In some embodiments, the host animal is non-human. In some embodiments, the fragment of SEQ ID NO: 30 comprises at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive amino acids of SEQ ID NO: 30. In some embodiments, the fragment of SEQ ID NO: 30 comprises 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive amino acids of SEQ ID NO: 30. In some embodiments, the N-terminal amino acid of the fragment of SEQ ID NO: 30 is alanine at position 1 of the N-terminus of SEQ ID NO: 30. In some embodiments, the method comprises immunizing the host animal with a peptide consisting of SEQ ID NO: 30. In some embodiments, the antibody from the immunized host animal is obtained from immune cells isolated from the host animal. In some embodiments, the method further comprises generating hybridomas using the immune cells. Thus, in some embodiments, the present disclosure provides hybridomas that produce the monoclonal antibodies described herein.

[0014] In one aspect, the present disclosure provides an antibody or fragment thereof that specifically binds to SCF248 and binds to an epitope comprising at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 consecutive amino acids of SEQ ID NO: 33, wherein the antibody inhibits the interaction between SCF248 and c-Kit. In a further embodiment, the epitope comprises SEQ ID NO: 33 or SEQ ID NO: 36. In yet a further embodiment, the epitope consists of SEQ ID NO: 33 or SEQ ID NO: 36.

[0015] In one aspect, the present disclosure provides compositions and methods for inhibiting the interaction between SCF and c-Kit. c-kit is expressed on immune cells, hematopoietic stem cells, and some stromal cells. SCF248, the ligand of c-kit, is upregulated on myofibroblasts, activated epithelium, endothelium, macrophages, eosinophils, mast cells, monocytes, etc. In some embodiments, the compositions and methods specifically inhibit the interaction between SCF248 and c-Kit. For example, in some embodiments, the compositions and methods specifically inhibit the interaction between SCF248 on myofibroblasts and c-Kit on immune cells. As another example, in some embodiments, the compositions and methods provided herein specifically inhibit the interaction between SCF248 on myofibroblasts, activated epithelium, endothelium, macrophages, eosinophils, mast cells, and / or monocytes and c-Kit on immune cells and / or stromal cells. In some embodiments, the method comprises contacting SCF248 on myofibroblasts with the antibody or fragment thereof provided herein. In some embodiments, the antibody or fragment thereof provided herein blocks the binding of SCF248 to c-Kit. In some embodiments, the blocking is via steric hindrance. In some embodiments, the antibody or fragment thereof provided herein internalizes SCF248.

[0016] In some embodiments, the present disclosure provides a method for inhibiting inflammation in a subject in need thereof, the method comprising administering to the subject an antibody or fragment thereof provided herein. In some embodiments, the present disclosure provides a method for inhibiting an inflammatory disease in a subject in need thereof, the method comprising administering to the subject an antibody or fragment thereof provided herein. In further embodiments, the inflammatory disease is a chronic inflammatory disease. In some embodiments, the present disclosure provides a method for treating inflammation and / or a chronic inflammatory disease in a subject in need thereof, the method comprising administering to the subject an antibody or fragment thereof provided herein.

[0017] In some embodiments, the present disclosure provides a method for inhibiting fibrosis in a subject in need thereof, the method comprising administering to the subject an antibody or fragment thereof provided herein. In some embodiments, the present disclosure provides a method for treating a fibrotic disease in a subject in need thereof, the method comprising administering to the subject an antibody or fragment thereof provided herein. In embodiments, the method further comprises administering one or more additional therapies and / or therapeutic agents.

[0018] In some embodiments, the inflammatory disease or fibrotic disease is selected from the group consisting of urticaria, atopic dermatitis, bullous pemphigoid, scleroderma, systemic sclerosis, non-alcoholic steatohepatitis (NASH), primary sclerosing cholangitis, cirrhosis, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis (IPF), scleroderma lung fibrosis), chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, asthma, bleomycin lung, endomyocardial fibrosis, fibromyalgia, eosinophilic esophagitis, radiation fibrosis, rheumatoid arthritis, and inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019]

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Mode for Carrying Out the Invention

[0020] Stem cell factor (SCF) is an important mediator of acute and chronic inflammation, fibrotic diseases, and tissue remodeling diseases. Inflammation and fibrosis are initiated and sustained by the interaction of SCF with c-Kit on immune cells. The present disclosure provides compositions and methods for inhibiting the interaction of SCF with c-Kit. In one aspect, the present disclosure provides compositions and methods for preventing the interaction of SCF248, an inflammatory form of SCF, with c-Kit to reduce and / or prevent the activation of immune cells. Accordingly, the present disclosure provides methods for treating chronic inflammation as well as fibrotic and tissue remodeling diseases. In one aspect, the present disclosure provides compositions and methods for reducing the accumulation (e.g., proliferation and / or retention) of immune cells within an organ or tissue. For example, the present disclosure provides compositions and methods for preventing the interaction of SCF248 with c-Kit to reduce and / or prevent the accumulation of immune cells within an organ or tissue. In some embodiments, the present disclosure provides compositions and methods for reducing and / or preventing the activation and / or accumulation of mast cells, eosinophils, type 2 innate lymphoid (ILC2) cells, and type 3 innate lymphoid (ILC3) cells within an organ or tissue.

[0021] Specifically, the present disclosure provides antibodies and fragments thereof that specifically bind to SCF and block or inhibit its interaction with c-Kit. In some embodiments, the antibodies and fragments provided herein bind to SCF and inhibit the activity of c-Kit and c-Kit+ cells. The present disclosure also provides methods for generating antibodies and fragments thereof that specifically bind to SCF, as well as diagnostic and therapeutic methods for their use. In one aspect, the antibodies and fragments provided herein specifically bind to SCF248, an SCF isoform that promotes inflammation. Accordingly, the present disclosure provides specific and effective compositions and methods for inhibiting inflammation and fibrosis and treating chronic inflammatory and fibrotic diseases.

[0022] Definitions As used herein, the term "antibody" refers to a binding protein having at least one antigen-binding domain. The antibodies and fragments thereof of the present invention may be whole antibodies or any fragment thereof. Thus, the antibodies and fragments of the present invention include monoclonal antibodies or fragments thereof, antibody variants or fragments thereof, and immunoconjugates. Antigen-binding fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, bispecific Fab dimers (Fab2), trispecific Fab trimers (Fab3), Fv, single-chain Fv proteins ("scFv"), bis-scFv, (scFv)2, minibodies, diabodies, triabodies, tetra-bodies, disulfide-stabilized Fv proteins ("dsFv"), single-domain antibodies (sdAb, nanobodies), heavy-chain-only antibodies (e.g., camelid VHH, camelid nanobodies, shark Ig NAR), and portions of full-length antibodies responsible for antigen binding. An isolated antibody or its antigen-binding fragment is an antibody that has been identified and separated from and / or recovered from components of its natural environment.

[0023] In some embodiments, the antibody and its antigen-binding fragment are an isolated antibody and its fragment. Thus, the present invention provides an isolated antibody and its antigen-binding fragment, as well as nucleic acids encoding such antibodies and fragments, and compositions comprising such isolated antibodies, fragments, and nucleic acids. The term "isolated" refers to a target compound (e.g., an antibody or nucleic acid) that has been separated from its natural environment. The present invention further provides a pharmaceutical composition comprising an isolated antibody or its fragment, or a nucleic acid encoding such an antibody or fragment, further comprising one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include, for example, excipients, diluents, encapsulating materials, fillers, buffers, or other agents.

[0024] As used herein, the term "derived from" when used to refer to a molecule or polypeptide relative to a reference antibody or other binding protein means a molecule or polypeptide that is specific for and capable of binding to the same epitope as the reference antibody or other binding protein.

[0025] As used herein, the expression "specific for" may mean that an antibody does not bind to a target only by non-specific interactions, and this property can be determined by comparison with an isotype control or the like. Specific binding may include, but does not necessarily require, exclusive binding to a single target. In various embodiments, the antibodies provided herein specifically bind to SCF248 and do not bind to SCF220.

[0026] The term "host cell" means a cell that has been transformed with, or is capable of being transformed with, a nucleic acid sequence and expresses a gene of interest. The term includes the progeny of the parent cell, whether or not the progeny are identical to the original parent cell in morphology or genetic composition, as long as the gene of interest is present.

[0027] A "variant" of a polypeptide (e.g., an antigen-binding protein, or an antibody) includes an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted in the amino acid sequence as compared to another polypeptide sequence. Variants include antibodies or fragments provided herein, or antibodies or fragments having the recited DNA or amino acid sequences, as well as antibodies and their fragments having the recited percent identity.

[0028] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules determined by alignment and comparison of the sequences. "Percent identity", "percent homology", "sequence identity", "sequence homology", etc. mean the percentage of identical residues between amino acids or nucleotides in the molecules being compared, and are calculated based on the size of the smallest of the molecules being compared. For such calculations, gaps in the alignment (if any) are preferably handled by a particular mathematical model or computer program (i.e., "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, A.M., ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, D.W., ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, A.M., and Griffin, H.G., eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073. In calculating the percent identity, the sequences being compared are typically aligned so as to maximize the degree of match between the sequences.

[0029] The term "light chain" includes a full-length light chain and fragments thereof having a variable region sequence sufficient to confer binding specificity. A full-length light chain includes a variable region domain and a constant region domain. The variable region domain of the light chain is at the amino terminus of the polypeptide. Light chains include kappa chains and lambda chains.

[0030] The term "heavy chain" includes a full-length heavy chain and fragments thereof having a variable region sequence sufficient to confer binding specificity. A full-length heavy chain includes a variable region domain and three constant region domains (C H 1, C H 2, and C H 3). The variable heavy chain domain is at the amino terminus of the polypeptide, the C H domain is at the carboxyl terminus, and CH3 is closest to the carboxyl terminus of the polypeptide. Heavy chains can be of any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE. The term "isotype" refers to the antibody class encoded by the heavy chain constant region gene. In some embodiments, the antibodies provided herein have an IgG4 heavy chain, or an IgG4 heavy chain containing certain amino acid mutations. For example, in some embodiments, IgG4 contains a mutation at position 228 to inhibit Fab arm exchange (EU numbering scheme (Kabat et al. Sequence of proteins of immunologic interest, 5th ed Bethesda, MD, NIH 1991)). For example, in some embodiments, the IgG4 heavy chain is an IgG4 S228P heavy chain. In some embodiments, the heavy chain reduces or eliminates the effector function of the antibody by including one or more amino acid mutations that reduce binding to Fc receptors. For example, the heavy chain can contain a mutation at one or more of positions 233, 234, 235, 236, 237, 265, 309, 331, and 409 (EU numbering).

[0031] The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chains of an antibody, and typically includes approximately 120-130 amino acids at the amino terminus within the heavy chain and approximately 100-110 amino acids at the amino terminus within the light chain. In certain embodiments, the variable regions of different antibodies vary widely in amino acid sequence even among antibodies of the same species. The variable region of an antibody typically determines the specificity of that particular antibody for its target. As used herein, the term "target" refers to a molecule or a portion of a molecule to which binding by an antigen-binding protein is possible. In certain embodiments, the target may have one or more epitopes. In certain embodiments, the target is an antigen. The use of "antigen" in the expression "antigen-binding protein" simply means that the protein sequence constituting the antigen can be bound by an antibody. In this context, it is not required that the protein be foreign or capable of inducing an immune response.

[0032] The term "epitope" includes any determinant (e.g., an antibody or T cell receptor) to which binding by an antigen-binding protein is possible. An epitope is the region of an antigen that is bound by an antigen-binding protein targeting the antigen, and when the antigen is a protein, the epitope includes specific amino acids that directly contact the antigen-binding protein. Epitopes are most often present on proteins, but may in some cases be present on other types of molecules (e.g., nucleic acids). Epitope determinants can include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl, and can have specific three-dimensional structural characteristics and / or specific charge characteristics. Generally, an antibody specific for a particular target antigen preferentially recognizes the epitope on the target antigen present in a complex mixture of proteins and / or macromolecules. Antibody epitopes can be linear or conformational. In embodiments, the epitopes provided herein are linear epitopes.

[0033] The use of the singular form includes the plural unless otherwise specified. The words "a" or "an" mean "at least one" unless otherwise specified. The use of "or" means "and / or" unless otherwise stated. The meaning of the expression "at least one" is equivalent to the meaning of the expression "one or more". Further, the use of the term "including" and other forms (e.g., "includes" and "included") is not limiting. Also, terms such as "element" or "component" include both an element or component that includes one unit and an element or component that includes two or more units unless otherwise specified. As used herein, the term "about" refers to an amount that is more or less than the recited parameter value, e.g., plus or minus 5% or 10% of the object modified by "about", or an amount that can be recognized from the context by one of ordinary skill in the art (e.g., approximately 50% of the interval between values). Also, the term "about" includes the value being referred to.

[0034] Stem cell factor There are at least two forms of SCF in humans, which have different structures and activities. SCF220 functions in several homeostatic functions including hematopoiesis and spermatogenesis and is found in the bone marrow, testis, as well as other tissues and organs. SCF220 is slowly cleavable and is sometimes called "membrane SCF". In contrast, SCF248 is rapidly cleavable and contains a cleavage site in exon 6 located between the N-terminal c-kit binding domain and the transmembrane domain. SCF248 is sometimes referred to as "soluble SCF". Exon 6 is excluded from SCF220 by alternative splicing, thus this cleavage site is absent in SCF220. The monomeric extracellular domain (SCF165) is a cleavage product and functions as a biomarker in plasma for chronic inflammatory diseases. Plasma may also contain detectable levels of the SCF extracellular domain derived from SCF220, but most of the detectable extracellular domain is expected to be SCF165. SCF248 is an isoform found in myofibroblasts, activated epithelial cells, and other cells, activates immune cells during inflammation, and contributes to the perpetuation of fibrosis. More specifically, SCF248 binds to c-Kit on immune cells, initiates cytokine production, activates fibroblasts to transform into myofibroblasts, and secretes extracellular matrix proteins, collagen, and fibronectin. In addition to activated myofibroblasts, activated epithelial, endothelial, macrophage, eosinophil, mast cell, monocyte, and other cells also express SCF on the cell surface, activate more c-Kit+ immune cells, resulting in further cytokine release and immune activation and fibrotic responses.

[0035] The antibodies and antigen-binding fragments thereof disclosed herein are specific for SCF. In some embodiments, the antibodies and fragments thereof are specific for human SCF. In some embodiments, the antibodies and fragments thereof are specific for SCF248. In some embodiments, the antibody binds to SCF248 and does not bind to other isoforms of SCF. In some embodiments, the antibody binds to SCF248 and does not bind to SCF220. In some embodiments, the present disclosure provides a method for generating an antibody or fragment thereof that is specific for SCF248. The present disclosure provides exemplary antibodies and fragments that are specific for SCF248, and methods for generating and using such antibodies and fragments. In some embodiments, the antibodies and fragments thereof provided herein bind to SCF248 and disrupt the positive feedback loop between SCF248-expressing cells and cKit+ immune cells by blocking the interaction between SCF248 and c-Kit.

[0036] Antibodies and fragments The present disclosure provides antibodies (including monoclonal antibodies) and fragments thereof. Antibody fragments specific to SCF provided herein (e.g., SCF248), which may be referred to herein as antigen-binding fragments, mean that they contain a part of the parent antibody capable of binding to the target antigen (SCF (e.g., SCF248)). As used herein, "antibody fragment", "antigen-binding fragment", etc. are used interchangeably. Examples of antigen fragments include Fab fragment, Fab' fragment, F(ab') fragment, Fv fragment, isolated CDR region, bispecific Fab dimer (Fab2), trispecific Fab trimer (Fab3), single-chain Fv protein ("scFv"), bis-scFv, (scFv)2, minibody, diabody, triabody, tetrabody, disulfide-stabilized Fv protein ("dsFv"), single-domain antibody (sdAb, nanobody), heavy-chain-only antibody (e.g., camelid VHH, camelid nanobody, shark Ig NAR), and a part of the full-length antibody responsible for antigen binding.

[0037] A "Fab fragment" contains one light chain and the C H 1 and variable regions of one heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A "Fab' fragment" contains one light chain and a part of one heavy chain, and this part of the heavy chain contains the VH domain and C H 1 domain, and further the region between the C H 1 domain and the C H 2 domain, so that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule. An "F(ab')2 fragment" contains two light chains and two heavy chains, and these heavy chains contain the C H 1 domain and the C HIt includes a part of the constant region between the two domains, as a result, an inter-chain disulfide bond is formed between the two heavy chains. Therefore, the F(ab’)2 fragment is composed of two Fab’ fragments, which are held together by the disulfide bond between the two heavy chains. The Fv fragment contains the variable regions of both the heavy and light chains and lacks the constant region. "scFv" is an Fv molecule in which the variable regions of the heavy and light chains are connected by a flexible linker to form a single polypeptide chain that forms the antigen-binding region.

[0038] In some embodiments, the antibodies and fragments thereof provided herein are defined by these complementarity-determining regions (CDRs). CDRs are part of the variable chains within the antibody, and each of the light-chain variable region and the heavy-chain variable region contains three CDRs: CDR1, CDR2, and CDR3. The CDRs of the antibody determine antigen specificity. In certain embodiments, the definitive definition of the CDRs and the identification of the residues comprising the binding site of the antibody are accomplished by elucidating the structure of the antibody and / or the structure of the antibody-ligand complex. In certain embodiments, it can be accomplished by any of a variety of techniques known to those of skill in the art (e.g., X-ray crystallography). In certain embodiments, various analytical methods can be used to identify or approximate the CDR regions. Examples of such methods include, but are not limited to, the Kabat definition, the Chothia definition, the AbM definition, and the contact definition.

[0039] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, for example, Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but in the Chothia definition, the positions of certain structural loop regions are considered. See, for example, Chothia et al., J. Mol. Biol., 196:901-17 (1986); Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses an integrated package of computer programs for modeling antibody structures created by Oxford Molecular Group. See, for example, Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989); “AbM (trademark), A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK; Oxford Molecular, Ltd. The AbM definition models the tertiary structure of antibodies from the primary sequence using a combination of a knowledge database and first principles methods as described in Samudrala et al., “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach” (PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999)). The contact definition is based on the analysis of available complex crystal structures. See, for example, MacCallum et al., J. Mol. Biol., 5:732-45 (1996).

[0040] Antibodies and their fragments can also include recombinant polypeptides, fusion proteins, and bispecific antibodies. The anti-SCF antibodies and their fragments disclosed herein can be of the IgG1, IgG2, IgG3, or IgG4 isotype. In one embodiment, the anti-SCF antibodies and their fragments disclosed herein are of the IgG1 or IgG4 isotype. The anti-SCF antibodies and their fragments of the present invention can be derived from any species (including but not limited to mouse, rat, rabbit, primate, llama, camel, goat, shark, chicken, and human). The SCF antibodies and their fragments can be chimeric antibodies, humanized antibodies, or fully human antibodies. In one embodiment, the anti-SCF antibody is a mouse antibody. In another embodiment, the anti-SCF antibody is a chimeric antibody. In a further embodiment, the chimeric antibody is a mouse-human chimeric antibody. In another embodiment, the antibody is derived from a mouse and is humanized.

[0041] A "chimeric antibody" is an antibody having at least a portion of the heavy chain variable region and at least a portion of the light chain variable region derived from one species, and at least a portion of the constant region derived from another species. For example, in one embodiment, a chimeric antibody can include a mouse variable region and a human constant region.

[0042] A "humanized antibody" is an antibody that includes complementarity-determining regions (CDRs) derived from a non-human antibody and framework regions and constant regions derived from a human antibody. For example, the anti-SCF antibodies provided herein can include CDRs derived from one or more mouse antibodies and human framework regions and constant regions. Thus, in one embodiment, the humanized antibodies provided herein bind to the same epitope on SCF as the mouse antibody from which the CDRs of the antibody are derived.

[0043] In some embodiments, the antibodies and fragments thereof provided herein include heavy and light chains, each of which includes three CDRs. The amino acid sequences of exemplary heavy chain CDR1, CDR2, and CDR3 (HCDR1, HCDR2, and HCDR3, respectively) and light chain CDR1, CDR2, and CDR3 (LCDR1, LCDR2, and LCDR3, respectively) are shown in Table 1 below. Also shown in Table 1 are the amino acid sequences of exemplary heavy and light chain variable regions. In some embodiments, the present disclosure provides antibodies referred to herein as "5H10" and "2G8". The humanized heavy chain variable regions of humanized 5H10 or 2G8 are referred to herein as VH1, VH2, VH3, VH4, and VH5. 5H10 VH0 is the variable heavy chain of the mouse parental antibody generated by the methods described herein. VH1, VH2, VH3, VH4, VH5 are each humanized heavy chain variable regions derived from 5H10 VH0 or 2G8 VH0. The 5H10 antibody includes a kappa light chain. The mouse parental antibody variable light chain is referred to herein as 5H10 VK0. VK1, VK2, VK3, and VK4 are each humanized light chain variable regions derived from VK0. The 2G8 antibody includes a lambda light chain. The mouse parental antibody variable light chain is referred to herein as 2G8 VL0. VL1, VL2, VL3, and VL4 are each humanized light chain variable regions derived from VL0.

Table 1-1

Table 1-2

[0044] One of ordinary skill in the art will understand that the variable heavy chain and variable light chain may be independently selected from the antibodies provided herein, or may be mixed and matched. Thus, in some embodiments, the antibodies and fragments thereof provided herein include combinations of heavy and light chains selected from the group consisting of VH0 / VK0, VH0 / VK1, VH0 / VK2, VH0 / VK3, VH0 / VK4, VH1 / VK0, VH1 / VK1, VH1 / VK2, VH1 / VK3, VH1 / VK4, VH2 / VK0, VH2 / VK1, VH2 / VK2, VH2 / VK3, VH2 / VK4, VH3 / VK0, VH3 / VK1, VH3 / VK2, VH3 / VK3, VH3 / VK4, VH4 / VK0, VH4 / VK1, VH4 / VK2, VH4 / VK3, VH4 / VK4, VH5 / VK0, VH5 / VK1, VH5 / VK2, VH5 / VK3, and VH5 / VK4.

[0045] In some embodiments, the present disclosure provides an antibody or fragment thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-12. In some embodiments, the present disclosure provides an antibody or fragment thereof comprising a heavy chain variable region as set forth in a sequence selected from the group consisting of SEQ ID NOs: 7-12. In some embodiments, the present disclosure provides an antibody or fragment thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 7-11 and comprising heavy chain CDR1, CDR2, and CDR3 that are identical to SEQ ID NOs: 1, 2, and 3, respectively. In some embodiments, the present disclosure provides an antibody or fragment thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to the amino acid sequence of SEQ ID NO: 12 and comprising heavy chain CDR1, CDR2, and CDR3 that are identical to SEQ ID NOs: 1, 37, and 3, respectively.

[0046] In some embodiments, the present disclosure provides an antibody or fragment thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 17. In some embodiments, the present disclosure provides an antibody or fragment thereof comprising a light chain variable region as set forth in a sequence selected from the group consisting of SEQ ID NOs: 13 to 17. In some embodiments, the present disclosure provides an antibody or fragment thereof comprising an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 17, and comprising light chain CDR1, CDR2, and CDR3 that are identical to SEQ ID NOs: 4, 5, and 6, respectively.

[0047] In some embodiments, the present disclosure provides an antibody or fragment thereof having an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% homology to the following: SEQ ID NO: 7 and SEQ ID NO: 13; SEQ ID NO: 7 and SEQ ID NO: 14; SEQ ID NO: 7 and SEQ ID NO: 15; SEQ ID NO: 7 and SEQ ID NO: 16; SEQ ID NO: 7 and SEQ ID NO: 17; SEQ ID NO: 7 and SEQ ID NO: 13; SEQ ID NO: 7 and SEQ ID NO: 14; SEQ ID NO: 7 and SEQ ID NO: 15; SEQ ID NO: 7 and SEQ ID NO: 16; SEQ ID NO: 7 and SEQ ID NO: 17; SEQ ID NO: 8 and SEQ ID NO: 13; SEQ ID NO: 8 and SEQ ID NO: 14; SEQ ID NO: 8 and SEQ ID NO: 15; SEQ ID NO: 8 and SEQ ID NO: 16; SEQ ID NO: 8 and SEQ ID NO: 17; SEQ ID NO: 9 and SEQ ID NO: 13; SEQ ID NO: 9 and SEQ ID NO: 14; SEQ ID NO: 9 and SEQ ID NO: 15; SEQ ID NO: 9 and SEQ ID NO: 16; SEQ ID NO: 9 and SEQ ID NO: 17; SEQ ID NO: 10 and SEQ ID NO: 13; SEQ ID NO: 10 and SEQ ID NO: 14; SEQ ID NO: 10 and SEQ ID NO: 15; SEQ ID NO: 10 and SEQ ID NO: 16; SEQ ID NO: 10 and SEQ ID NO: 17; SEQ ID NO: 11 and SEQ ID NO: 13; SEQ ID NO: 11 and SEQ ID NO: 14; SEQ ID NO: 11 and SEQ ID NO: 15; SEQ ID NO: 11 and SEQ ID NO: 16; SEQ ID NO: 11 and SEQ ID NO: 17; SEQ ID NO: 12 and SEQ ID NO: 13; SEQ ID NO: 12 and SEQ ID NO: 14; SEQ ID NO: 12 and SEQ ID NO: 15; SEQ ID NO: 12 and SEQ ID NO: 16; or SEQ ID NO: 12 and SEQ ID NO: 17.

[0048] In certain embodiments, the antibody and its fragments comprise a combination of heavy and light chains selected from the group consisting of VH1 / VK1, VH1 / VK2, VH1 / VK3, VH2 / VK1, VH2 / VK2, VH2 / VK3, VH3 / VK1, VH3 / VK2, VH3 / VK3, VH4 / VK1, VH4 / VK2, VH4 / VK3, VH5 / VK1, VH5 / VK2, and VH5 / VK3. In some embodiments, the antibody comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 9, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 16. In some embodiments, the antibody or its fragment comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 9, and an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 16, and the antibody or fragment comprises heavy chain CDR1, CDR2, and CDR3 identical to SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 identical to SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the antibody or its fragment comprises an amino acid sequence having at least 95% or at least 99% sequence identity to SEQ ID NO: 8 or SEQ ID NO: 9, and an amino acid sequence having at least 95% or at least 99% sequence identity to SEQ ID NO: 16, and the antibody or fragment comprises heavy chain CDR1, CDR2, and CDR3 identical to SEQ ID NOs: 1, 2, and 3, respectively, and light chain CDR1, CDR2, and CDR3 identical to SEQ ID NOs: 4, 5, and 6, respectively. The antibody or its fragment may specifically bind to SCF248 but not to SCF220. In some embodiments, the antibody comprises the heavy chain variable region set forth in SEQ ID NO: 8 and the light chain variable region set forth in SEQ ID NO: 16.In some embodiments, the antibody comprises the heavy chain variable region set forth in SEQ ID NO: 9 and the light chain variable region set forth in SEQ ID NO: 16.

[0049] In some embodiments, the antibodies and fragments provided herein comprise the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, or 12 or a variant thereof, and / or the light chain variable region amino acid sequence set forth in SEQ ID NO: 13, 14, 15, 16, or 17 or a variant thereof. The variant can include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions or deletions, or combinations thereof. In some embodiments, the amino acid substitutions are conservative substitutions. An anti-SCF antibody disclosed herein having one or more amino acid substitutions, insertions, deletions, or combinations thereof in a CDR or variable light or heavy chain region retains the biological activity of the corresponding anti-SCF antibody having no amino acid substitutions, insertions, or deletions relative to the sequences provided herein. Thus, the variant anti-SCF antibodies provided herein retain specific binding to SCF248. As used herein, the terms percent homology, sequence identity, sequence homology, etc. are used interchangeably and refer to the number of identical amino acid sequences shared by two reference sequences divided by the total number of amino acid positions and multiplied by 100.

[0050] In some embodiments, the present invention provides an antibody that binds to the same epitope as any one of the exemplary antibodies disclosed herein. Thus, in some embodiments, the present invention provides an antibody whose binding to SCF competes with the exemplary antibodies provided herein. For example, in some embodiments, the present disclosure provides an antibody that specifically binds to a region of the amino acid sequence shown as SEQ ID NO: 29 herein. In some embodiments, the antibodies provided herein specifically bind to an epitope comprising the amino acid sequence of SEQ ID NO: 33 (ASSLRNDSSSSNRK) or SEQ ID NO: 36 (ASSLRNDSSSSNR). In some embodiments, the present disclosure provides an antibody that specifically binds to an epitope consisting of the amino acid sequence set forth in SEQ ID NO: 33 or SEQ ID NO: 36. In some embodiments, the present disclosure provides an antibody that specifically binds to an epitope comprising at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 contiguous amino acids of SEQ ID NO: 33.

[0051] In some embodiments, the antibodies and fragments thereof provided herein comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3 of the heavy and light chain variable regions provided herein, or variants thereof. Thus, in some embodiments, the antibodies and fragments thereof provided herein include antibodies in which the HCDR is the HCDR of SEQ ID NO: 7, 8, 9, 10, 11, or 12, and / or antibodies in which the LCDR is the LCDR of SEQ ID NO: 13, 14, 15, 16, or 17. For example, in some embodiments, the antibody and its fragment comprise amino acids 31-35, 50-65, and 95-102 of any one of the heavy chain variable regions provided herein as defined by the Kabat numbering scheme. In some embodiments, the antibody and its fragment comprise amino acids 24-34, 50-56, and 89-97 of any one of the light chain variable regions provided herein as defined by the Kabat numbering scheme.

[0052] This specification provides exemplary humanized antibodies. Additional anti-SCF antibodies comprising the heavy and light chain CDRs or variants thereof provided herein can be generated using any human framework sequence and are encompassed by the present invention. In one embodiment, framework sequences suitable for use in the present invention include framework sequences that are structurally similar to the framework sequences provided herein. Further modifications can be made in the framework region to improve the properties of the antibodies provided herein. Such further framework modifications can include chemical modifications, point mutations to reduce immunogenicity or remove T cell epitopes, or back mutations to residues within the original germline sequence.

[0053] In some embodiments, such framework modifications include modifications corresponding to the mutations exemplified herein, including back mutations to the germline sequence. For example, in one embodiment, one or more amino acids within the VH and / or VL human framework regions of the humanized antibodies provided herein have been back mutated to the corresponding amino acids in the parental mouse antibody. The present invention also encompasses humanized antibodies that bind to SCF (e.g., SCF248) and that include framework modifications corresponding to the exemplary modifications described herein for any suitable framework sequence, as well as other framework modifications that improve the properties of the antibody in other ways. In other embodiments, the antibodies provided herein include one or more mutations that improve stability, improve solubility, modify glycosylation, and / or reduce immunogenicity by targeted amino acid changes that, for example, reduce deamidation or oxidation, reduce isomerization, optimize hydrophobic core and / or charge cluster residues, remove hydrophobic surface residues, optimize residues involved in the interface between the variable heavy chain and the variable light chain, and / or modify the isoelectric point.

[0054] The anti-SCF antibodies and fragments thereof provided herein may further include Fc region modifications for modifying effector functions. Fc modifications can be in the form of amino acid insertions, deletions, or substitutions, or chemical modifications. For example, Fc region modifications can be made to increase or decrease complement binding, increase or decrease antibody-dependent cellular cytotoxicity, or increase or decrease the half-life of the antibody. Some Fc modifications increase or decrease the affinity of the antibody for Fcγ receptors (e.g., FcγRI, FcγRII, FcγRIII, or FcRn). A variety of Fc modifications have been described in the art (e.g., Shields et al., J Biol. Chem 276;6591(2001); Tai et al., Blood 119;2074(2012); Spiekermann et al., J Exp. Med 196;303(2002); Moore et al., mAbs 2:2;181(2010); Medzihradsky, Methods in Molecular Biology 446;293(2008); Mannan et al., Drug Metabolism and Disposition 35;86(2007); and Idusogie et al., J Immunol 164;4178(2000). In some embodiments, the glycosylation pattern of the Fc region is modified. In other embodiments, the Fc region is modified by pegylation (e.g., by reacting the antibody or fragment thereof with polyethylene glycol (PEG)). Exemplary Fc modifications include modifications at one or more amino acid positions selected from the group consisting of 228, 233, 234, 235, 236, 241, 248, 265, 297, 309, 331, and 409 (Kabat numbering; Kabat et al., Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). In embodiments, the antibody has a modification for reducing or eliminating effector function.In some embodiments, the antibody is an IgG1 antibody having one or more Fc modifications selected from the group consisting of E233P, L234V, L234A, L235V, L235A, G236 (deletion), D265A, D270A, N297A, and N297Q. In some embodiments, the antibody is an IgG4 antibody having one or more Fc modifications selected from the group consisting of S228P, E233P, F234A, F234V, L235A, L235V, S241P, L248E, D265A, D265T, L309L, and R409K. In some embodiments, the anti-SCF antibody provided herein comprises the S241P mutation and the L248E mutation.

[0055] In some embodiments, the present disclosure provides the antibodies provided herein that comprise the human IgG4 constant regions set forth in SEQ ID NOs: 40 and 41. In some embodiments, the present disclosure provides antibodies comprising at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 99% sequence identity to SEQ ID NO: 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50. In some embodiments, the present disclosure provides an antibody comprising the heavy chain set forth in SEQ ID NO: 40 and the light chain set forth in SEQ ID NO: 41. In some embodiments, the present disclosure provides an antibody comprising the heavy chain set forth in SEQ ID NO: 42, 43, 44, 45, or 46 and the light chain set forth in SEQ ID NO: 47, 48, 49, or 50. In some embodiments, the present disclosure provides an antibody comprising the heavy chain set forth in SEQ ID NO: 42 and the light chain set forth in SEQ ID NO: 49. In some embodiments, the present disclosure provides an antibody comprising the heavy chain set forth in SEQ ID NO: 43 and the light chain set forth in SEQ ID NO: 49. In some embodiments, the present disclosure provides an antibody comprising the heavy chain set forth in SEQ ID NO: 44 and the light chain set forth in SEQ ID NO: 49. In some embodiments, the present disclosure provides an antibody comprising the heavy chain set forth in SEQ ID NO: 45 and the light chain set forth in SEQ ID NO: 49. In some embodiments, the present disclosure provides an antibody comprising the heavy chain set forth in SEQ ID NO: 46 and the light chain set forth in SEQ ID NO: 49.

[0056] In some embodiments, the present disclosure provides a method for generating an antibody that specifically binds to SCF248. The SCF248 isoform of SCF contains exon 6. This contains a cleavage site between two alanine residues (amino acids 16 and 17 of SEQ ID NO: 34, which shows the amino acid sequence of exon 6). Previous anti-SCF antibodies were generated by immunizing mice with a peptide spanning exon 6 and part of exon 7 (see, e.g., U.S. Patent No. 8,911,729, which is incorporated herein by reference in its entirety for all purposes). Since SCF220 is associated with constitutive activity, any cross-reactivity with SCF220 is considered detrimental as it can cause various off-target effects in the subject. Advantageously, the antibodies provided by the present disclosure bind to SCF248 with very high specificity. In some embodiments, the antibodies provided herein are specific for SCF248 and do not bind to SCF220. Thus, the antibodies provided herein can specifically inhibit the interaction between SCF248 and c-Kit that induces and perpetuates the chronic inflammatory response and fibrosis. Furthermore, the antibodies provided herein can reduce the interaction between SCF248 and c-Kit by specifically inducing the internalization of SCF. Thus, in some embodiments, the present disclosure provides antibodies that are specific for SCF248 and are safe and effective for various inflammatory and fibrotic diseases discussed herein and known in the art.

[0057] For the preparation of monoclonal antibodies, any technique can be used that results in the production of antibody molecules by a continuous cell line in culture (see, e.g., Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Such techniques include, but are not limited to, the hybridoma technique first developed by Kohler and Milstein, as well as the trioma technique, human B cell hybridoma techniques (see, e.g., Kozbor et al., Immunol. Today, 4:72 (1983)), and the EBV-hybridoma technique for producing human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). Alternatively, the antibodies may be made by recombinant DNA methods. In some embodiments, the antibodies according to the present disclosure can be made, for example, by isolating monoclonal antibodies from phage display libraries using the techniques described in Clackson et al., Nature 352:624-28 (1991) and Marks et al., J. Mol. Biol. 222(3):581-97 (1991). In some embodiments, the antibodies are fully human antibodies constructed by combining Fv clone variable domain sequences (s) selected from a human-derived phage display or yeast display library (ies) with known human constant domain sequences (s).

[0058] In some embodiments provided herein, the antibody is prepared from a hybridoma. Using the hybridoma method, a mouse, hamster, or other suitable host animal is immunized by injecting an immunizing peptide, thereby inducing the production of antibodies that specifically bind to the immunizing antigen by lymphocytes. Alternatively, the lymphocytes may be immunized in vitro. After immunization, the lymphocytes are isolated and fused with a suitable myeloma cell line, for example using polyethylene glycol, to form hybridoma cells, which can then be selected from other than unfused lymphocytes and myeloma cells. The hybridoma produces monoclonal antibodies that specifically target the selected antigen as determined by immunoprecipitation, immunoblotting, or in vitro binding assays (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)), and the hybridoma can then be propagated in vitro (e.g., under culture) or in vivo as an animal ascites tumor using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 1986). The monoclonal antibody can then be purified from the culture medium or ascites as described above for polyclonal antibodies.

[0059] In some embodiments, the antibodies provided herein are generated using a mouse hybridoma system. Hybridoma production in mice is a well-established procedure. Immunization protocols and techniques for isolating immunized spleen cells for fusion are known in the art. Also known are fusion partners (e.g., mouse myeloma cells) and fusion procedures. Embodiments of the techniques herein provide antibodies (e.g., monoclonal antibodies) produced from hybridomas prepared by immunizing mice with a peptide that is a part or fragment of the SCF protein.

[0060] In some embodiments, the antibodies specific for SCF248 provided herein are generated by immunizing mice with peptides having amino acid sequences that are primarily or exclusively within exon 6. For example, the immunogenic peptide comprises any contiguous five or more amino acids within SEQ ID NO: 34. As another example, the immunogenic peptide comprises any contiguous five or more amino acids starting from amino acid position 20 of SEQ ID NO: 29. As another example, the immunogenic peptide comprises any contiguous five or more amino acids starting at amino acid position 20 of SEQ ID NO: 29 and ending at any one of positions 25-38 of SEQ ID NO: 29. Thus, in some embodiments, the immunogenic peptide comprises the amino acid sequence of exon 6 after the cleavage site, and is either completely contained within exon 6 or contains only 1, 2, 3, 4, or 5 amino acids of exon 7. In some embodiments, the immunogenic peptide comprises or consists of SEQ ID NO: 30. In some embodiments, the immunogenic peptide comprises any of the peptides provided herein or conservative variants thereof. Conservative variants can include 1, 2, 3, 4, or 5 amino acid substitutions or deletions, or combinations thereof. As shown above, in some embodiments, the antibodies generated using the immunogenic peptides provided herein have epitopes that are completely or mostly contained within exon 6. "Mostly within" means that at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the peptide is contained within exon 6. In some embodiments, the epitope starts at the cleavage site of exon 6 (i.e., between the alanines at amino acid positions 19 and 20 of SEQ ID NO: 29) and extends to the end of exon 6. In some embodiments, the epitope starts at the cleavage site of exon 6 and extends to the first, second, third, fourth, or fifth n-terminal amino acids of the transmembrane domain. In some embodiments, the epitope comprises or consists of SEQ ID NO: 33. In some embodiments, the antibodies referred to herein as 5H10 (including mouse, chimeric, and humanized 5H10 antibodies) bind to an epitope of SCF that comprises or consists of SEQ ID NO: 33.

[0061] In some embodiments, the methods provided herein were used to generate an antibody referred to herein as 5H10. In some embodiments, the antibody “5H10” is also referred to herein as “OpSCF”. Advantageously, antibody 5H10 binds with high specificity to SCF248 and does not bind to SCF220. The amino acid sequences of the mouse parent antibody 5H10 and its humanized variants are provided herein (see Table 1).

[0062] In one embodiment, the invention provides a bispecific or multispecific antibody specific for SCF and at least one other antigen or epitope. The anti-SCF antibodies and fragments thereof provided herein can be tested for binding to SCF using the binding assays provided herein or any other binding assay known in the art.

[0063] Unless otherwise specified, the practice of the present invention will employ conventional techniques of molecular biology, cell biology, biochemistry, and immunology that are well known in the art, such as those described in Methods in Molecular Biology, Humana Press; Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989); Current Protocols in Immunology (J.E. Coligan et al., eds., 1991); Immunobiology (C.A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Phage display: a laboratory manual (C. Barbas III et al, Cold Spring Harbor Laboratory Press, 2001); and Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999).

[0064] Therapeutic method In one aspect, the present disclosure provides a method for treating and / or preventing any disease or condition associated with the migration, activation, and / or proliferation of immune cells via the interaction of SCF248 with c-Kit on immune cells. Thus, in some embodiments, the present disclosure provides a method for inhibiting or preventing the activation of immune cells, as well as a method for treating or preventing various diseases and disorders associated with inflammation by reducing or preventing the accumulation of immune cells in an organ or tissue. In some embodiments, the immune cells are selected from the group consisting of mast cells, natural lymphocytes (ILCs (e.g., ILC2 or ILC3 cells)), and eosinophils.

[0065] As used herein, the terms "treatment" or "treating" refer to both therapeutic treatment and prophylactic treatment. Subjects in need of treatment include, in addition to subjects already having a disease or condition, subjects who may be at risk of developing a disease or condition and who are targeted for prevention, delay, or alleviation of that disease or condition. As used herein, the term "subject" means a mammal (e.g., rodents, cats, dogs, and primates). Preferably, the subject described in the present invention is a human. As used herein, the term "therapeutically effective amount" refers to the amount of a compound or composition necessary to provide a therapeutic and / or prophylactic benefit to a subject.

[0066] In one aspect, the present invention provides a method for treating an inflammatory disease, a fibrotic disease, and / or a tissue remodeling disease in a subject. In some embodiments, the inflammatory disease is a chronic inflammatory disease.

[0067] Chronic inflammatory diseases, fibrotic diseases, and tissue remodeling diseases include diseases of the lung, kidney, liver, heart, skin, connective tissue, and other tissues. Exemplary inflammatory, fibrotic, or tissue remodeling diseases include, but are not limited to, pulmonary fibrosis (e.g., idiopathic pulmonary fibrosis (IPF), scleroderma pulmonary fibrosis, scleroderma-related interstitial lung disease (SSc-ILD), pulmonary fibrosis associated with lung infection or pneumonia, pulmonary fibrosis associated with systemic lupus erythematosus and / or rheumatoid arthritis, sarcoidosis), chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), cystic fibrosis, peribronchial fibrosis, bleomycin lung, hypersensitivity pneumonitis, asthma, fibrothorax, mediastinal fibrosis, chronic rhinosinusitis, urticaria (e.g., chronic idiopathic urticaria), atopic dermatitis, dermatomyositis, nodular subepidermal fibrosis, scleroderma, keloid, renal fibrosis, chronic kidney disease, glomerulonephritis, chronic renal allograft rejection, nephropathy (e.g., IgA nephropathy, focal segmental glomerulosclerosis, rapidly progressive glomerulonephritis, crescentic glomerulonephritis, lupus nephritis, hypertensive nephropathy, or diabetic nephropathy), non-alcoholic steatohepatitis (NASH), cirrhosis, liver fibrosis, primary sclerosing cholangitis, primary biliary cirrhosis, fibromyalgia, gingival fibrosis, radiation-induced fibrosis, eosinophilic esophagitis, articular fibrosis, and atrial fibrosis, endomyocardial fibrosis, parenchymal fibrosis, fibrous histiocytoma, or glial scarring.

[0068] In some embodiments, the antibodies and fragments thereof disclosed herein can be administered to a subject by at least one route selected from parenteral, subcutaneous, intramuscular, intravenous, intra-articular, intra-bronchial, intraperitoneal, intra-capsular, intra-cartilaginous, intra-cavity, intracerebral, intraventricular, intra-colonic, intra-cervical, intra-gastric, intra-hepatic, intra-myocardial, intra-osseous, intra-pelvic, intra-pericardial, intra-peritoneal, intra-pleural, intra-prostatic, intra-pulmonary, intra-rectal, intra-renal, intra-retinal, intra-spinal, intra-synovial, intra-thoracic, intra-tympanic, intra-uterine, intra-vesical, intra-vitreal, bolus, sub-conjunctival, oral, vaginal, rectal, buccal, sub-lingual, intra-nasal, intra-tumoral, and transdermal.

[0069] In various embodiments, the antibodies and fragments thereof disclosed herein can be administered to a subject in need thereof in combination with one or more additional therapies. The one or more additional therapies can be a procedure such as a surgical procedure or, in some cases, a therapeutic agent (e.g., an agent designed to reduce or mitigate the symptoms of a disease or disorder associated with fibrosis and / or inflammation).

[0070] The present invention will be further described by reference to the following examples. It should be noted, however, that these examples are illustrative only, as are the above-described embodiments, and are not to be construed as limiting the scope of the present invention in any way.

Examples

[0071] The following examples are presented for the purpose of illustrating various embodiments of the present disclosure and are not intended to limit the present disclosure in any way. It will be recognized by those skilled in the art that variations and other uses of these examples are subsumed within the spirit of the present disclosure as defined by the claims.

[0072] An overview of the tissue injury / disease process is summarized in FIG. 1. The disease process initiates inflammation. c-Kit+ immune cells produce cytokines, causing fibroblasts to change into activated myofibroblasts that express SCF248 on their surface. When SCF248 is expressed on the surface of myofibroblasts and other cells, more immune cells are activated, resulting in the release of IL-4, IL-9, IL-13, IL-25, TGFβ, and other cytokines, perpetuating the inflammation. Myofibroblasts secrete extracellular matrix proteins, collagen, fibronectin, causing fibrosis and remodeling diseases (e.g., pulmonary fibrosis, skin fibrosis, severe asthma, and other diseases).

[0073] An exemplary mechanism of the antibodies of the present disclosure that target SCF248 (this antibody is referred to herein as OpSCF and / or 5H10) is summarized in FIG. 2.

[0074] As shown above, SCF has two isoforms resulting from alternative splicing: SCF248 and SCF220. SCF248 and SCF220 differ by exon 6. SCF220 is associated with a constitutive function, and SCF248 is associated with inflammation and fibrosis. SCF248 activates immune cells during inflammation and is sometimes called "soluble SCF". SCF248 is expressed in various cell types including myofibroblasts, activated epithelium, endothelium, macrophages, eosinophils, mast cells, and monocytes (Figure 3). The SCF248 isoform results in cleavage of a monomeric truncated extracellular domain called SCF165. The amino acid sequence of exon 6 is shown herein as SEQ ID NO: 34.

[0075] Example 1: Production of anti-SCF mAbs Using Hybridoma Technology Antibodies that bind to SCF248 were generated using a peptide containing ASSLRNDSSSSNRKAKNPPGD (SEQ ID NO: 30). The immunopeptide included a portion of exon 6, i.e., the SCF248 isoform of stem cell factor. Specifically, the immunopeptide included a portion of exon 6 starting after the cleavage site as defined herein. Mice were immunized with the peptide of SEQ ID NO: 30 according to a standard protocol. Quantification of high-titer serum antibodies showed that appropriate immunization and fusion hybridomas had been generated. Culture supernatants were analyzed for SCF-specific antibodies from individual clones and selected based on specificity. Hybridomas producing monoclonal antibodies specific for the peptide were grown and then the most highly titered monoclonal was tested in biologically relevant cultures. Antibody 5H10 had high specificity for SCF248 and no cross-reactivity with SCF220. None of the other monoclonal antibodies produced by the hybridomas had high specificity for SCF248 without cross-reactivity with SCF220. Therefore, 5H10 was selected for further characterization, development, and chimerization, and subsequent humanization.

[0076] Example 2. Binding of 5H10 to the Full-Length Extracellular Domain of SCF248 The mouse 5H10 antibody obtained by the method described in Example 1 was directly conjugated with a fluorescent marker, and this labeled antibody was incubated with Sl / Sl4 hSCF248 cells expressing SCF248, Sl / Sl4 hSCF220 cells expressing SCF220, or control cells not expressing SCF. The binding of the labeled antibody to the cells was evaluated by flow cytometry. It is shown in Figure 4A that 5H10 has specificity for SCF248 and lacks cross-reactivity with SCF220.

[0077] The binding of the mouse 5H10 antibody to the truncated extracellular domain (ECD) containing only amino acids 1 to 165 of SCF and the full-length ECD containing amino acids 1 to 194 of SCF was evaluated by ELISA. This antibody bound to the full-length SCF ECD but not to the truncated SCF ECD (Figure 4B), indicating that this antibody is specific for the full-length extracellular domain and does not bind to the monomeric truncated ECD circulating in the blood.

[0078] To evaluate the ability of the 2G8 and 5H10 antibodies to internalize SCF248 on myofibroblasts, these antibodies were labeled with pHrodo red, which is colorless at neutral pH and fluoresces red at low pH within endosomes. The labeled antibodies were incubated with cultured human IPF myofibroblasts for 45 minutes, and the red fluorescence was visualized by microscopy. As shown in Figure 5, the dye-labeled antibodies were rapidly internalized, while the control IgG was not internalized. 5H10 was internalized more rapidly than 2G8, and higher fluorescence was obtained.

[0079] SCF induces c-kit to signal through two different pathways: the MEK / ERK pathway and the P13K / AKT pathway. A test was conducted to examine whether the mouse 5H10 antibody inhibits intracellular signaling of c-kit positive cells in one or both of these pathways. In the presence of either 5H10 or IgG control, eosinophils were incubated with the SCF248-expressing cell line, and the expression of phosphorylated proteins was measured using the BioRad Bio-Plex assay system. 5H10 significantly decreased the levels of phospho-MEK and phospho-AKT, indicating that the antibody significantly reduces intracellular signaling via c-kit (Figure 6).

[0080] Collectively, the results of these tests showed that the antibody 5H10 specifically binds to and internalizes SCF248 and does not cross-react with the SCF220 isoform or the cleaved ECD. Furthermore, 5H10 significantly inhibits the intracellular signaling pathway that perpetuates inflammation within c-kit positive cells.

[0081] Example 3: Humanization of the mouse antibody 5H10 Chimeric antibodies derived from 5H10 were produced by subcloning the variable domains of the heavy and light chains into vectors with a human IgG4 backbone. The chimeric antibodies were expressed and purified using standard protocols. 2G8 is a previously developed antibody that binds to SCF248 and SCF220 and contains a lambda light chain. The chimeric heavy and light chains of 2G8 were named VH0 and VL0, respectively. The SCF248-specific antibody 5H10 provided herein contains a kappa light chain. The chimeric heavy and light chains of 5H10 were named VH0 and VK0, respectively.

[0082] This chimeric antibody was humanized. The humanized heavy chain had the same complementarity-determining regions (CDRs) but retained a more "human-like" framework region. Several humanized variants of each of the 2G8 and 5H10 variable heavy chains (designated herein as VH1, VH2, VH3, VH4, and VH5) were generated. Humanized kappa light chain variants of 5H10 (designated herein as VK1, VK2, VK3, and VK4) were also generated. The humanized lambda light chain of 2G8 was named VL1, VL2, VL3, and VL4. The combinations of chimeric and humanized light and heavy chains in 2G8 and 5H10 tested are shown in Tables 2 and 3, respectively. As shown in Table 2, certain heavy and light chain combinations of the 5H10 antibody variant resulted in high binding to hSCF248. The binding data used to determine the binding scores are shown in Example 5 below.

Table 2

Table 3

[0083] Binding affinity was also evaluated using BiaCore analysis. From the BiaCore data, it was shown that the affinity of all humanized 5H10 antibodies having a VK1, VK2, or VK3 light chain for the immobilized SCF248 peptide antigen was very similar to the binding affinity of the parental mouse 5H10 using this assay. The humanized 5H10 antibody having a VK4 light chain did not bind to this peptide.

Table 4

[0084] Example 4. Evaluation of Anti-SCF Chimeric Antibody Binding by Flow Cytometry Using the transfected cell line Sl / Sl4 hSCF248 that expresses SCF248, the binding of chimeric antibodies 2G8 and 5H10 was tested. An anti-SCF antibody was used as a positive control for SCF binding. A human IgG4 antibody was used as a negative isotype control antibody. Early passage (P3) Sl / Sl4 hSCF248 cells were compared with late passage (P5) cells. As expected, the negative control (human IgG4 antibody) did not bind to any of the cell populations. 5H10 bound to the cells in the case of early passage (Figure 7A), but was not detected in the case of late passage (Figure 7B). This is because the expression of SCF248 was lost after multiple passages. Similarly, the maximum mean fluorescence intensity (MFI) detected with 10 μg / mL of 2G8 was approximately 4-fold lower in P5 compared to P3.

[0085] Similar observations were made in cells treated with hygromycin B. Hygromycin selection was used to enrich the fraction of Sl / Sl4 cells expressing the corresponding human SCF sequence. The binding of two chimeric anti-SCF mAbs 2G8 and 5H10 and a humanized anti-SCF antibody was evaluated by flow cytometry. Using Sl / Sl4 hSCF248 cells and Sl / Sl4 hSCF220 cells (SCF248+ and SCF220+ respectively), the binding and specificity of chimeric antibodies 2G8 and 5H10 were tested. An anti-SCF antibody was used as a positive control for SCF binding. A human IgG4 antibody was used as a negative isotype control antibody. Cells treated with hygromycin B were compared with early passage (P3) cells. The maximum mean fluorescence intensity (MFI) and the antibody dose-response curve were the same as those of early passage (P3) cells (Figure 8A, Figure 8B).

[0086] Example 5. Evaluation of the Binding of 2G8 and 5H10 Humanized mAbs by Flow Cytometry Using Sl / Sl4 hSCF248 cells and Sl / Sl4 hSCF220 cells (SCF248+ and SCF220+ respectively), the binding and specificity of chimeric antibodies 2G8 and 5H10, as well as their humanized variants, were tested. In both experimental sets, a human IgG4 antibody was used as a negative isotype control antibody. The isotype control did not bind to either Sl / Sl4 hSCF248 cells or Sl / Sl4 hSCF220 cells (Figures 9A, 9B, 10A, and 10B). A commercially available anti-SCF antibody (Abcam, catalog number EP665Y / ab52603) was found to bind to Sl / Sl4 hSCF220 cells and bind slightly to Sl / Sl4 hSCF248 cells (only at a 1:25 dilution) (Figures 9A, 9B, 10A, and 10B). 2G8 VH0 / VL0 (chimeric 2G8) showed stronger binding than its humanized clone. However, 2G8 VH0 / VL0 also showed binding to Sl / Sl4 hSCF220 cells at antibody concentrations of 3.3 and 10 μg / mL (Figure 9A, Figure 9B). 5H10 VH0 / VK0 showed higher binding compared to the humanized clones 5H10 VH3 / VK2, 5H10 VH3 / VK3, 5H10 VH4 / VK2, 5H10 VH4 / VK3, 5H10 VH5 / VK2, and 5H10 VH5 / VK3 (Figure 10A). No binding of 5H10 or any of its humanized variants to Sl / Sl4 hSCF220 cells was observed (Figure 10B). From the perspective of 50% maximum binding (BC 50 ) in this test, the differences between the shown 5H10 humanized variants are presented in Table 5. The binding of the 5H10 clone reached saturation at 3.3 μg / mL on Sl / Sl4 hSCF248 cells. (Figure 10A).

Table 5

[0087] Using Sl / Sl4 hSCF248 cells, the binding of further humanized variants of 5H10 and 2G8 was tested. 2G8 VH0 / VL0 (chimeric) showed stronger cell binding than the humanized variants (Figure 11A). The binding profiles of the humanized 5H10 clones 5H10 VH1 / VK1, 5H10 VH1 / VK2, 5H10 VH1 / VK3, 5H10 VH2 / VK2, and 5H10 VH2 / VK3 were equivalent to 5H10 VH0 / VK0 (Figure 11B). Consistent with the Biacore data presented above, the humanized variant 5H10 VH1 / VK4 lost target binding (Figure 11B). Isotype controls did not bind to Sl / Sl4 hSCF248 cells. Based on the data presented in these tests, binding scores to Sl / Sl4 hSCF248 cells were assigned to the humanized 2G8 and 5H10 mAbs and are presented in Tables 2 and 3 above.

[0088] In another experiment, the binding of 5H10 clones VH1 / VK3, VH2 / VK3, VH3 / VK3, VH4 / VK3, and VH5 / VK3 to SCF248-expressing cell lines was evaluated by flow cytometry. As shown in Figures 11C and 11D, VH1 / VK3 and VH2 / VK3 showed high binding, which reached a maximum at 1 μg / mL. The negative control was the secondary antibody only. No binding was observed for the control SCF220-expressing cell line (not shown).

[0089] Example 6. In Vitro Blockade of the Interaction between SCF and c-kit The humanized 5H10 antibody was tested for its ability to inhibit the SCF-c-kit interaction and the inflammatory feed-forward loop in vitro. Cultured human IPF myofibroblasts (Mfb) expressing surface SCF248 were overlaid with the SCF-responsive cell line, LAD2 mast cells. In the absence of any other intervention, Mfb stimulates LAD2 cells, and the cytokines produced thereby stimulate Mfb, resulting in the production of additional cytokines and extracellular matrix proteins. In this assay, the readouts for inflammation and the feed-forward loop were the mRNAs for CCL11, collagen 1 and 3, and fibronectin.

[0090] Mouse 5H10 antibody and humanized (VH1 / VK3, VH2 / VK3, VH3 / VK3, VH4 / VK3, and VH5 / VK3) 5H10 antibodies were pre-incubated with Mfb at concentrations of 1 μg / mL and 10 μg / mL to evaluate their ability to inhibit these feed-forward loops. The results are shown in Figures 12A - 12D. The humanized VH1 / VK3 antibody consistently showed inhibition of the SCF-c-kit interaction even at low concentrations.

[0091] To evaluate the ability of humanized antibodies (5H10, VH1 / VK3, and VH2 / VK3) to internalize SCF248 on myofibroblasts, these antibodies were labeled with pHrodo red, which is colorless at neutral pH and fluoresces red at the low pH within endosomes. The labeled antibodies were incubated with cultured human IPF myofibroblasts for 45 minutes, and the red fluorescence was visualized by microscopy. As shown in Figure 13, similar to the mouse parental antibody 5H10 and the chimeric antibody (VH0 / VK0), the humanized antibodies were also rapidly internalized.

[0092] Example 7. Immunogenicity of Chimeric Antibody 5H10 and Humanized Lead Candidates The immunogenicity of five humanized antibodies 5H10 VH1 / VK3, 5H10 VH2 / VK3, 5H10 VH3 / VK3, 5H10 VH4 / VK3, and 5H10 VH5 / VK3 was compared with that of the chimeric antibody 5H10 VH0 / VK0.

[0093] The initial evaluation of any cytotoxic effects of the samples on PBMC viability was performed on five donors used in the EpiScreen™ time-course assay. CD8+ T cell-depleted PBMC were incubated with the samples, and cell viability was quantified using a Luna-FL™ Automated Cell Counter on day 7. As a result, the average viability of PBMC from five donors treated with anti-SCF mAb was similar to that of cells treated with medium alone, and was shown to be in the range of 83% - 90% (Figure 14). KLH (Pierce, Life Technologies, UK) was used as the neoantigen. Exenatide (Bydueon, AstraZeneca, UK) was used as a clinical benchmark control. Figures 15A - H and Table 6 illustrate the results obtained from the EpiScreen™ time-course T cell proliferation assay of CD4+ T cell responses induced by the samples and controls. Both the clinical benchmark exenatide and the neoantigen KLH induced positive proliferation responses. 5H10 VH1 / VK3, 5H10 VH2 / VK3, and 5H10 VH3 / VK3 induced low frequencies of positive response (SI ≥ 1.90, p < 0.05) rates, which were in the range of 4% - 8%. Sample 5H10 VH5 / VK3 induced a higher positive response in 17% of the donor cohort. 5H10 VH0 / VK0 and 5H10 VH4 / VK3 did not induce any positive response. The average magnitude of the positive T cell proliferation response was 2.14 - 3.61 across all samples (Table 7).

[0094] Analysis of variance (ANOVA) of the entire proliferation dataset (using the maximum magnitude of proliferation on days 5 - 8) was used to determine whether there were statistically significant differences in the maximum magnitude of the CD4+ T cell response to the test conditions compared to each other and compared to the clinical benchmark exenatide (Figure 16). The maximum magnitude of the T cell proliferation response to exenatide was statistically higher than the response to all samples (Table 8).

Table 6

Table 7

Table 8

[0095] In summary, the risk of clinical immunogenicity was determined by measuring ex vivo T cell responses using peripheral blood mononuclear cells (PBMCs) isolated from 24 healthy donors representative of European and North American populations (based on HLA allotypes) in the EpiScreen™ time-course T cell assay. T cell responses were measured using a proliferation assay ( 3 H]-thymidine incorporation). As a result, four of the lead humanized antibodies were shown to have low potential for clinical immunogenicity.

[0096] Example 8. Evaluation of Mouse 5H10 in the Bleomycin Model of Lung Inflammation and Fibrosis The in vivo effect of 5H10 was evaluated using an animal model of bleomycin-induced pulmonary fibrosis. Bleomycin is a chemotherapeutic agent that causes pulmonary fibrosis in humans and animals. C57BL6 mice were administered bleomycin intratracheally on day 1, and 20 mg / kg of 5H10 or an isotype-matched control antibody was administered intraperitoneally on days 8 and 12. Samples were collected on day 17. In 5H10-treated animals, significant improvement in lung tissue appearance (Figure 17), reduction in lung hydroxyproline (a quantitative measure of fibrosis; Figure 18), maintenance of body weight over time (Figure 19), reduction in mRNA in inflammatory cytokines and myofibroblast activation markers (Figure 20), and reduction in lung mast cells, eosinophils, and ILC2 lymphocytes (Figure 21) were observed. In addition, pulmonary function tests were also significantly improved (Figure 22). Thus, this study demonstrated that 5H10 is effective in reducing fibrosis and inflammation and improving lung function in an in vivo model of pulmonary fibrosis.

[0097] Example 9. Evaluation of the Humanized 5H10 Antibody in a Chronic Allergic Asthma Model The humanized 5H10 antibody was tested in an in vivo model of chronic allergic asthma. Mice were sensitized intraperitoneally and subcutaneously with cockroach antigen (CRA), and then intranasal boosting was performed on days 14, 18, 22, and 26. On days 26, 29, 32, and 34, the indicated humanized 5H10 antibody or PBS control was administered intraperitoneally at 20 mg / kg, or an irrelevant antibody as a control was administered. On days 30 and 34, CRA was also administered intratracheally. Samples were collected on day 35. A schematic diagram of the study design is shown in Figure 23.

[0098] In animals treated with antibody 5H10 VH1 / VK3, airway resistance was significantly less compared to the PBS control and other humanized variant antibodies (Figure 24A). Furthermore, in animals treated with VH1 / VK3 or VH2 / VK3, a significant decrease in lung IL-13 mRNA was observed compared to the chronic asthma control (i.e., animals administered CRA without any antibody treatment (PBS control)) (Figure 24B). Furthermore, in animals treated with the VH1 / VK3 antibody, the expression of matrix genes was significantly reduced (collagen 1 mRNA shown in Figure 24C, collagen 3 mRNA shown in Figure 24D). Also, the expression of SCF248 mRNA was significantly reduced in animals treated with VH1 / VK3 (Figure 24E). Figures 25A, 25B, and 25C show that administration of VH1 / VK3 reduced the mRNA levels of mucin protein Gob5 and cytokines IL-13 and IL-5 at concentrations of 1 mg / kg and 5 mg / kg. Therefore, this data indicates that the VH1 / VK3 antibody blocks the expression of cytokines and matrix genes in vivo in the chronic asthma model.

[0099] Example 12. Phase 1a Clinical Trial to Evaluate the Safety, Pharmacokinetics, and Pharmacodynamics of Anti-SCF248 Antibody In the Phase 1a trial, 110 healthy volunteers were enrolled. The primary objectives were to obtain a safety assessment for the humanized anti-SCF248 antibody 5H10, as well as accurate pharmacokinetic and pharmacodynamic data.

[0100] Administer humanized 5H10 or placebo in single or multiple escalating doses either intravenously or subcutaneously. Group 6 - 8 subjects together. The starting dose is based on toxicity tests conducted in accordance with GLP (Good Laboratory Practice). Obtain baseline pharmacokinetics for all development and product life. To evaluate pharmacodynamics, in addition to serum inflammatory markers such as SCF165, evaluate the number of circulating c-kit+ cells including mast cell progenitor cells and type 2 natural lymphocyte (ILC2) cells.

[0101] Example 13. Clinical trial to evaluate the safety and efficacy of anti-SCF248 antibody in patients In a clinical trial, enroll patients suffering from inflammatory disorders (e.g., atopic dermatitis, chronic urticaria, pulmonary fibrosis, and / or others). One objective of this trial is to establish the dose-response relationship between the humanized 5H10 antibody and pharmacodynamic markers (e.g., the number of circulating c-kit+ cells (e.g., mast cell progenitor cells and type 2 natural lymphocyte (ILC2) cells)) in affected patients. Also measure inflammatory biomarkers (e.g., ADAM8, CCL17, EPX, RNASE3, CCL2, CCL5, tryptase, histamine, and SCF165).

[0102] Administer the 5H10 antibody to each subject group in single-dose escalations. The starting dose is based on the pharmacodynamic biomarkers of the Phase 1a trial. Patients are treated for 1 month, 2 months, 3 months, or more months. The results of this trial will show that the humanized 5H10 antibody is effective in stabilizing and / or treating and / or preventing the progression of inflammatory disorders and fibrotic diseases.

[0103] All publications, patents, and patent applications cited in this specification are hereby specifically incorporated by reference in their entirety. Although the described invention has been explained with reference to specific embodiments thereof, those skilled in the art should understand that various changes may be made and equivalents may be substituted without departing from the spirit and scope of the invention. In addition, many modifications may be made to a particular situation, material, composition of matter, process, process step(s) to adapt them to the objectives, spirit, and scope of the described invention. All such changes are intended to be within the scope of the claims appended hereto. The present invention provides, for example, the following items. (Item 1) An antibody or a fragment thereof that specifically binds to stem cell factor (SCF), wherein the antibody comprises heavy chain CDR1, CDR2, and CDR3 each containing SEQ ID NO: 1, 2, and 3, respectively, the antibody or the fragment thereof. (Item 2) The antibody or the fragment thereof according to Item 1, wherein the antibody further comprises light chain CDR1, CDR2, and CDR3 each containing SEQ ID NO: 4, 5, and 6, respectively. (Item 3) The antibody or the fragment thereof according to Item 1 or Item 2, wherein the antibody comprises a heavy chain variable region having at least 80% identity to a sequence selected from SEQ ID NO: 7, 8, 9, 10, and 11. (Item 4) The antibody or the fragment thereof according to Item 3, wherein the antibody comprises a heavy chain variable region having at least 90% identity to a sequence selected from SEQ ID NO: 7, 8, 9, 10, and 11. (Item 5) The antibody or the fragment thereof according to Item 1 or Item 2, wherein the antibody comprises a light chain variable region having at least 80% identity to a sequence selected from SEQ ID NO: 13, 14, 15, and 16. (Item 6) The antibody or the fragment thereof according to Item 5, wherein the antibody comprises a light chain variable region having at least 90% identity to a sequence selected from SEQ ID NO: 13, 14, 15, and 16. (Item 7) The antibody or the fragment thereof according to Item 1, wherein the antibody or the fragment thereof comprises a heavy chain variable region amino acid sequence selected from SEQ ID NO: 7, 8, 9, 10, and 11 and a light chain variable region amino acid sequence selected from SEQ ID NO: 13, 14, 15, and 16. (Item 8) The antibody or the fragment thereof according to Item 1, wherein the antibody or the fragment thereof comprises the heavy chain variable region amino acid sequence of SEQ ID NO: 7 and the light chain variable region amino acid sequence of SEQ ID NO: 16. (Item 9) The antibody or fragment thereof according to item 1, wherein the antibody or fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 8 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 16. (Item 10) The antibody or fragment thereof according to item 1, wherein the antibody or fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 9 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 16. (Item 11) The antibody or fragment thereof according to item 1, wherein the antibody or fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 10 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 16. (Item 12) The antibody or fragment thereof according to item 1, wherein the antibody or fragment thereof comprises the heavy chain variable region amino acid sequence set forth in SEQ ID NO: 11 and the light chain variable region amino acid sequence set forth in SEQ ID NO: 16. (Item 13) The antibody or fragment thereof according to item 1 or 2, wherein the antibody is humanized. (Item 14) The antibody or fragment thereof according to any one of items 1 to 13, wherein the antibody is a monoclonal antibody. (Item 15) The antibody according to item 14, wherein the antibody comprises a human IgG4 domain. (Item 16) The antibody according to item 15, wherein the IgG4 domain comprises an S241P mutation at amino acid residue 241 and an L248E mutation at amino acid residue 248, and the numbering of the residues is according to the Kabat numbering system. (Item 17) The antibody according to any one of items 14 to 16, wherein the antibody comprises the heavy chain set forth in SEQ ID NO: 42 and the light chain set forth in SEQ ID NO: 49. (Item 18) The antibody according to any one of items 14 to 16, wherein the antibody comprises the heavy chain set forth in SEQ ID NO: 43 and the light chain set forth in SEQ ID NO: 49. (Item 19) The antibody or fragment thereof according to any one of items 1 to 18, wherein the antibody or fragment thereof blocks the interaction between SCF and c-Kit. (Item 20) The antibody or fragment thereof according to any one of items 1 to 18, wherein the antibody or fragment thereof causes internalization of SCF. (Item 21) The antibody or fragment thereof according to any one of items 1 to 20, wherein the antibody specifically binds to SCF248. (Item 22) The antibody or fragment thereof according to any one of items 1 to 21, wherein the antibody does not bind to SCF220. (Item 23) The fragment is selected from Fab, F(ab’ )2 , Fab’, scFv, and single domain antibody (sdAb), the antibody or fragment thereof according to any one of items 1 to 14 or 17 to 22. (Item 24) An isolated nucleic acid molecule encoding the antibody or fragment thereof according to any one of items 1 to 23. (Item 25) An expression vector comprising a nucleic acid segment encoding the antibody or fragment thereof according to any one of items 1 to 23. (Item 26) A recombinant host cell comprising the expression vector according to item 25. (Item 27) A method for producing an antibody that specifically binds to stem cell factor isoform 248 (SCF248), the method comprising immunizing a host animal with a peptide comprising SEQ ID NO: 30 or a fragment thereof, and obtaining an antibody from the immunized host animal. (Item 28) The method according to item 27, wherein the fragment comprises at least 5 amino acids. (Item 29) The method according to item 27, wherein the fragment comprises at least 10 amino acids. (Item 30) The method according to item 27, wherein the peptide consists of SEQ ID NO: 30. (Item 31) The method according to item 27, wherein the peptide comprises at least 5 amino acids, and the N-terminal amino acid of the peptide is alanine at position 1 of SEQ ID NO: 30. (Item 32) The method according to item 31, wherein the peptide comprises at least 10 amino acids. (Item 33) The method according to any one of items 27 to 32, wherein the antibody from the immunized host animal is obtained from immune cells isolated from the host animal. (Item 34) The method according to item 33, further comprising generating a hybridoma using the immune cells. (Item 35) An antibody prepared by the method according to any one of items 27 to 34, which specifically binds to SCF248. (Item 36) A method for inhibiting chronic inflammation in a subject in need thereof, the method comprising administering to the subject the antibody or fragment thereof according to any one of items 1 to 23. (Item 37) A method for inhibiting fibrosis in a subject in need thereof, comprising administering to the subject an antibody or a fragment thereof according to any one of items 1 to 23, said method. (Item 38) A method for treating a chronic inflammatory disease or a fibrotic disease in a subject in need thereof, comprising administering to the subject an antibody according to any one of items 1 to 23, said method. (Item 39) The method according to item 38, wherein the chronic inflammatory disease or the fibrotic disease is selected from the group consisting of urticaria, atopic dermatitis, non-alcoholic steatohepatitis (NASH), primary sclerosing cholangitis, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, asthma, bleomycin lung, scleroderma, cirrhosis, endomyocardial fibrosis, fibromyalgia, and eosinophilic esophagitis. (Item 40) The method according to item 39, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis or scleroderma-related pulmonary fibrosis. (Item 41) The method according to item 37, wherein the method further comprises administering an additional therapeutic agent to the subject. (Item 42) An antibody or a fragment thereof that specifically binds to SCF248, binds to an epitope comprising at least 8 consecutive amino acids of SEQ ID NO: 33, and inhibits the interaction between SCF248 and c-Kit, said antibody or fragment thereof. (Item 43) The antibody or a fragment thereof according to item 42, wherein the epitope comprises SEQ ID NO: 33. (Item 44) The antibody or a fragment thereof according to item 42, wherein the epitope consists of SEQ ID NO: 33. (Item 45) The antibody or a fragment thereof according to item 42, wherein the antibody or a fragment thereof blocks the binding of SCF248 to c-Kit on immune cells. (Item 46) A method for inhibiting the activation of immune cells, comprising contacting the immune cells with an antibody or a fragment thereof that specifically binds to SCF248, wherein the antibody or a fragment thereof binds to an epitope comprising at least 8 consecutive amino acids of SEQ ID NO: 33, and inhibits the interaction between SCF248 and c-Kit, said method. (Item 47) The method according to item 46, wherein the method is performed in vitro, ex vivo, or in vivo. (Item 48) A method for inhibiting inflammation in a subject in need thereof, comprising administering to the subject an antibody or a fragment thereof that specifically binds to SCF248, wherein the antibody or the fragment thereof binds to an epitope comprising at least 8 consecutive amino acids of SEQ ID NO: 33, and the antibody inhibits the interaction between SCF248 and c-Kit, said method. (Item 49) A method for inhibiting fibrosis in a subject in need thereof, comprising administering to the subject an antibody or a fragment thereof that specifically binds to SCF248, wherein the antibody or the fragment thereof binds to an epitope comprising at least 8 consecutive amino acids of SEQ ID NO: 33, and the antibody inhibits the interaction between SCF248 and c-Kit, said method. (Item 50) A method for treating an inflammatory disease or disorder in a subject in need thereof, comprising administering to the subject an antibody or a fragment thereof that specifically binds to SCF248, wherein the antibody or the fragment thereof binds to an epitope comprising at least 8 consecutive amino acids of SEQ ID NO: 33, and the antibody inhibits the interaction between SCF248 and c-Kit, said method. (Item 51) The method according to item 50, wherein the inflammatory disease or the inflammatory disorder is a chronic inflammatory disease or disorder. (Item 52) A method for treating a fibrotic disease in a subject in need thereof, comprising administering to the subject an antibody or a fragment thereof that specifically binds to SCF248, wherein the antibody or the fragment thereof binds to an epitope comprising at least 8 consecutive amino acids of SEQ ID NO: 33, and the antibody inhibits the interaction between SCF248 and c-Kit, said method. (Item 53) The method according to any one of items 50 to 52, wherein the inflammatory disease or the inflammatory disorder or the fibrotic disease is selected from the group consisting of urticaria, atopic dermatitis, non-alcoholic steatohepatitis (NASH), primary sclerosing cholangitis, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, asthma, bleomycin lung, scleroderma, liver cirrhosis, endomyocardial fibrosis, fibromyalgia, and eosinophilic esophagitis. (Item 54) The method according to item 53, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis or scleroderma-related pulmonary fibrosis. (Item 55) An antibody or fragment thereof that specifically binds to SCF248 for use as a medicament, which binds to an epitope comprising at least 8 consecutive amino acids of SEQ ID NO: 33, and wherein the antibody inhibits the interaction between SCF248 and c-Kit, said antibody or fragment thereof. (Item 56) An antibody or fragment thereof that specifically binds to SCF248 for use in a method of treatment, which binds to an epitope comprising at least 8 consecutive amino acids of SEQ ID NO: 33, wherein the antibody inhibits the interaction between SCF248 and c-Kit, and wherein the method comprises inhibiting activation of immune cells of a subject, inhibiting inflammation of a subject, inhibiting fibrosis of a subject, treating an inflammatory disease or disorder, treating a chronic inflammatory disease or disorder, and / or treating a fibrotic disease, said method. (Item 57) An antibody or fragment thereof according to any one of items 1 to 21, 34, or 42 to 45, or a pharmaceutical composition according to item 22, for use as a medicament. (Item 58) An antibody or fragment thereof according to any one of items 1 to 21, 34, or 42 to 45, or a pharmaceutical composition according to item 22, for use in a method of inhibiting chronic inflammation of a subject, inhibiting fibrosis of a subject, treating a chronic inflammatory disease of a subject, and / or treating a fibrotic disease of a subject. (Item 59) The antibody or fragment thereof for use according to item 56 or item 58, wherein the chronic inflammatory disease or the fibrotic disease is selected from the group consisting of urticaria, atopic dermatitis, non-alcoholic steatohepatitis (NASH), primary sclerosing cholangitis, pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS), cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, asthma, bleomycin lung, scleroderma, cirrhosis, endomyocardial fibrosis, fibromyalgia, and eosinophilic esophagitis. (Item 60) The antibody or fragment thereof for use according to item 59, wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis or scleroderma-related pulmonary fibrosis. (Item 61) The antibody or fragment thereof for use according to any one of items 55 to 60, wherein the use further comprises administering an additional therapeutic agent to the subject.

Claims

1. An antibody that specifically binds to stem cell factor 248 (SCF248), wherein the antibody comprises a heavy chain CDR1, CDR2, and CDR3 each containing SEQ ID NO: 1, 2, and 3, and a light chain CDR1, CDR2, and CDR3 each containing SEQ ID NO: 4, 5, and 6, the antibody is humanized, and the antibody (a) has a heavy chain variable region having the amino acid sequence of SEQ ID NO: 8, and (b) has a light chain variable region having the amino acid sequence of SEQ ID NO: 16 The antibody as described above.

2. The antibody according to claim 1, wherein the antibody is a monoclonal antibody.

3. The antibody according to claim 2, wherein the antibody contains a human IgG4 domain.

4. The antibody according to claim 3, wherein the IgG4 domain contains an S241P mutation at amino acid residue 241 and an L248E mutation at amino acid residue 248, and the numbering of the residues is according to the Kabat numbering system.

5. The antibody according to claim 2, wherein the antibody comprises a heavy chain as described in SEQ ID NO: 42 and a light chain as described in SEQ ID NO:

49.

6. (a) The antibody blocks the interaction between SCF and c-Kit, (b) the antibody causes internalization of SCF, and / or (c) the antibody does not bind to SCF220, The antibody according to claim 1.

7. The antibody according to claim 1, wherein the antibody binds to an epitope containing at least 8 consecutive amino acids of SEQ ID NO: 33, and the antibody inhibits the interaction between SCF248 and c-Kit.

8. The antibody according to claim 7, wherein the epitope consists of SEQ ID NO:

33.

9. The antibody according to claim 3, wherein the human IgG4 domain contains the amino acid sequence of SEQ ID NO:

40.

10. The antibody according to claim 1, comprising a light chain human Ig kappa constant region.

11. The antibody according to claim 10, wherein the light chain human Ig kappa constant region contains the amino acid sequence of SEQ ID NO:

41.

12. The antibody according to claim 1, comprising a light chain human Ig lambda constant region.

13. An anti-SCF248 antibody that specifically binds to stem cell factor 248 (SCF248), (i) comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 42, and (ii) a light chain containing the amino acid sequence of SEQ ID NO: 49 The antibody as described above.

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