Method for detecting phosphorylation of DDR1

Abstract

The present disclosure provides methods of detecting discoid domain receptor tyrosine kinase 1 (DDR1) phosphorylation to determine the effectiveness, or possibly the effectiveness, of DDR1 antagonistic therapy. The present disclosure provides antibodies that specifically bind to DDR1 for use in the treatment of DDR1-related disorders. Also provided herein are compositions comprising these antibodies for detecting phosphorylated DDR1, nucleic acids encoding these antibodies, expression vectors, and host cells for making these antibodies.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application Serial No. 63 / 366,567, filed on June 17, 2022, the entire contents of which are incorporated herein by reference.

[0003] 2. Reference to sequence listing

[0004] This application contains a sequence listing, which is submitted electronically in ST.26 format and is incorporated herein by reference in its entirety (the ST.26 copy was created on June 16, 2023, is named "201000_seqlist.xml", and is 151,659 bytes in size).

[0005] 3. Field

[0006] The present disclosure relates to methods of detecting discoidin domain receptor tyrosine kinase 1 (DDR1) phosphorylation to determine the effectiveness or potential effectiveness of DDR1 inhibitors, such as anti-DDR1 antibodies, or methods of screening for such DDR1 inhibitors.

[0007] 4. Background

[0008] Receptor tyrosine kinases (RTKs) play a key role in the communication between cells and their microenvironment. These molecules are involved in the regulation of cell growth, differentiation and metabolism. The DDR1 protein, encoded by the DDR1 gene, is an RTK that is widely expressed in normal and transformed epithelial cells and activated by various types of collagen. The DDR1 protein belongs to a subfamily of tyrosine kinase receptors and has a homology region to the Dictyostelium discoideum protein discoidin I in its extracellular domain. Its autophosphorylation is achieved by all collagens (types I to VI) tested so far. In situ studies and Northern blot analysis have shown that the expression of the protein encoded by DDR1 is restricted to epithelial cells, particularly in the kidney, lung, gastrointestinal tract and brain. In addition, the DDR1 protein is significantly overexpressed in several human tumors from the breast, ovary, esophagus and pediatric brain. In addition to being expressed in cancer, DDR1 protein is also expressed in organs such as the kidney, lung, gastrointestinal tract, skin, and brain, and has been associated with fibrosis in the skin, lung, and liver.

[0009] Therefore, methods of determining the effectiveness or potential effectiveness of DDR1 inhibitors in treating or preventing DDR1-related disorders are greatly needed. 5. Summary of the invention

[0010] The present disclosure demonstrates that anti-DDR1 antibodies can inhibit DDR1 phosphorylation. DDR1-related disorders are associated with collagen-mediated DDR1 phosphorylation and subsequent increase in downstream signaling. Therefore, methods for detecting DDR1 phosphorylation can be used to determine the effectiveness or potential effectiveness of anti-DDR1 antibodies in treating DDR1-related disorders.

[0011] If the anti-DDR1 antibody is able to inhibit DDR1 phosphorylation and / or the ability of DDR1 to interact with collagen, the anti-DDR1 antibody may be considered effective for treating DDR1-related disorders. In addition, if the anti-DDR1 antibody is able to inhibit DDR1 phosphorylation and / or the ability of DDR1 to interact with collagen, the anti-DDR1 antibody may be effective for treating DDR1-related disorders. In one aspect, the present disclosure provides a method for monitoring the effectiveness of an anti-discoid domain receptor tyrosine kinase 1 (DDR1) antibody or an antigen-binding fragment thereof in a subject in need thereof, the method comprising: a) administering an effective amount of the anti-DDR1 antibody to the subject; and b) detecting the level of DDR1 phosphorylation in a sample from the subject, wherein a reduction in DDR1 phosphorylation in the sample from the subject compared to a positive reference sample indicates that the administration of the anti-DDR1 antibody is effective.

[0012] In some embodiments, the subject has cancer. In some embodiments, the cancer is selected from: pancreatic cancer; lung cancer, including small cell lung cancer and non-small cell lung cancer; colon cancer and colorectal cancer; head and neck cancer; stomach (gastric) cancer; ovarian cancer; breast cancer; kidney cancer; liver cancer; prostate cancer; cervical cancer; brain cancer; skin cancer, including melanoma; sarcoma; bile duct cancer; and bone cancer. In some embodiments, the subject has a fibrotic disorder. In some embodiments, the fibrotic disorder is selected from: hypertrophic scarring of the skin, scleroderma, lung scarring, idiopathic pulmonary fibrosis, cirrhotic liver fibrosis, renal fibrosis, and interstitial lung disease.

[0013] In one aspect, the present disclosure provides a method for treating a DDR1-related disorder in a subject in need thereof, the method comprising: a) administering to the subject an effective amount of an anti-DDR1 antibody or an antigen-binding fragment thereof; and b) detecting the level of DDR1 phosphorylation in a sample from the subject, wherein a decrease in DDR1 phosphorylation in the sample from the subject compared to a positive reference sample indicates that the treatment is effective.

[0014] In one aspect, the present disclosure provides a method for screening a subject suffering from a DDR1-related disorder that may be effectively treated with an anti-DDR1 antibody, the method comprising detecting the level of DDR1 phosphorylation in a sample from the subject, wherein if DDR1 phosphorylation is higher in the sample from the subject compared to a negative reference sample, then the DDR1-related disorder may be effectively treated with the anti-DDR1 antibody.

[0015] In one aspect, the present disclosure provides a method for treating a DDR1-related disorder in a subject in need thereof, the method comprising: a) detecting the level of DDR1 phosphorylation in a sample from the subject; and b) if DDR1 phosphorylation is higher in the sample from the subject compared to a negative reference sample, administering to the subject an effective amount of an anti-DDR1 antibody or an antigen-binding fragment thereof.

[0016] In some embodiments, the DDR1-related disorder is cancer. In some embodiments, the cancer is selected from: pancreatic cancer; lung cancer, including small cell lung cancer and non-small cell lung cancer; colon cancer and colorectal cancer; head and neck cancer; stomach (gastric) cancer; ovarian cancer; breast cancer; kidney cancer; liver cancer; prostate cancer; cervical cancer; brain cancer; skin cancer, including melanoma; sarcoma; bile duct cancer; and bone cancer.

[0017] In some embodiments, the DDR1-associated disorder is a fibrotic disorder. In some embodiments, the fibrotic disorder is selected from the group consisting of hypertrophic scarring of the skin, scleroderma, lung scarring, idiopathic pulmonary fibrosis, cirrhotic liver fibrosis, renal fibrosis, and interstitial lung disease.

[0018] In some embodiments, the sample from the subject described herein comprises tumor tissue. In some embodiments, the sample from the subject described herein comprises one or more selected from blood cells, skin tissue, lung tissue, kidney tissue, and liver tissue. In some embodiments, the sample from the subject described herein comprises a skin puncture biopsy sample.

[0019] In one aspect, the present disclosure provides a method for screening an anti-DDR1 antibody or an antigen-binding fragment thereof that is effective in treating a DDR1-related disorder, the method comprising: a) administering an effective amount of an anti-DDR1 antibody or an antigen-binding fragment thereof to a cell; and b) detecting the level of DDR1 phosphorylation in the cell, wherein a decrease in DDR1 phosphorylation in the cell compared to a positive reference cell indicates that the anti-DDR1 antibody or the antigen-binding fragment thereof is effective in treating the DDR1-related disorder.

[0020] In one aspect, the present disclosure provides a method for screening an anti-DDR1 antibody or an antigen-binding fragment thereof that is effective in reducing the interaction between collagen and cells, the method comprising: a) administering an effective amount of an anti-DDR antibody or an antigen-binding fragment thereof to the cells; and b) detecting the level of DDR1 phosphorylation in the cells, wherein a reduction in DDR1 phosphorylation in the cells compared to positive reference cells indicates that the anti-DDR1 antibody or the antigen-binding fragment thereof is effective in reducing the interaction between collagen and the cells.

[0021] In some embodiments, the cells described herein are cancer cells. In some embodiments, the cancer cells are derived from a cancer selected from the group consisting of pancreatic cancer, lung cancer, including small cell lung cancer and non-small cell lung cancer, colon cancer and colorectal cancer, head and neck cancer, stomach (gastric) cancer, ovarian cancer, breast cancer, kidney cancer, liver cancer, prostate cancer, cervical cancer, brain cancer, skin cancer, including melanoma, sarcoma, bile duct cancer, and bone cancer.

[0022] In some embodiments, the cells described herein are one or more selected from skin cells, lung cells, kidney cells, and liver cells.

[0023] In some embodiments, the anti-DDR1 antibodies or antigen-binding fragments thereof described herein comprise a heavy chain variable domain (VH) and a light chain variable region (VL), wherein the VH comprises the CDRH1, CDRH2, and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 4 or 13, and the VL comprises the CDRL1, CDRL2, and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 3, 11, or 12.

[0024] In some embodiments, a) the CDRL1 comprises the amino acid sequence of SEQ ID NO:5; b) the CDRL2 comprises the amino acid sequence QAS; c) the CDRL3 comprises the amino acid sequence of SEQ ID NO:7; d) the CDRH1 comprises the amino acid sequence of SEQ ID NO:8; e) the CDRH2 comprises the amino acid sequence of SEQ ID NO:9; and f) the CDRH3 comprises the amino acid sequence of SEQ ID NO:10.

[0025] In some embodiments, a) the CDRL1 comprises the amino acid sequence of SEQ ID NO: 17; b) the CDRL2 comprises the amino acid sequence GVF; c) the CDRL3 comprises the amino acid sequence of SEQ ID NO: 19; d) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 20; e) the CDRH2 comprises the amino acid sequence of SEQ ID NO: 21; and f) the CDRH3 comprises the amino acid sequence of SEQ ID NO: 22.

[0026] In some embodiments, the anti-DDR1 antibody comprises a) a VL domain comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 3, 11, and 12; and b) a VH domain comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from SEQ ID NOs: 4 and 13.

[0027] In some embodiments, the anti-DDR1 antibody comprises a) a VL domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11, and 12; and b) a VH domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 13.

[0028] In some embodiments, the anti-DDR1 antibody comprises a VL domain and a VH domain selected from the group consisting of: a) SEQ ID NOs: 3 and 4, respectively; b) SEQ ID NOs: 11 and 13, respectively; and c) SEQ ID NOs: 12 and 13, respectively.

[0029] In some embodiments, the anti-DDR1 antibody comprises a VL domain and a VH domain, and the VL domain and the VH domain comprise the amino acid sequences of SEQ ID NOs: 3 and 4, respectively. In some embodiments, the anti-DDR1 antibody comprises a VL domain and a VH domain, and the VL domain and the VH domain comprise the amino acid sequences of SEQ ID NOs: 11 and 13, respectively. In some embodiments, the anti-DDR1 antibody comprises a VL domain and a VH domain, and the VL domain and the VH domain comprise the amino acid sequences of SEQ ID NOs: 12 and 13, respectively.

[0030] In some embodiments, detecting the phosphorylation level of DDR1 comprises detecting the phosphorylation level of a sheared form of DDR1. In some embodiments, the sheared form of DDR1 has a molecular weight of about 65 kDa.

[0031] 6. Brief description of the accompanying drawings

[0032] Figures 1A to 1DThis indicates that 9H1-WT monoclonal antibody mediated inhibition of collagen I-induced discoidin domain receptor tyrosine kinase 1 (DDR1) phosphorylation. Figure 1A Depicted are phosphorylated DDR1 normalized to vinculin levels in cancer cells pretreated with increasing concentrations of 9H1-WT (or IgG1-WT as a negative control) and stimulated with human collagen I. Figure 1B Depicted are phosphorylated DDR1 normalized to vinculin levels in cancer cells pretreated with increasing concentrations of 9H1-WT (or IgG1-WT as a negative control) and stimulated with rat collagen I. Figure 1C to Figure 1D Depicted with Figure 1A to Figure 1B Representative immunoblots corresponding to the quantitative results described in .

[0033] FIG. 2A to FIG. 2J This indicates that 9H1-WT and Ab#33 monoclonal antibodies have no effect on cancer cell proliferation or cell death. Figure 2A Representative fluorescent images of T47D cells treated with 9H1-WT or control IgG1-WT are depicted. Figure 2B Representative fluorescent images of T47D cells treated with Ab #33 or control inert IgG1 are depicted. Figure 2C to Figure 2F is the nuclear count as a measure of cell proliferation in response to 9H1-WT, IgG1-WT as a negative control, and paclitaxel as a positive control ( Figure 2C ) and total cell area ( Figure 2D ), and Annexin V-positive cells as a measure of cell death ( Figure 2E ) and Cytox Green positive cells ( Figure 2F ) time course diagram. Figure 2G to Figure 2J is the nuclear count as a measure of cell proliferation in response to Ab#33, inert IgG1 as a negative control, and paclitaxel as a positive control ( Figure 2G ) and total cell area ( Figure 2H ), and Annexin V-positive cells as a measure of cell death ( Fig.2I ) and Cytox Green positive cells ( Figure 2J ) time course diagram.

[0034] Figure 3A and Figure 3B These results suggest that collagen I and V effectively induce DDR1 phosphorylation. Figure 3A Depicted are representative immunoblots (left) and quantification (right) of DDR1 phosphorylation (pDDR1) in response to stimulation with 25 μg / ml of different types of rat and human collagen. Figure 3B Responses to 50 μg / ml Figure 3ARepresentative immunoblots (left) and quantification (right) of pDDR1 stimulated with rat and human collagens of the types used.

[0035] FIG. 4A to FIG. 4E This indicates that anti-DDR1 mAbs reach levels sufficient to bind DDR1 and inhibit collagen I-induced phosphorylation in vivo. Figure 4A A schematic diagram depicting the study protocol is shown. FIG. 4B to FIG. 4D Depicted is the administration of humanized mAb #9H1 ( Figure 4B ), humanized mAb #9H1 with wt IgG1Fc ( Figure 4C ) and chimeric rabbit / human mAb #33 with an inert IgG1 Fc ( Figure 4D ) in vivo antibody concentration over the course of 168 hours. Figure 4E Depicted FIG. 4B to FIG. 4D The average antibody concentration over the course of 168 hours for each antibody described in .

[0036] Figure 5A and Figure 5B Indicates the inhibition of DDR1 phosphorylation induced by 9H1-WT mAb on collagen I and V. Figure 5A Depicted are pDDR1 normalized to vinculin in T47D cells stimulated with 50 μg / ml human collagen I or collagen V following pretreatment with 9H1-WT, negative control IgG1, or positive control 2.45-IN. Figure 5B Depicted in Figure 5A Total DDR1 normalized to vinculin in pretreated and stimulated cells.

[0037] FIG. 6A to FIG. 6D The calculated IC50 indicated that 9H1-WT (PRTH-101) inhibited DDR1 phosphorylation. FIG. 6A to FIG. 6C Inhibition curves from three independent experiments and their calculated IC50 values ​​+ / - standard error of the mean are depicted. Fig.6D Combined curves of all three experiments were plotted and IC50 values ​​+ / - standard error of the mean were calculated.

[0038] 7A to 7C This indicates that 9H1-WT inhibits the adhesion of DRR1-expressing cells to human collagen I. Fig. 7A Depicted are representative fluorescent images of WT or DDR1-overexpressing HEK293 cells treated with increasing concentrations of 9H1-WT (PRTH-101) or control IgG1-WT and incubated on collagen I-coated adhesion plates. FIG. 7B to FIG. 7C Depicted are two independent experiments Fig. 7A The number of adherent cells for the experimental groups described.

[0039] Figure 8Calculated IC50s for inhibition of DDR1 expressing cell adhesion by 9H1-WT (PRTH-101) are shown. The inhibition curves, which include pooled values ​​from four independent experiments, depict the number of adherent cells at increasing concentrations of 9H1-WT (PRTH-101).

[0040] Fig.9A and Fig. 9B These results suggest that 9H1-WT inhibits collagen II- and collagen III-mediated DDR1 phosphorylation in T47D cells. Fig.9A Depicted are phosphorylated DDR1 normalized to vinculin expression in T47D cells pretreated with increasing concentrations of 9H1-WT (PRTH-101) or control IgG1-WT and stimulated with 50 μg / ml human collagen II or collagen III. Fig. 9B Depicted with Fig.9A Depicted is an experiment identical to the one described above but further including a 50 μg / ml human collagen I stimulation group for comparison.

[0041] Fig. 10A and Fig. 10B Demonstrating rabbit mAb#33 and chimeric mAb#33-mediated inhibition of collagen I-induced DDR1 phosphorylation. Fig. 10A Depicted are phosphorylated DDR1 normalized to vinculin expression in T47D cells pretreated with increasing concentrations of rabbit mAb #33, control IgG57, 9H1-WT (PRTH-101), or control IgG1-WT and stimulated with 50 μg / ml human collagen I. Fig. 10B Depicted are phosphorylated DDR1 normalized to vinculin expression in T47D cells pretreated with increasing concentrations of chimeric mAb #33 or IgG inert Fc and in the presence or absence of 50 μg / ml human collagen I stimulation.

[0042] Fig.11A and Fig. 11B showed that both full-length and cleaved forms of DDR1 could be detected by Western blotting in skin samples from healthy human donors, but only the cleaved form was phosphorylated. Fig.11A Depicted is a Western blot of skin sample lysates prepared by different methods from 2 healthy human subjects using pDDR1-specific antibodies. Collagen-stimulated and unstimulated T47D breast cancer cells are also included. Full-length pDDR1 (about 125 kDa) is indicated by a black arrow. Sheared pDDR1 (about 60 kDa) is indicated by a white arrow. Fig. 11BDepicted is a Western blot of skin sample lysates using pDDR1 specific and universal DDR1 antibodies. The antibodies used are shown along the bottom. A representative universal DDR1 antibody lane is depicted by a black arrow. A representative pDDR1 specific lane is depicted by a white arrow.

[0043] 7. Detailed description

[0044] The present disclosure provides methods for detecting DDR1 phosphorylation for monitoring the effectiveness or potential effectiveness of DDR1 inhibitors (eg, anti-DDR1 antibodies) in inhibiting DDR1-mediated collagen interactions and / or treating DDR1-related disorders.

[0045] 7.1 Definitions

[0046] As used herein, the term "DDR1" refers to the discoidin domain receptor tyrosine kinase 1 encoded by the DDR1 gene. Unless otherwise indicated, the term "DDR1" refers to the wild-type DDR1 gene (e.g., GenBank TM Accession number NM_013993.3) encoded DDR1 protein. An exemplary DNA sequence and amino acid sequence of human DDR1 are provided in Table 1 below. As used herein, "DDR1 phosphorylation" refers to the attachment of a phosphate group to any residue of DDR1. For example, phosphorylation can occur on serine, threonine, or tyrosine residues of the DDR1 protein, and can occur by intermolecular interactions (e.g., by a separate kinase) or intramolecular interactions (e.g., autophosphorylation). Relative to the amino acid sequence of SEQ ID NO:2 shown in Table 1 below, exemplary DDR1 phosphorylation sites include, but are not limited to, Y484, Y513, Y520, S631, Y740, Y792, Y796, and Y797.

[0047] Table 1. Exemplary human DDR1 DNA and amino acid sequences.

[0048]

[0049]

[0050] As used herein, the term "antibody" includes full-length antibodies, antigen-binding fragments of full-length antibodies, and molecules comprising antibody CDRs, VH regions, and / or VL regions. Examples of antibodies include, but are not limited to, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain molecules and two light chain molecules, antibody light chain monomers, antibody heavy chain monomers, antibody light chain dimers, antibody heavy chain dimers, antibody light chain-antibody heavy chain pairs, intrabodies, heteroconjugate antibodies, antibody-drug conjugates, single domain antibodies, monovalent antibodies, single-chain antibodies or single-chain Fv (scFv), camelized antibodies, affibodies, Fab fragments, F(ab')2 fragments, disulfide-linked Fv (sdFv), anti-idiotypic (anti-Id) antibodies (including, for example, anti-anti-Id antibodies), and antigen-binding fragments of any of the above. In certain embodiments, antibodies described herein refer to polyclonal antibody colonies.Antibodies can be any type (e.g., IgG, IgE, IgM, IgD, IgA, or IgY) of immunoglobulin molecules, any category (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2), or any subclass (e.g., IgG2a or IgG2b).In certain embodiments, antibodies described herein are IgG antibodies or their categories (e.g., human IgG1 or IgG4) or subclasses.In embodiments, the antibody is a humanized monoclonal antibody.In embodiments, the antibody is a human monoclonal antibody.

[0051] As used herein, the term "CDR" or "complementarity determining region" refers to the non-contiguous antigen binding sites present within the variable regions of the heavy and light chain polypeptides. These specific regions have been described, for example, in Kaba et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), Chothia et al., J. Mol. Biol. 196: 901-917 (1987), and MacCallum et al., J. Mol. Biol. 262: 732-745 (1996), all of which are incorporated herein by reference in their entirety, wherein the definitions include overlapping or subsets of amino acid residues when compared to each other. In certain embodiments, the term "CDR" is a CDR as defined by MacCallum et al., J. Mol. Biol. 262: 732-745 (1996) and Martin A "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In certain embodiments, the term "CDR" is a CDR as defined by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest (1991). In certain embodiments, different conventions are used to define the heavy chain CDR and light chain CDR of an antibody. In certain embodiments, heavy chain CDRs and / or light chain CDRs are defined by structural analysis of the antibody and identifying residues in the variable region predicted to contact an epitope region of a target molecule (e.g., human and / or mouse DDR1). CDRH1, CDRH2, and CDRH3 represent heavy chain CDRs, and CDRL1, CDRL2, and CDRL3 represent light chain CDRs.

[0052] As used herein, the terms "variable region" and "variable domain" are used interchangeably and are common in the art. A variable region generally refers to a part of an antibody, generally a part of a light chain or a heavy chain, typically about 110 to 120 amino acids at the amino terminal, or 110 to 125 amino acids in a mature heavy chain, and about 90 to 115 amino acids in a mature light chain, which are very different in the sequence of the antibody and are used for the binding and specificity of a specific antibody to its specific antigen. The variability of the sequence is concentrated in those regions known as complementary determining regions (CDRs), while the more highly conserved regions in the variable region are known as framework regions (FRs). It is not desirable to be bound by any particular mechanism or theory, and it is believed that the CDRs of light and heavy chains are primarily responsible for the interaction and specificity of antibodies with antigens. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region includes rodents, such as murines or lagomorphs, such as rabbit CDRs and human framework regions (FRs). In an embodiment, the variable region is a primate (e.g., non-human primate) variable region. In an embodiment, the variable region includes rodent, eg, murine, or lagomorph, eg, rabbit CDRs and primate (eg, non-human primate) framework regions (FRs).

[0053] As used herein, the terms "VH" and "VL" refer to antibody heavy and light chain variable regions, respectively, as described in Kabat et al., (1991) Sequences of Proteins of Immunological Interest (NIH Publication No. 91-3242, Bethesda), which is incorporated herein by reference in its entirety.

[0054] As used herein, the term "constant region" is common in the art. The constant region is an antibody portion, e.g., the carboxyl terminal portion of a light chain and / or a heavy chain, which is not directly involved in binding of the antibody to an antigen, but which may exhibit various effector functions, such as interaction with an Fc receptor (e.g., an Fcγ receptor).

[0055] As used herein, the term "heavy chain" when referring to antibodies can refer to any of the different types based on the amino acid sequence of the constant region, such as alpha (α), delta (δ), epsilon (ε), gamma (γ), and mu (μ), which give rise to IgA, IgD, IgE, IgG, and IgM class antibodies, respectively, including subclasses of IgG, such as IgG1, IgG2, IgG3, and IgG4.

[0056] As used herein, the term "light chain" when referring to an antibody can refer to any of the different types based on the amino acid sequence of the constant region, such as kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In an embodiment, the light chain is a human light chain.

[0057] As used herein, the term "cancer" refers to any condition characterized by the uncontrolled division of abnormal cells in the body. For example, a mutation may occur in a cell that makes it unable to regulate cell division and leads to the formation of one or more tumors. Cancer can be benign, precancerous or malignant. Cancer occurs in various cells and tissues, including, but not limited to, the oral cavity (e.g., mouth, tongue, pharynx, etc.), digestive system (e.g., esophagus, stomach, small intestine, colon, rectum, liver, bile duct, gallbladder, pancreas, etc.), respiratory system (e.g., larynx, lungs, bronchi, etc.), bones, joints, skin (e.g., basal cell, squamous cell, meningioma, etc.), breast, reproductive system (e.g., uterus, ovaries, prostate, testicles, etc.), urinary system (e.g., bladder, kidneys, ureters, etc.), eyes, nervous system (e.g., brain, etc.), endocrine system (e.g., thyroid, etc.), soft tissue (e.g., muscle, fat, etc.), and hematopoietic system (e.g., lymphoma, myeloma, leukemia, acute lymphocytic leukemia, chronic lymphocytic leukemia, acute myeloid leukemia, chronic myeloid leukemia, etc.).

[0058] As used herein, "cell" refers to the basic structural and functional unit of an organism. A cell includes a membrane-bounded cytoplasm containing biomacromolecules (e.g., nucleic acids, carbohydrates, lipids, and proteins) and organelles required to sustain life. As used herein, "cancer cell" refers to an abnormal cell that undergoes uncontrolled cell division (e.g., a cell that has accumulated one or more harmful mutations). Healthy cells can be derived from healthy tissues, such as skin tissue, while cancer cells can be derived from pathological tissues, such as tumors.

[0059] As used herein, the term "interaction" refers to a non-covalent chemical bond formed between biomacromolecules for a functionally relevant duration. "Collagen interaction" as used herein with reference to DDR1 refers to a non-covalent chemical bond that occurs between the extracellular domain of the DDR1 protein and collagen, the strength and duration of which is sufficient to promote the autophosphorylation of the intracellular domain of DDR1.

[0060] As used interchangeably herein, the terms "DDR1-related disease," "DDR1-related disorder," or "DDR1-related condition" refer to any pathological condition associated with or directly caused by abnormal expression and / or function of DDR1. For example, a DDR1-related disorder may include cancer, in which overexpression of DDR1 suppresses anti-tumor immunity, thereby hindering the recognition and clearance of tumors. By further example, a DDR1-related disorder may include a fibrotic disorder, in which overexpression of DDR1 is associated with excessive accumulation of extracellular matrix components (e.g., collagen) and impaired tissue function.

[0061] The terms "effective", "effective amount", "therapeutically effective amount" and "pharmaceutically effective amount" are used interchangeably herein in reference to treatment, and refer to an amount of an agent sufficient to achieve a desired biological result. The result may be a reduction and / or alleviation of the severity, duration and / or frequency of one or more signs, symptoms, side effects and / or causes of the disease or disorder being treated.

[0062] As used herein, the terms "fibrosis" and "fibrotic disorder" refer to any disorder characterized by the replacement of normal parenchymal tissue by connective tissue. For example, damage or inflammation of tissue can lead to excessive accumulation of extracellular matrix components (e.g., collagen). When severe enough, this accumulation can interfere with the normal structure and / or function of the tissue.

[0063] As used herein, "reference sample" refers to one or more biological samples containing DDR1 or its derivatives, which can be compared with the subject sample. "Positive reference sample" used herein refers to a sample known to exist and / or to meet the conditions for the purpose of comparison. For example, a positive reference sample may include a sample derived from a cancer tissue known to overexpress phosphorylated DDR1. In contrast, "negative reference sample" used herein refers to a sample known to not exist and / or not meet the conditions for the purpose of comparison. For example, a negative reference sample may include a sample derived from a healthy tissue (e.g., skin tissue) known to express normal levels of phosphorylated DDR1. As used herein, "reference cell" refers to one or more cells containing phosphorylated DDR1, which can be compared with the subject cell. "Positive reference cell" used herein refers to a cell known to exist and / or to meet the conditions for the purpose of comparison. For example, a positive reference cell may include a cell derived from a cancer tissue known to overexpress phosphorylated DDR1. In contrast, "negative reference cell" used herein refers to a cell known to not exist and / or not meet the conditions for the purpose of comparison. For example, a negative reference cell may include a cell derived from a healthy tissue (e.g., skin tissue) known to express normal levels of phosphorylated DDR1.

[0064] As used herein, the terms "specific binding," "specific recognition," "immunospecific binding," and "immunospecific recognition" are analogous terms in the context of antibodies and refer to molecules that bind to an antigen (e.g., an epitope or immune complex) as such binding is understood by those skilled in the art. For example, a molecule that specifically binds to an antigen may bind other peptides or polypeptides, generally with lower affinity, as determined by, for example, immunoassays, KinExA 3000 instrument (Sapidyne Instruments, Boise, ID) or other assays known in the art. In embodiments, the K of a molecule that specifically binds to an antigen is A than the K when the molecule nonspecifically binds to another antigen A At least 2 log (eg, multiple of 10), 2.5 log, 3 log, 4 log or more higher.

[0065] As used herein, the term "tissue" refers to a group of interconnected cells that share a common biological origin in an organism. As an example, the tissue may perform a physiological function in the body (e.g., lung tissue that allows gas transfer) or be the result of a pathological state (e.g., tumor tissue as a product of cancer, fibrotic tissue as a product of excessive inflammation, etc.).

[0066] As used herein, the term "EU numbering system" refers to the EU numbering convention for antibody constant regions as described in Edelman GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969) and Kabat et al., Sequences of Proteins of Immunological Interest, U. Sept. Health and Human Services, 5th edition, 1991, each of which is incorporated herein by reference in its entirety.

[0067] As used herein, the terms "treat", "treating" and "treatment" refer to therapeutic or preventive measures described herein. The method of "treating" employs administering an antibody to a subject suffering from a disease or disorder or susceptible to such a disease or disorder to prevent, cure, delay, reduce the severity of the disease or disorder or a recurring disease or disorder, or to ameliorate one or more symptoms of the disease or disorder or a recurring disease or disorder, or to prolong the survival of the subject beyond the expected survival time in the absence of such treatment.

[0068] As used herein, the term "effective amount" in the context of administering a treatment to a subject refers to that amount of the treatment that achieves the desired prophylactic or therapeutic effect.

[0069] As used herein, the term "subject" includes any human or non-human animal. In certain embodiments, the subject is a human or non-human mammal. In certain embodiments, the subject is a human.

[0070] As used herein, for antibodies or polynucleotides, the term "isolated" refers to antibodies or polynucleotides that are separated from one or more contaminants (e.g., polypeptides, polynucleotides, lipids or carbohydrates, etc.) that are present in the natural source of the antibodies or polynucleotides. All examples of "isolated antibodies" described herein are additionally considered as antibodies that may, but need not, be separated. All examples of "isolated polynucleotides" described herein are additionally considered as polynucleotides that may, but need not, be separated. All examples of "antibodies" described herein are additionally considered as antibodies that may, but need not, be separated. All examples of "polynucleotides" described herein are additionally considered as polynucleotides that may, but need not, be separated.

[0071] The determination of "percent identity" between two sequences (e.g., amino acid sequences or nucleic acid sequences) can be accomplished using a mathematical algorithm. A non-limiting example of a mathematical algorithm for comparing two sequences is the algorithm in Karlin S & Altschul SF (1990) PNAS 87:2264-2268, improved by Karlin S & Altschul SF (1993) PNAS 90:5873-5877, each of which is incorporated herein by reference in its entirety. This algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215:403, which is incorporated herein by reference in its entirety. BLAST nucleotide searches can be performed using the NBLAST nucleotide program parameter set, e.g., score = 100, word length = 12, to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program parameter set, e.g., score 50, word length = 3, to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul SF et al., (1997) Nuc Acids Res 25:3389-3402, which is incorporated herein by reference in its entirety. Alternatively, an iterated search can be performed using PSI-BLAST, which detects long-range relationships between molecules (supra). When using BLAST, gapped BLAST, and PSIBLAST programs, the default parameters of the corresponding programs (e.g., XBLAST and NBLAST) can be used (e.g., see the National Center for Biotechnology Information (NCBI) on the World Wide Web, NCBI.nlm.nih.gov). Another non-limiting example of a mathematical algorithm for sequence comparison is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is incorporated herein by reference in its entirety. This algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package.When using the ALIGN program to align amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0072] The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. When calculating percent identity, typically only exact matches are counted.

[0073] 7.2 Anti-DDR1 Antibody

[0074] In one aspect, the present disclosure provides methods of screening, determining the effectiveness, or determining the possible effectiveness of one or more inhibitors of DDR1. In some embodiments, the inhibitor of DDR1 comprises an antibody specific for DDR1 (ie, an anti-DDR1 antibody).

[0075] In one embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1 (anti-DDR1). The amino acid sequences of CDRs and VH / VL of exemplary antibodies that specifically bind to DDR1 are shown in Table 2 and Table 3, respectively.

[0076] Table 2. CDR amino acid sequences of exemplary anti-DDR1 antibodies

[0077]

[0078] Table 3. VH / VL amino acid sequences of exemplary anti-DDR1 antibodies

[0079]

[0080]

[0081] In various embodiments, the present disclosure provides an isolated antibody that specifically binds to DDR1 (anti-DDR1), the anti-DDR1 antibody comprising CDRL1, CDRL2, and CDRL3 shown in Table 2. In one embodiment, the anti-DDR1 antibody comprises CDRL1 shown in Table 2. In one embodiment, the anti-DDR1 antibody comprises CDRL2 shown in Table 2. In one embodiment, the anti-DDR1 antibody comprises CDRL3 shown in Table 2.

[0082] In one embodiment, the anti-DDR1 antibody comprises a VL domain comprising one, two or all three CDRs of the VL domain disclosed in Table 3. In one embodiment, the anti-DDR1 antibody comprises CDRL1 of the VL domain shown in Table 3. In one embodiment, the anti-DDR1 antibody comprises CDRL2 of the VL domain shown in Table 3. In one embodiment, the anti-DDR1 antibody comprises CDRL3 of the VL domain shown in Table 3. In one embodiment, the anti-DDR1 antibody comprises a VL domain shown in Table 3.

[0083] In various embodiments, the present disclosure provides an isolated antibody (anti-DDR1) that specifically binds to DDR1, the anti-DDR1 antibody comprising a CDRH1, a CDRH2, and a CDRH3 shown in Table 2. In one embodiment, the anti-DDR1 antibody comprises a CDRH1 shown in Table 2. In one embodiment, the anti-DDR1 antibody comprises a CDRH2 shown in Table 2. In one embodiment, the anti-DDR1 antibody comprises a CDRH3 shown in Table 2.

[0084] In one embodiment, the anti-DDR1 antibody comprises a VH domain comprising one, two or all three CDRs of a VH domain disclosed in Table 3. In one embodiment, the anti-DDR1 antibody comprises a CDRH1 of a VH domain shown in Table 3. In one embodiment, the anti-DDR1 antibody comprises a CDRH2 of a VH domain shown in Table 3. In one embodiment, the anti-DDR1 antibody comprises a CDRH3 of a VH domain shown in Table 3. In one embodiment, the anti-DDR1 antibody comprises a VH domain shown in Table 3.

[0085] In one embodiment, the anti-DDR1 antibody comprises CDRL1 of SEQ ID NO: 5 or 17. In one embodiment, the anti-DDR1 antibody comprises CDRL2 of QAS or GVF. In one embodiment, the anti-DDR1 antibody comprises CDRL3 of SEQ ID NO: 7 or 19. In one embodiment, the anti-DDR1 antibody comprises at least two of CDRL1, CDRL2 or CDRL3 shown in SEQ ID NO: 5-7 or 17-19. In one embodiment, the anti-DDR1 antibody comprises CDRL1, CDRL2 and CDRL3 of SEQ ID NO: 5-7 or SEQ ID NO: 17-19.

[0086] In one embodiment, the anti-DDR1 antibody comprises CDRL1 of SEQ ID NO:5. In one embodiment, the anti-DDR1 antibody comprises CDRL2 of QAS. In one embodiment, the anti-DDR1 antibody comprises CDRL3 of SEQ ID NO:7. In one embodiment, the anti-DDR1 antibody comprises at least two of CDRL1, CDRL2 or CDRL3 as shown in SEQ ID NO:5-7. In one embodiment, the anti-DDR1 antibody comprises CDRL1, CDRL2 and CDRL3 of SEQ ID NO:5-7.

[0087] In one embodiment, the anti-DDR1 antibody comprises CDRL1 of SEQ ID NO: 17. In one embodiment, the anti-DDR1 antibody comprises CDRL2 of GVF. In one embodiment, the anti-DDR1 antibody comprises CDRL3 of SEQ ID NO: 19. In one embodiment, the anti-DDR1 antibody comprises at least two of CDRL1, CDRL2 or CDRL3 of SEQ ID NO: 17-19. In one embodiment, the anti-DDR1 antibody comprises CDRL1, CDRL2 and CDRL3 of SEQ ID NO: 17-19.

[0088] In one embodiment, the anti-DDR1 antibody comprises a VL domain comprising one, two or all three CDRs of the VL domain of SEQ ID NO: 3. In one embodiment, the anti-DDR1 antibody comprises a VL domain of SEQ ID NO: 3. In one embodiment, the anti-DDR1 antibody comprises a VL domain comprising one, two or all three CDRs of the VL domain of SEQ ID NO: 11. In one embodiment, the anti-DDR1 antibody comprises a VL domain comprising one, two or all three CDRs of the VL domain of SEQ ID NO: 12. In one embodiment, the anti-DDR1 antibody comprises a VL domain of SEQ ID NO: 12.

[0089] In one embodiment, the anti-DDR1 antibody comprises a light chain (LC) comprising a VL of the LC sequence shown in Table 4 below. In one embodiment, the anti-DDR1 antibody comprises a LC as shown in Table 4.

[0090] Table 4. LC / HC amino acid sequences of exemplary anti-DDR1 antibodies

[0091]

[0092] In one embodiment, the anti-DDR1 antibody comprises a light chain comprising the VL of the light chain of SEQ ID NO: 23. In one embodiment, the anti-DDR1 antibody comprises the light chain of SEQ ID NO:23.

[0093] In one embodiment, the anti-DDR1 antibody comprises a light chain comprising the VL of the light chain of SEQ ID NO: 161. In one embodiment, the anti-DDR1 antibody comprises the light chain of SEQ ID NO:161.

[0094] In one embodiment, the anti-DDR1 antibody comprises a light chain comprising the VL of the light chain of SEQ ID NO: 164. In one embodiment, the anti-DDR1 antibody comprises the light chain of SEQ ID NO:164.

[0095] In one embodiment, the anti-DDR1 antibody comprises a CDRH1 of SEQ ID NO: 8 or 20. In one embodiment, the anti-DDR1 antibody comprises a CDRH2 of SEQ ID NO: 9 or 21. In one embodiment, the anti-DDR1 antibody comprises a CDRH3 of SEQ ID NO: 10 or 22. In one embodiment, the anti-DDR1 antibody comprises at least two of the CDRH1, CDRH2, or CDRH3 of SEQ ID NO: 8-10 or 20-22. In one embodiment, the anti-DDR1 antibody comprises a CDRH1, CDRH2, and CDRH3 of SEQ ID NO: 8-10 or SEQ ID NO: 20-22.

[0096] In one embodiment, the anti-DDR1 antibody comprises a CDRH1 of SEQ ID NO: 8. In one embodiment, the anti-DDR1 antibody comprises a CDRH2 of SEQ ID NO: 9. In one embodiment, the anti-DDR1 antibody comprises a CDRH3 of SEQ ID NO: 10. In one embodiment, the anti-DDR1 antibody comprises at least two of the CDRH1, CDRH2 or CDRH3 of SEQ ID NO: 8-10. In one embodiment, the anti-DDR1 antibody comprises a CDRH1, CDRH2 and CDRH3 of SEQ ID NO: 8-10.

[0097] In one embodiment, the anti-DDR1 antibody comprises a CDRH1 of SEQ ID NO: 20. In one embodiment, the anti-DDR1 antibody comprises a CDRH2 of SEQ ID NO: 21. In one embodiment, the anti-DDR1 antibody comprises a CDRH3 of SEQ ID NO: 22. In one embodiment, the anti-DDR1 antibody comprises at least two of the CDRH1, CDRH2 or CDRH3 of SEQ ID NO: 20-22. In one embodiment, the anti-DDR1 antibody comprises a CDRH1, CDRH2 and CDRH3 of SEQ ID NO: 20-22.

[0098] In one embodiment, the anti-DDR1 antibody comprises a VH domain comprising one, two or all three CDRs of the VH domain of SEQ ID NO: 4. In one embodiment, the anti-DDR1 antibody comprises a VH domain of SEQ ID NO: 4. In one embodiment, the anti-DDR1 antibody comprises a VH domain comprising one, two or all three CDRs of the VH domain of SEQ ID NO: 13. In one embodiment, the anti-DDR1 antibody comprises a VH domain of SEQ ID NO: 13.

[0099] In one embodiment, the anti-DDR1 antibody comprises a heavy chain comprising the VH of the heavy chain of SEQ ID NO: 24. In one embodiment, the anti-DDR1 antibody comprises the heavy chain of SEQ ID NO:24.

[0100] In one embodiment, the anti-DDR1 antibody comprises a heavy chain comprising the VH of the heavy chain of SEQ ID NO: 162. In one embodiment, the anti-DDR1 antibody comprises the heavy chain of SEQ ID NO:162.

[0101] In one embodiment, the anti-DDR1 antibody comprises a heavy chain comprising the VH of the heavy chain of SEQ ID NO: 163. In one embodiment, the anti-DDR1 antibody comprises the heavy chain of SEQ ID NO:163.

[0102] In one embodiment, the anti-DDR1 antibody comprises a heavy chain comprising the VH of the heavy chain of SEQ ID NO: 165. In one embodiment, the anti-DDR1 antibody comprises the heavy chain of SEQ ID NO:165.

[0103] In one embodiment, the anti-DDR1 antibody comprises CDRL1, CDRL2 and CDRL3 of SEQ ID NO: 5, QAS and SEQ ID NO: 7, respectively; and CDRH1, CDRH2 and CDRH3 of SEQ ID NO: 8, SEQ ID NO: 9 and SEQ ID NO: 10. In one embodiment, the anti-DDR1 antibody comprises the VL domain of SEQ ID NO: 3 and the VH domain of SEQ ID NO: 4.

[0104] In one embodiment, the anti-DDR1 antibody comprises CDRL1, CDRL2, and CDRL3 of SEQ ID NO: 17, GVF, and SEQ ID NO: 19, respectively; and CDRH1, CDRH2, and CDRH3 of SEQ ID NO: 20, SEQ ID NO: 21, and SEQ ID NO: 22. In one embodiment, the anti-DDR1 antibody comprises the VL domain of SEQ ID NO: 11; and the VH domain of SEQ ID NO: 13. In one embodiment, the anti-DDR1 antibody comprises the VL domain of SEQ ID NO: 12; and the VH domain of SEQ ID NO: 13.

[0105] In one embodiment, the anti-DDR1 antibody comprises a light chain of SEQ ID NO: 23; and a heavy chain of SEQ ID NO: 24. In one embodiment, the anti-DDR1 antibody comprises a light chain of SEQ ID NO: 161; and a heavy chain of SEQ ID NO: 162. In one embodiment, the anti-DDR1 antibody comprises a light chain of SEQ ID NO: 161; and a heavy chain of SEQ ID NO: 163. In one embodiment, the anti-DDR1 antibody comprises a light chain of SEQ ID NO: 164; and a heavy chain of SEQ ID NO: 165.

[0106] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3 selected from the CDRL1, CDRL2 and CDRL3 sequences of each mAb shown in Table 5 below; and a heavy chain variable region having CDRH1, CDRH2 and CDRH3 selected from the CDRH1, CDRH2 and CDRH3 sequences of each mAb shown in Table 6 below, or a variant thereof, wherein one or more CDRL and / or CDRH have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0107] Table 5. CDRs of light chain amino acid variable region sequences of DDR1 antibodies

[0108]

[0109] Table 6. CDRs of heavy chain amino acid variable region sequences of DDR1 antibodies

[0110]

[0111]

[0112] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QNIYSN (SEQ ID NO:25), GAS and QSGYYSSSTDIA (SEQ ID NO:44); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFSLSRYA (SEQ ID NO:63), IGSSGLT (SEQ ID NO:82) and ARGMYDDSDDYEDYFNL (SEQ ID NO:101), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0113] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QTISSW (SEQ ID NO:26), YAF and QQGISSSNVDNV (SEQ ID NO:45); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GIDLSSYA (SEQ ID NO:64), INIGGGT (SEQ ID NO:83) and ARDVDAHTLTYFTL (SEQ ID NO:102), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0114] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QTISSW (SEQ ID NO:27), YAF and QCTYGSGSSSSYGCA (SEQ ID NO:46); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFTLSNNA (SEQ ID NO:65), IYASGRT (SEQ ID NO:84) and ARGDTETDYGIPYFDL (SEQ ID NO:103), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0115] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QSVYSNY (SEQ ID NO:28), ETS and QGGYSEIIENT (SEQ ID NO:47); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFSFSSSYY (SEQ ID NO:66), IYASSGST (SEQ ID NO:85) and AILGADYRLTRLDL (SEQ TD NO:104), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0116] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QSIGSTY (SEQ ID NO:29), KAS and LYGGFGSSTGDA (SEQ ID NO:48); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFSFSSGYY (SEQ ID NO:67), IYTGRTDFT (SEQ ID NO:86) and ARGDYSGGVGGNYWLDL (SEQ ID NO:105), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0117] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QTIYSN (SEQ ID NO:30), QAS and QSYYGADDYT (SEQ ID NO:49); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GIDLSNTW (SEQ ID NO:68), ITDSGTT (SEQ ID NO:87) and GRDPGDITSGTNDL (SEQ ID NO:106), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0118] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of KSVYNNNA (SEQ ID NO:31), GVS and AGDYSDISDNN (SEQ ID NO:50); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of SGFSLNNY (SEQ ID NO:69), IFNNGDI (SEQ ID NO:88) and ARTGYRTGGWL (SEQ ID NO:107), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0119] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QSISSY (SEQ ID NO:32), EAS and QNNNGFSGSNFNN (SEQ ID NO:51); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GIDLSYYA (SEQ ID NO:70), INGRGDT (SEQ ID NO:89) and AREDSATPFTVGNYYGMDL (SEQ ID NO:108), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0120] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QTIYSS (SEQ ID NO:33), KAS and QQGSSISNVDKNA (SEQ ID NO:52); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of TFSFNSRYW (SEQ ID NO:71), INGDIS (SEQ ID NO:90) and AKGGNLAGDCYGL (SEQ ID NO:109), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0121] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QSIGSY (SEQ ID NO:34), EAS and QNNNGMTVSDFNA (SEQ ID NO:53); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFSLNRYA (SEQ ID NO:72), IGSSGST (SEQ ID NO:91) and ARDLDDSYGYTYATGMDIRLDL (SEQ ID NO:110), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0122] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QIIDHDH (SEQ ID NO:35), RAS and QNNNGMTVSDFNA (SEQ ID NO:54); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFSLSDYA (SEQ ID NO:73), INSRDDT (SEQ ID NO:92) and AREDSSIPFIVGNYYGMDL (SEQ ID NO:111), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0123] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QSVVDKNW (SEQ ID NO:36), EAS and AGDFESGVSG (SEQ ID NO:55); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFSLSSYG (SEQ ID NO:74), IYPSGSI (SEQ ID NO:93) and VRYLTGSSDLHL (SEQ ID NO:112), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0124] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of KNIYNNNA (SEQ ID NO:37), GAS and AADYSDISDNN (SEQ ID NO:56); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFSLSDYA (SEQ ID NO:75), INNGDIY (SEQ ID NO:94) and ARPGYRTGIWL (SEQ ID NO:113), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0125] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QSVYSNNY (SEQ ID NO: 38), AAS and LGGYNDDAN (SEQ ID NO: 57); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFDLRSYYY (SEQ ID NO: 76), IHGGEGNT (SEQ ID NO: 95) and RGGWTNYF (SEQ ID NO: 114), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0126] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of ESVYSNNH (SEQ ID NO:39), AAS and LGGYNDDAN (SEQ ID NO:58); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFDLSSNYY (SEQ ID NO:77), IYSSNTRT (SEQ ID NO:96) and RGGWTNYL (SEQ ID NO:115), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0127] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QSIDNND (SEQ ID NO:40), RTS and QSYCVNTYGYT (SEQ ID NO:59); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFSLSSHD (SEQ ID NO:78), IISSGNT (SEQ ID NO:97) and ARDVYSGASP (SEQ ID NO:116), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0128] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of QSISNH (SEQ ID NO:41), RAS and QSYYIINRSNY ANS (SEQ ID NO:60); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of TFSFNSRYW (SEQ ID NO:79), INNGDIT (SEQ ID NO:98) and AKGGNLAGDCYGL (SEQ ID NO:117), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0129] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of ESINSW (SEQ ID NO:42), DAS and QSYYIINRSNYGNS (SEQ ID NO:61); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFSLSSYY (SEQ ID NO:80), ITTAGPL (SEQ ID NO:99) and ARGHAGSIYYSYFDL (SEQ ID NO:118), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0130] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having CDRL1, CDRL2 and CDRL3, wherein the CDRL1, CDRL2 and CDRL3 respectively comprise the amino acid sequences of ENLYKDNY (SEQ ID NO:43), GAS and AGGYDSVVD (SEQ ID NO:62); and a heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1, CDRH2 and CDRH3 respectively comprise the amino acid sequences of GFDLSSYYY (SEQ ID NO:81), IYTSSGAT (SEQ ID NO:100) and RGGWCDFNL (SEQ ID NO:119), or variants thereof, wherein one or more CDRLs and / or CDRHs have one, two or three amino acid substitutions, additions, deletions or a combination thereof.

[0131] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having a light chain variable region amino acid sequence selected from the sequence shown in Table 7 below (e.g., SEQ ID NOs: 120-139). In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a heavy chain variable region amino acid sequence selected from the sequence shown in Table 8 below (e.g., SEQ ID NOs: 140-159).

[0132] Table 7. Light chain variable region amino acid sequences of anti-DDR1 antibodies

[0133]

[0134]

[0135] Table 8. Anti-DDR1 antibody heavy chain variable region amino acid sequence

[0136]

[0137]

[0138] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having a light chain variable region amino acid sequence selected from SEQ ID NOs: 120 to 139, and a heavy chain variable region amino acid sequence selected from SEQ ID NOs: 140 to 159. In various embodiments, any one of the variable light chain amino acid sequences corresponding to SEQ ID NOs: 120 to 139 can be used in combination with any one of the variable heavy chain amino acid sequences corresponding to SEQ ID NOs: 140 to 159.

[0139] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 120 (DDR1-1K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 140 (DDR1-1H).

[0140] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 121 (DDR1-3K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 141 (DDR1-3H).

[0141] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 122 (DDR1-5K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 142 (DDR1-5H).

[0142] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 123 (DDR1-6K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 143 (DDR1-6H).

[0143] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 124 (DDR1-9K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 144 (DDR1-9H).

[0144] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 125 (DDR1-11K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 145 (DDR1-11H).

[0145] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 126 (DDR1-12K), and a heavy chain variable region having an amino acid sequence of SEQ ID NO: 146 (DDR1-12H).

[0146] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 127 (DDR1-13K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 147 (DDR1-13H).

[0147] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 128 (DDR1-14K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 148 (DDR1-14H).

[0148] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 129 (DDR1-15K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 149 (DDR1-15H).

[0149] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 130 (DDR1-17K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 150 (DDR1-17H).

[0150] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 131 (DDR1-20K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 151 (DDR1-20H).

[0151] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 132 (DDR1-21K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 152 (DDR1-21H).

[0152] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 133 (DDR1-22K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 153 (DDR1-22H).

[0153] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 134 (DDR1-23K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 154 (DDR1-23H).

[0154] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 135 (DDR1-26K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 155 (DDR1-26H).

[0155] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having an amino acid sequence of SEQ ID NO: 136 (DDR1-28K), and a heavy chain variable region having an amino acid sequence of SEQ ID NO: 156 (DDR1-28H).

[0156] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 137 (DDR1-29K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 157 (DDR1-29H).

[0157] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 138 (DDR1-32K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 158 (DDR1-32H).

[0158] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a light chain variable region having the amino acid sequence of SEQ ID NO: 139 (DDR1-34K), and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 159 (DDR1-34H).

[0159] The individual CDRs of the antibodies disclosed herein can be identified according to any CDR numbering scheme known in the art.

[0160] In some embodiments, one or more CDRs of an antibody disclosed herein can be determined according to Kabat et al., J. Biol. Chem 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest (1991), each of which is incorporated herein by reference in its entirety.

[0161] In some embodiments, one or more CDRs of an antibody disclosed herein can be identified according to the Chothia numbering scheme, which refers to the positions of immunoglobulin structural loops (e.g., see Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226, all of which are incorporated herein by reference in their entirety).

[0162] In some embodiments, one or more CDRs of the antibodies disclosed herein can be determined according to MacCallum RM et al., (1996) J Mol Biol 262: 732-745, which is incorporated herein by reference in its entirety. See also, for example, Martin A "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Dubel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001), which is incorporated herein by reference in its entirety.

[0163] In some embodiments, the CDRs of the antibodies disclosed herein can be identified according to the IMGT numbering system as described in Lefranc MP, (1999) The Immunologist 7: 132-136; Lefranc MP et al., (1999) Nucleic Acids Res 27: 209-212, each of which is incorporated herein by reference in its entirety; and Lefranc MP et al., (2009) Nucleic Acids Res 37: D1006-D1012.

[0164] In some embodiments, the CDRs of the antibodies disclosed herein can be identified according to the AbM numbering scheme, which refers to the AbM hypervariable regions, which represents a compromise between Kabat CDRs and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.), which is incorporated herein by reference in its entirety.

[0165] In some embodiments, the CDRs of the antibodies disclosed herein can be identified according to the AHo numbering system as described in Honegger and Pluckthun A J. Mol. Biol. 309:657-670 (2001), which is incorporated herein by reference in its entirety.

[0166] In some embodiments, the individual CDRs of the antibodies disclosed herein are each independently determined according to one of the Kabat, Chothia, MacCallum, IMGT, AHo, or AbM numbering schemes, or by structural analysis of multispecific molecules, wherein the structural analysis identifies residues in the variable region predicted to contact the epitope region of DDR1.

[0167] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof is a variant, wherein the light chain variable region sequence and / or the heavy chain variable region sequence of the variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid substitutions, additions, deletions or combinations thereof compared to the parent light chain variable region sequence or heavy chain variable region sequence, wherein the variant retains binding specificity and / or other functional properties to the DDR1 protein. In some embodiments, the light chain variable region sequence and / or the heavy chain variable region sequence of the variant has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative or non-conservative amino acid substitutions. In some embodiments, the variant has 1, 2 or 3 amino acid substitutions, additions, deletions or combinations thereof in one or more CDRLs and / or CDRHs of the variant light chain variable region or the variant heavy chain variable region compared to the parent CDRL or CDRH. In some embodiments, compared with the parent light chain variable region sequence or heavy chain variable region sequence, the variant antibody or its antigen-binding fragment has 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions, deletions or combinations thereof in the framework region sequence of the light chain variable region and / or the heavy chain variable region. In some embodiments, the antibody or its antigen-binding fragment has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative or non-conservative amino acid substitutions in the framework region sequence of the light chain variable region and / or the heavy chain variable region. As shown in Table 3, Table 7 and Table 8, the aforementioned changes apply to each of the light chain variable region and the heavy chain variable region.

[0168] In some embodiments, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VL that comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 3, 11 or 12. In embodiments, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VL that comprises an amino acid sequence set forth in SEQ ID NO: 3, 11 or 12. In embodiments, the amino acid sequence of the VL consists of an amino acid sequence set forth in SEQ ID NO: 3, 11 or 12.

[0169] In some embodiments, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VL that comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 3. In embodiments, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VL that comprises the amino acid sequence set forth in SEQ ID NO: 3. In embodiments, the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 3.

[0170] In some embodiments, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VL that comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 11. In embodiments, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VL that comprises the amino acid sequence set forth in SEQ ID NO: 11. In embodiments, the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 11.

[0171] In some embodiments, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VL that comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95%, or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 12. In embodiments, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VL that comprises the amino acid sequence set forth in SEQ ID NO: 12. In embodiments, the amino acid sequence of the VL consists of the amino acid sequence set forth in SEQ ID NO: 12.

[0172] In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VH, wherein the VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence shown in SEQ ID NO: 4. In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 4. In an embodiment, the amino acid sequence of the VH consists of the amino acid sequence shown in SEQ ID NO: 4.

[0173] In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VH, wherein the VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence shown in SEQ ID NO: 13. In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VH, wherein the VH comprises the amino acid sequence shown in SEQ ID NO: 13. In an embodiment, the amino acid sequence of the VH consists of the amino acid sequence shown in SEQ ID NO: 13.

[0174] In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VH and a VL, wherein the VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence shown in SEQ ID NO: 4 or 13, and the VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence shown in SEQ ID NO: 3, 11 or 12. In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 4 or 13, and the VL comprises the amino acid sequence of SEQ ID NO: 3, 11 or 12. In an embodiment, the amino acid sequence of the VH consists of the amino acid sequence shown in SEQ ID NO: 4 or 13; and the amino acid sequence of the VL consists of the amino acid sequence shown in SEQ ID NO: 3, 11 or 12.

[0175] In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VH and a VL, wherein the VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 4, and the VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g., 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 3. In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 4, and the VL comprises the VL of the amino acid sequence of SEQ ID NO: 3. In an embodiment, the amino acid sequence of the VH consists of the amino acid sequence shown in SEQ ID NO:4; and the amino acid sequence of the VL consists of the amino acid sequence shown in SEQ ID NO:3.

[0176] In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VH and a VL, wherein the VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 13, and the VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g., 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 11. In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 13, and the VL comprises the amino acid sequence of SEQ ID NO: 11. In an embodiment, the amino acid sequence of the VH consists of the amino acid sequence shown in SEQ ID NO:13; and the amino acid sequence of the VL consists of the amino acid sequence shown in SEQ ID NO:11.

[0177] In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VH and a VL, wherein the VH comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g., at least 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 13, and the VL comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, 95% or 100% (e.g., 91, 92, 93, 94, 95, 96, 97, 98 or 99%) identical to the amino acid sequence set forth in SEQ ID NO: 12. In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising a VH and a VL, wherein the VH comprises the amino acid sequence of SEQ ID NO: 13, and the VL comprises the amino acid sequence of SEQ ID NO: 12. In an embodiment, the amino acid sequence of the VH consists of the amino acid sequence shown in SEQ ID NO:13; and the amino acid sequence of the VL consists of the amino acid sequence shown in SEQ ID NO:12.

[0178] In an embodiment, the present disclosure provides an isolated antibody that specifically binds to DDR1, comprising VH and VL amino acid sequences as shown in SEQ ID NOs: 3 and 4; 11 and 13; or 12 and 13, respectively. In an embodiment, the amino acid sequences of VH and VL consist of the amino acid sequences shown in SEQ ID NOs: 3 and 4; 11 and 13; or 12 and 13, respectively.

[0179] In an embodiment, the present disclosure provides an isolated antibody that cross-competes for binding to DDR1 with an antibody comprising the VH and VL amino acid sequences set forth in SEQ ID NOs: 3 and 4; 11 and 13; or 12 and 13, respectively.

[0180] In embodiments, the disclosure provides isolated antibodies that bind to the same or overlapping epitope of DDR1 as an antibody described herein (eg, an antibody comprising VH and VL amino acid sequences as shown in SEQ ID NOs: 3 and 4; 11 and 13; or 12 and 13, respectively).

[0181] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 120. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 140. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 120, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 140.

[0182] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 121. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 141. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 121, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 141.

[0183] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 122. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 142. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 122, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 142.

[0184] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 123. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 143. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 123, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 143.

[0185] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 124. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 144. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 124, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 144.

[0186] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 125. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 145. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 125, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 145.

[0187] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 126. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 146. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 126, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 146.

[0188] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 127. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 147. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 127, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 147.

[0189] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 128. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 148. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 128, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 148.

[0190] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 129. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 149. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 129, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 149.

[0191] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 130. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 150. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 130, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 150.

[0192] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 131. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 151. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 131, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 151.

[0193] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 132. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 152. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 132, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 152.

[0194] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 133. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 153. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 133, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 153.

[0195] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 134. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 154. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 134, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 154.

[0196] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 135. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 155. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 135, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 155.

[0197] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 136. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 156. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 136, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 156.

[0198] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 137. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 157. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 137, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 157.

[0199] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 138. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 158. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 138, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 158.

[0200] In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 139. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 159. In some embodiments, the anti-DDR1 antibody or antigen-binding fragment thereof comprises a VL amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VL amino acid sequence of SEQ ID NO: 139, and a VH amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the VH amino acid sequence of SEQ ID NO: 159.

[0201] In embodiments, the epitope of the antibody can be identified by, for example, NMR spectroscopy, surface plasmon resonanceX-ray diffraction crystallographic studies, ELISA assays, hydrogen / deuterium exchange coupled to mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping) are used to determine. For X-ray crystallography, crystallization can be achieved using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50 (Pt 4): 339-350; McPherson A (1990) Eur J Biochem 189: 1-23; Chayen NE (1997) Structure 5: 1269-1274; McPherson A (1976) J Biol Chem 251: 6300-6303, all of which are incorporated herein by reference in their entirety). Antibody:antigen crystals can be studied using well-known X-ray diffraction techniques and can be refined using computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.; U.S. Patent Application No. 2004 / 0014194) and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49 (Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 276A: 361-423, ed. Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56 (Pt 10): 1316-1323, all of which are incorporated herein by reference in their entirety). Mutagenesis mapping studies can be completed using any method known to those skilled in the art. For example, for a description of mutagenesis techniques including alanine scanning mutagenesis techniques, see, Champe M et al., (1995) above and Cunningham BC & Wells JA (1989) above. In an embodiment, alanine scanning mutagenesis studies are used to determine the epitope of the antibody. In addition, or antibodies that recognize and bind to the same or overlapping epitopes of DDR1 (e.g., human DDR1 or mouse DDR1) can be identified using conventional techniques such as immunoassays, for example, by showing the ability of one antibody to block the binding of another antibody to the target antigen, i.e., competitive binding assays.Competitive binding assays can also be used to determine if two antibodies have similar binding specificities for an epitope. Competitive binding can be determined in an assay in which a test immunoglobulin inhibits specific binding of a reference antibody to a common antigen, such as DDR1 (eg, human DDR1 or mouse DDR1). Various types of competitive binding assays are known, for example: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137:3614-9); solid phase direct labeling assay, solid phase direct labeling sandwich assay (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct labeling RIA using I-125 labeling (see Morel GA et al., (1988) Mol Immunol 25(1):7-15); solid phase direct biotin-avidin EIA (see Cheung RC et al., (1990) Virology 176:546-52); and direct labeling RIA (see Moldenhauer G et al., (1990) Scand J Immunol 32:77-82), all of which are incorporated herein by reference in their entirety. Typically, such an assay involves the use of a purified antigen (e.g., DDR1, such as human DDR1 or mouse DDR1) bound to a solid surface or a cell carrying one of these antigens, an unlabeled test immunoglobulin, and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to a solid surface or cell in the presence of a test immunoglobulin. Typically, the test immunoglobulin is present in excess. Typically, when the competing antibody is present in excess, it will inhibit the specific binding of the reference or antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75% or more. Competitive binding assays can be configured in a large number of different formats using labeled antigens or labeled antibodies. In a common version of this assay, the antigen is fixed to a 96-well plate. A radioactive or enzymatic label is then used to measure the ability of the unlabeled antibody to block the binding of the labeled antibody to the antigen.For further details, see, e.g., Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68:269-274; Kuroki M et al., (1990) Cancer Res 50:4872-4879; Kuroki M et al., (1992) Immunol Invest 21:523-538; Kuroki M et al., (1992) Hybridoma 11:391-407 and Antibodies: A Laboratory Manual, ed. Harlow E & Lane D, eds., supra, pp. 386-389, all of which are incorporated herein by reference in their entirety.

[0202] In embodiments, the antibody inhibits the binding of human DDR1 to human collagen. In embodiments, the binding of human DDR1 to human collagen is reduced by more than 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% in the presence of the antibody relative to the binding of human DDR1 to human collagen in the absence of the antibody.

[0203] In embodiments, the antibodies disclosed herein are coupled to a cytotoxic agent, a cytostatic agent, a toxin, a radionuclide, or a detectable label. In embodiments, the cytotoxic agent is capable of inducing cell death or destruction in contact with it. In embodiments, the cytostatic agent is capable of preventing or substantially reducing cell proliferation in contact with it and / or inhibiting the activity or function of cells in contact with it. In embodiments, the cytotoxic agent or cytostatic agent is a chemotherapeutic agent. In embodiments, the radionuclide is selected from an isotope 3 H. 14 C. 32 P. 35 S. 36 Cl, 51 Cr, 57 Co. 58 Co. 59 Fe, 67 Cu, 90 Y. 99 Tc, 111 In, 117 Lu, 121 I. 124 I. 125 I. 131 I. 198 Au,211 At 213 Bi, 225 Ac and 186 Re. In embodiments, the detectable label comprises a fluorescent moiety or a click chemistry handle.

[0204] Any immunoglobulin (Ig) constant region can be used in the antibodies disclosed herein. In embodiments, the Ig region is a human IgG, IgE, IgM, IgD, IgA or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecules.

[0205] In an embodiment, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., CH2 domain (residues 231-340 of human IgG1)) and / or CH3 domain (residues 341-447 of human IgG1 numbered according to the EU numbering system) and / or hinge region (residues 216-230 numbered according to the EU numbering system) of an antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or antigen-dependent cellular cytotoxicity.

[0206] In an embodiment, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of an antibody described herein such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described, e.g., in U.S. Pat. No. 5,677,425, which is incorporated herein by reference in its entirety. The number of cysteine ​​residues in the hinge region can be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody.

[0207] In an embodiment, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into an IgG constant region or an FcRn binding fragment thereof (preferably an Fc or hinge-Fc fragment) to change (e.g., reduce or increase) the half-life of the antibody in vivo. See, e.g., WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631 International Publication; and U.S. Pat. Nos. 5869046; 6,121,022; 6,277,375; and 6165745, all of which are incorporated herein by reference in their entirety as examples of mutations that change (e.g., reduce or increase) the half-life of an antibody in vivo. In certain embodiments, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into an IgG constant region or an FcRn binding fragment thereof (preferably an Fc or hinge-Fc fragment) to reduce the half-life of the antibody in vivo. In certain embodiments, one, two or more amino acid mutations (e.g., substitutions, insertions or deletions) are introduced into the IgG constant region or its FcRn binding fragment (preferably Fc or hinge-Fc fragment) to increase the half-life of the antibody in vivo. In embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU numbering system. In embodiments, the constant region of the IgG1 of the antibody described herein comprises a substitution of methionine (M) at position 252 numbered according to the EU numbering system to tyrosine (Y), a substitution of serine (S) at position 254 to threonine (T), and a substitution of threonine (T) at position 256 to glutamic acid (E). See U.S. Pat. No. 7,658,921, which is incorporated herein by reference in its entirety. This type of mutant IgG, called a "YTE mutant," has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24, which is incorporated herein by reference in its entirety). In certain embodiments, the antibody comprises an IgG constant region comprising one, two, three or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389 and 428-436 numbered according to the EU numbering system.

[0208] In certain embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., CH2 domain (residues 231-340 of human IgG1) and / or CH3 domain (residues 341-447 of human IgG1 numbered according to the EU numbering system) and / or hinge region (residues 216-230 numbered according to the EU numbering system)) of an antibody described herein to increase or decrease the affinity of the antibody for an Fc receptor on the surface of an effector cell (e.g., an activated Fc receptor). Mutations in the Fc region of an antibody that decrease or increase the affinity of the antibody for an Fc receptor and techniques for introducing such mutations into the Fc receptor or fragment thereof are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be used to alter the affinity of the antibody for the Fc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, all of which are incorporated herein by reference in their entirety.

[0209] In an embodiment, the antibody comprises a heavy chain constant region, and the heavy chain constant region is a variant of a wild-type heavy chain constant region, wherein the variant heavy chain constant region binds to FcγRIIB with a higher affinity than the wild-type heavy chain constant region binds to FcγRIIB. In certain embodiments, the variant heavy chain constant region is a variant human heavy chain constant region, such as a variant human IgG1, a variant human IgG2, or a variant human IgG4 heavy chain constant region. In certain embodiments, the variant human IgG heavy chain constant region comprises one or more of the following amino acid mutations according to the EU numbering system: G236D, P238D, S239D, S267E, L328F, and L328E. In certain embodiments, the variant human IgG heavy chain constant region comprises a group of amino acid mutations selected from the group consisting of S267E and L328F according to the EU numbering system; P238D and L328E; P238D and one or more substitutions selected from the group consisting of E233D, G237D, H268D, P271G and A330R; P238D, E233D, G237D, H268D, P271G and A330R; G236D and S267E; S239D and S267E; V262E, S267E and L328F; and V264E, S267E and L328F. In embodiments, FcγRIIB is expressed on cells selected from the group consisting of macrophages, monocytes, B cells, dendritic cells, endothelial cells and activated T cells.

[0210] In an embodiment, one, two or more amino acid substitutions are introduced into the IgG constant region Fc region to change the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 239, 243, 267, 292, 297, 300, 318, 320, 322, 328, 330, 332 and 396 numbered according to the EU numbering system can be replaced by different amino acid residues so that the antibody has a changed affinity for the effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand to which its affinity is changed can be, for example, the C1 component of Fc receptors or complement. This method is further described in detail in No. 5624821 and No. 5648260 U.S. Patents, each of which is incorporated herein by reference in its entirety. In certain embodiments, the deletion or inactivation of the constant region domain (by point mutation or otherwise) can reduce the Fc receptor binding of circulating antibodies, thereby enhancing tumor localization. Describing mutations that delete or inactivate the constant region to enhance tumor localization, see, e.g., U.S. Pat. Nos. 5,585,097 and 8,591,886, each of which is incorporated herein by reference in its entirety. In an embodiment, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on the Fc region, which may reduce Fc receptor binding (see, e.g., Shields R Let al., (2001) J Biol Chem 276: 6591-604, which is incorporated herein by reference in its entirety). In various embodiments, one or more of the following mutations, numbered according to the EU numbering system, can be made in the constant region of an antibody described herein: N297A substitution; N297Q substitution; L234A substitution; L234F substitution; L235A substitution; L235F substitution; L235V substitution; L237A substitution; S239D substitution; E233P substitution; L234V substitution; L235A substitution; C236 deletion; P238A substitution; S239D substitution; F243L substitution; D265A substitution; S267E substitution; L328F substitution; R292P substitution; Y300L substitution; A327Q substitution; P329A substitution; A330L substitution; I332E substitution; or P396L substitution. In some embodiments, the following mutations are generated in the constant region of the antibody: L234A, L235E, G237A, A330S and P331S. In some embodiments, the following mutations are generated in the constant region of the antibody: P329G, L234A and L235A.

[0211] In certain embodiments, mutations selected from D265A, P329A and combinations thereof numbered according to the EU numbering system may be produced in the constant region of antibodies described herein. In certain embodiments, mutations selected from L235A, L237A and combinations thereof numbered according to the EU numbering system may be produced in the constant region of antibodies described herein. In certain embodiments, mutations selected from S267E, L328F and combinations thereof numbered according to the EU numbering system may be produced in the constant region of antibodies described herein. In certain embodiments, mutations selected from S239D, I322E, optionally A330L and combinations thereof numbered according to the EU numbering system may be produced in the constant region of antibodies described herein. In certain embodiments, mutations selected from L235V, F243L, R292P, Y300L, P396L and combinations thereof numbered according to the EU numbering system may be produced in the constant region of antibodies described herein. In certain embodiments, a mutation selected from S267E, L328F, and combinations thereof, numbered according to the EU numbering system, may be made in the constant region of an antibody described herein.

[0212] In an embodiment, the antibody described herein comprises a constant region of IgG1 having an N297Q or N297A amino acid substitution numbered according to the EU numbering system. In certain embodiments, the antibody described herein comprises a constant region of IgG1 having a mutation selected from D265A, P329A and combinations thereof numbered according to the EU numbering system. In another embodiment, the antibody described herein comprises a constant region of IgG1 having a mutation selected from L234A, L235A and combinations thereof numbered according to the EU numbering system. In another embodiment, the antibody described herein comprises a constant region of IgG1 having a mutation selected from L234F, L235F, N297A and combinations thereof numbered according to the EU numbering system. In certain embodiments, the amino acid residues at the positions corresponding to the positions L234, L235 and D265 numbered according to the EU numbering system in the constant region of the antibody described herein are not L, L and D. This method is described in detail in WO 14 / 108483 International Publication, which is incorporated herein by reference in its entirety. In an embodiment, the amino acids corresponding to positions L234, L235 and D265, respectively, in a human IgGl heavy chain as numbered according to the EU numbering system are F, E and A; or A, A and A.

[0213] In an embodiment, the amino acids at positions 433, 434, and 436 of the heavy chain constant region according to the EU numbering system are K, F, and Y, respectively. In an embodiment, the amino acids at positions 252, 254, and 256 of the heavy chain constant region according to the EU numbering system are Y, T, and E, respectively. In an embodiment, the amino acids at positions 428 and 434 of the heavy chain constant region according to the EU numbering system are L and S, respectively. In an embodiment, the amino acids at positions 309, 311, and 434 of the heavy chain constant region according to the EU numbering system are D, H, and S, respectively.

[0214] In an embodiment, one or more amino acids selected from amino acid residues 329, 331 and 322 of the constant region of an antibody described herein, numbered according to the EU numbering system, may be replaced by a different amino acid residue such that the antibody has altered C1q binding and / or reduced or eliminated complement dependent cytotoxicity (CDC). This approach is further described in detail in U.S. Pat. No. 6,194,551 (Idusogie et al), which is incorporated herein by reference in its entirety. In an embodiment, one or more amino acid residues within amino acid positions 231 to 238 of the N-terminal region of the CH2 domain of an antibody described herein, numbered according to the EU numbering system, are altered, thereby altering the ability of the antibody to fix complement. This approach is further described in International Publication No. WO 94 / 29351, which is incorporated herein by reference in its entirety. In an embodiment, the Fc region of an antibody described herein is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody for an Fcγ receptor by mutating one or more amino acids at the following positions numbered according to the EU numbering system (e.g., introducing an amino acid substitution): 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315 6, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This method is further described in International Publication No. WO00 / 42072, which is incorporated herein by reference in its entirety.

[0215] In an embodiment, any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of an antibody described herein having two heavy chain constant regions.

[0216] In embodiments, the disclosure provides isolated antibodies that specifically bind to DDR1 and function as antagonists (eg, reduce or inhibit DDR1 activity).

[0217] In embodiments, the disclosure provides an isolated antibody that specifically binds to DDR1 and reduces or inhibits DDR1 activity by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% relative to the activity of DDR1 without any antibody or with an irrelevant antibody (e.g., an antibody that does not specifically bind to DDR1) as assessed by methods described herein and / or known to those of skill in the art. In embodiments, the present disclosure provides an isolated antibody that specifically binds to DDR1 and reduces or inhibits DDR1 activity by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, relative to DDR1 activity without any antibody or with an irrelevant antibody (e.g., an antibody that does not specifically bind to DDR1) as assessed by methods described herein and / or known to those of skill in the art. Non-limiting examples of DDR1 activity can include DDR1 signaling; DDR1 binding to collagen (e.g., collagen I, II, III, IV, or V); or DDR1 phosphorylation. In embodiments, the reduction in DDR1 activity is assessed as described in the Examples.

[0218] In embodiments, the disclosure provides an isolated antibody that specifically binds to DDR1 and reduces or inhibits DDR1 phosphorylation by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% relative to DDR1 phosphorylation without any antibody or with an irrelevant antibody (e.g., an antibody that does not specifically bind to DDR1) as assessed by methods described herein and / or known to those of skill in the art. In embodiments, the disclosure provides an isolated antibody that specifically binds DDR1 and reduces or inhibits DDR1 phosphorylation by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more relative to DDR1 phosphorylation without any antibody or with an irrelevant antibody (e.g., an antibody that non-specifically binds DDR1) as assessed by methods described herein and / or known to those of skill in the art.

[0219] In embodiments, the disclosure provides an isolated antibody that specifically binds to DDR1 and reduces or inhibits the binding of DDR1 to collagen by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% relative to the binding of DDR1 to collagen without any antibody or with an irrelevant antibody (e.g., an antibody that does not specifically bind to DDR1) as assessed by methods described herein and / or known to those of skill in the art. In embodiments, the disclosure provides an isolated antibody that specifically binds DDR1 and reduces or inhibits the binding of DDR1 to collagen by at least about 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more relative to the binding of DDR1 to collagen without any antibody or with an irrelevant antibody (e.g., an antibody that non-specifically binds DDR1) as assessed by methods described herein and / or known to those of skill in the art.

[0220] In embodiments, the present disclosure provides an isolated antibody that specifically binds to DDR1 with a dissociation constant (K) of less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, or less than 0.1 nM. D )value.

[0221] 7.3 Pharmaceutical Compositions

[0222] Provided herein are compositions comprising an isolated anti-DDR1 antibody disclosed herein having a desired degree of purity in a physiologically acceptable carrier, excipient or stabilizer (see, e.g., Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). Acceptable carriers, excipients or stabilizers are nontoxic to the receptor at the dosages and concentrations used, and include buffers such as phosphates, citrates and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as TWEEN®. TM 、PLURONTCS TM or polyethylene glycol (PEG).

[0223] In an embodiment, the pharmaceutical composition comprises an isolated anti-DDR1 antibody disclosed herein in a pharmaceutically acceptable carrier, and optionally one or more additional prophylactic or therapeutic agents. In an embodiment, the antibody is the only active ingredient contained in the pharmaceutical composition. In an embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated anti-DDR1 antibody disclosed herein for use as a medicament. In another embodiment, the present disclosure provides a pharmaceutical composition for use in a method for treating a DDR1-related disease. In some embodiments, the DDR1-related disease is cancer or a fibrotic disorder.

[0224] Pharmaceutically acceptable carriers for parenteral preparations include aqueous vehicles, non-aqueous vehicles, antibacterial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending agents and dispersants, emulsifiers, isolating agents or chelating agents, and other pharmaceutically acceptable substances. The example of aqueous vehicles includes sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, and glucose and lactated Ringer's injection. Non-aqueous parenteral vehicles include fixed oils, cottonseed oil, corn oil, sesame oil and peanut oil of plant origin. Antibacterial agents that inhibit bacteria or inhibit fungal concentrations can be added to parenteral preparations packaged in multiple dose containers, including phenols or cresols, mercurial preparations, benzyl alcohol, chlorobutanol, methylparaben and propylparaben, thimerosal, benzalkonium chloride and benzethonium chloride. Isotonic agents include sodium chloride and glucose. Buffers include phosphates and citrates. Antioxidants include sodium bisulfate. Local anesthetics include procaine hydrochloride. Suspending and dispersing agents include sodium carboxymethylcellulose, hydroxypropyl methylcellulose and polyvinyl pyrrolidone. Emulsifiers include polysorbate 80 ( 80). Metal ion separators or chelators include EDTA. Pharmaceutical carriers also include ethanol, polyethylene glycol and propylene glycol as water-miscible vehicles, and sodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.

[0225] Pharmaceutical compositions can be formulated for any route of administration for a subject. Specific examples of routes of administration include intranasal, oral, pulmonary, transdermal, intradermal and parenteral. Parenteral administration characterized by subcutaneous, intramuscular or intravenous injection is also contemplated herein. Injections can be prepared as follows: liquid solutions or suspensions in conventional form, solid forms suitable for dissolving or suspending in liquids before injection, or as emulsions. The injections, solutions and emulsions also contain one or more excipients. Suitable excipients are, for example, water, saline, glucose, glycerol or ethanol. In addition, if desired, the pharmaceutical composition to be administered may also contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, stabilizers, dissolution enhancers and other such agents, such as, for example, sodium acetate, sorbitan laurate, triethanolamine oleate and cyclodextrin.

[0226] Antibody preparations for parenteral antibody administration include sterile solutions that can be injected directly, sterile dry soluble products that can be mixed directly with solvents before use (such as lyophilized powders, including hypodermic tablets), sterile suspensions that can be injected directly, sterile dry insoluble products that can be mixed directly with vehicles before use, and sterile emulsions. The solution can be aqueous or non-aqueous.

[0227] If administered intravenously, suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol and polypropylene glycol, and mixtures thereof.

[0228] As described, topical mixtures containing antibodies are prepared for local and systemic administration. The resulting mixture can be a solution, suspension, emulsion, etc., and can be formulated as a cream, gel, ointment, emulsion, solution, elixir, lotion, suspension, tincture, paste, foam, aerosol, rinse, spray, suppository, bandage, skin patch or any other dosage form suitable for topical administration.

[0229] The isolated anti-DDR1 antibodies disclosed herein can be formulated for topical or external administration, such as topical application to the skin and mucous membranes of the eye, such as in the form of gels, creams and lotions, and for ocular administration or for intracisternal or intraspinal administration. External administration is contemplated for transdermal delivery, and also for ocular or mucosal administration, or for inhalation therapy. Nasal solutions of the antibodies may also be administered alone or in combination with other pharmaceutically acceptable excipients.

[0230] Transdermal patches, including iontophoresis devices and electrophoresis devices, are well known to those skilled in the art and can be used to administer antibodies. For example, such patches are disclosed in U.S. Pat. Nos. 6,267,983, 6,261,595; 6,256,533; 6,167,301; 6,024,975; 6,010,715; 5,985,317; 5,983,134; 5,948,433; and 5,860,957, all of which are incorporated herein by reference in their entirety.

[0231] In an embodiment, the pharmaceutical composition comprising the antibody described herein is a lyophilized powder, which can be reconstituted into solutions, emulsions and other mixtures for administration. It can also be reconstituted and formulated as a solid or gel. The lyophilized powder is prepared by dissolving the antibody described herein or a pharmaceutically acceptable derivative thereof in a suitable solvent. In an embodiment, the lyophilized powder is sterile. The solvent may contain an excipient that improves the stability of the powder or the reconstituted solution prepared from the powder or other pharmacological components. Excipients that can be used include, but are not limited to, glucose, sorbitol, fructose, corn syrup, xylitol, glycerol, glucose, sucrose or other suitable agents. The solvent may also contain a buffer, such as citrate, sodium phosphate or potassium phosphate, or other buffers known to those skilled in the art, in certain embodiments, at about neutral pH. The solution is then sterile filtered and then lyophilized under standard conditions known to those skilled in the art to obtain the desired dosage form. In an embodiment, the resulting solution is aliquoted into vials for lyophilization. Each vial contains a single dose or multiple doses of the compound. The lyophilized powder can be stored under appropriate conditions, such as at about 4°C to room temperature. Reconstitution of this lyophilized powder with water for injection provides a formulation for parenteral administration. For reconstitution, the lyophilized powder is added to sterile water or other suitable carrier. The exact amount depends on the compound selected. This amount can be determined empirically.

[0232] The isolated anti-DDR1 antibodies disclosed herein and other compositions provided herein can also be formulated to target specific tissues, receptors or other areas of the body of the subject to be treated. Many such targeting methods are well known to those skilled in the art. All of these targeting methods are contemplated for use in the present compositions. For non-limiting examples of targeting methods, see, e.g., U.S. Pat. Nos. 6,316,652, 6,274,552, 6,271,359, 6,253,872, 6,139,865, 6,131,570, 6,120,751, 6,071,495, 6,060,082, 6,048,736, 6,039,975, 6,004,534, 5,985,307, 5,972,366, 5,900,252, 5,840,674, 5,759,542, and 5,709,874, all of which are incorporated herein by reference in their entirety. In embodiments, the antibodies described herein target tumors.

[0233] Compositions for in vivo administration may be sterile. This is easily achieved, for example, by filtration through sterile filtration membranes.

[0234] 7.4 Polynucleotides, Vectors, and Methods for Producing Antibodies

[0235] In one aspect, polynucleotides are provided herein, comprising nucleotide sequences encoding antibodies or a portion thereof or fragments thereof (e.g., VL and / or VH; and light and / or heavy chains) that specifically bind to DDR1 antigens as described herein, and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., E. coli and mammalian cells). Polynucleotides are provided herein, comprising nucleotide sequences encoding the heavy and / or light chains of antibodies provided herein, and vectors comprising such polynucleotide sequences, e.g., expression vectors for effectively expressing them in host cells (e.g., mammalian cells).

[0236] As used herein, "isolated" polynucleotides or nucleic acid molecules refer to polynucleotides or nucleic acid molecules isolated from other nucleic acid molecules present in the natural source (e.g., mouse or mankind) of the nucleic acid molecules. In addition, "isolated" nucleic acid molecules, such as cDNA molecules, can be substantially free of other cellular materials or cell culture media when produced by recombinant technology, or substantially free of chemical precursors or other chemical substances when chemically synthesized. For example, the language of "substantially free" includes that the preparation of polynucleotides or nucleic acid molecules has less than about 15%, 10%, 5%, 2%, 1%, 0.5% or 0.1% (especially, less than about 10%) of other substances, for example, cellular materials, cell culture media, other nucleic acid molecules, chemical precursors and / or other chemical substances. In embodiments, the nucleic acid molecules encoding antibodies described herein are isolated or purified.

[0237] In one aspect, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody that specifically binds to a DDR1 polypeptide and comprises an amino acid sequence described herein, as well as antibodies that compete with such an antibody for binding to a DDR1 polypeptide (e.g., in a dose-dependent manner), or antibodies that bind to the same epitope as such an antibody.

[0238] On the one hand, polynucleotides are provided herein, which include nucleotide sequences encoding light chains or heavy chains of antibodies described herein. The polynucleotides may include nucleotide sequences encoding light chains comprising VL FR and CDR of antibodies described herein (see, e.g., Table 2-8) or nucleotide sequences encoding heavy chains comprising VH FR and CDR of antibodies described herein (see, e.g., Table 2-8). In embodiments, polynucleotides encode VH, VL, heavy chains and / or light chains of antibodies described herein. In embodiments, polynucleotides encode the first VH and the first VL of antibodies described herein. In embodiments, polynucleotides encode the second VH and the second VL of antibodies described herein. In embodiments, polynucleotides encode the first heavy chain and the first light chain of antibodies described herein. In embodiments, polynucleotides encode the second heavy chain and the second light chain of antibodies described herein. In embodiments, polynucleotides encode VH and / or VL, or heavy chains and / or light chains of separated antibodies described herein.

[0239] In some embodiments, the polynucleotides encoding the heavy chain and / or light chain of the isolated antibody described herein further encode one or more signal peptides. In some embodiments, the signal peptide includes a secretory signal peptide. In some embodiments, the secretory signal peptide includes an immunoglobulin secretory signal peptide. Exemplary signal peptides include but are not limited to heavy chain IgM, IgG, IgD, IgA and IgE signal peptides, and light chain κ and λ signal peptides. In certain embodiments, the signal peptide is a mammalian signal peptide. In certain embodiments, the signal peptide is a human signal peptide. In certain embodiments, the signal peptide includes rodents, such as murines or lagomorphs, such as rabbit signal peptides. In embodiments, the signal peptide is a primate (e.g., non-human primate) signal peptide.

[0240] Also provided herein are polynucleotides encoding optimized isolated anti-DDR1 antibodies, such as by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA unstable elements. The method for producing an optimized nucleic acid encoding an isolated anti-DDR1 antibody or fragment thereof (e.g., a light chain, a heavy chain, a VH domain, or a VL domain) for recombinant expression by introducing codon changes and / or eliminating recombinant expression of an inhibitory region in mRNA can be performed by changing, for example, the corresponding optimization methods described in U.S. Pat. Nos. 5,965,726; 6,174,666; 6,291,664; 6,414,132; and 6,794,498, each of which is incorporated herein by reference in its entirety. For example, potential splicing sites and unstable elements (e.g., A / T or A / U-rich elements) in RNA can be mutated without changing the amino acid encoded by the nucleic acid sequence to increase the stability of the RNA for recombinant expression. These changes take advantage of the degeneracy of the genetic code, for example, using alternative codons for the same amino acid. In embodiments, it may be desirable to change one or more codons to encode a conservative mutation, e.g., a similar amino acid having a similar chemical structure and properties and / or function as the original amino acid. Such methods can increase expression of an isolated anti-DDR1 antibody or fragment thereof by at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold or more relative to expression of an isolated anti-DDR1 antibody encoded by a non-optimized polynucleotide.

[0241] In an embodiment, an optimized polynucleotide sequence encoding an isolated anti-DDR1 antibody or fragment thereof described herein (e.g., a VL domain and / or a VH domain) can be hybridized with an antisense (e.g., complementary) polynucleotide of an unoptimized polynucleotide sequence encoding an isolated anti-DDR1 antibody or fragment thereof described herein (e.g., a VH domain and / or a VL domain). In an embodiment, an optimized nucleotide sequence encoding an isolated anti-DDR1 antibody or fragment thereof described herein hybridizes with an antisense polynucleotide of an unoptimized polynucleotide sequence encoding an isolated anti-DDR1 antibody or fragment thereof described herein under high stringency conditions. In an embodiment, an optimized nucleotide sequence encoding an isolated anti-DDR1 antibody or fragment thereof described herein hybridizes with an antisense polynucleotide of an unoptimized nucleotide sequence encoding an isolated anti-DDR1 antibody or fragment thereof described herein under high stringency, medium or low stringency hybridization conditions. Information about hybridization conditions has been described, see, for example, U.S. Patent Application Publication No. US2005 / 0048549 (e.g., paragraphs 72-73), which is incorporated herein by reference in its entirety.

[0242] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotides is determined. The nucleotide sequences encoding antibodies described herein (e.g., the antibodies described in Tables 2-8) and modified versions of these antibodies can be determined using methods well known in the art, that is, assembling the nucleotide codons of known encoding specific amino acids in this way to produce nucleic acids encoding antibodies. Such polynucleotides encoding the antibodies can be assembled by chemically synthesized oligonucleotides (e.g., as described in Kutmeier G et al., (1994), BioTechniques 17: 242-6, incorporated herein by reference in their entirety), in short, involving the synthesis of overlapping oligonucleotides containing the sequence portion encoding the antibody, annealing and connecting these oligonucleotides, and then amplifying the oligonucleotides connected by PCR.

[0243] Alternatively, polynucleotides encoding the antigen binding regions of antibodies described herein can be produced from nucleic acids from a suitable source (e.g., hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers that can hybridize with the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from hybridoma cells that produce the antibody of interest. This PCR amplification method can be used to obtain nucleic acids containing sequences encoding antibody light chains and / or heavy chains. This PCR amplification method can be used to obtain nucleic acids containing sequences encoding variable light chain regions and / or variable heavy chain regions of antibodies. The amplified nucleic acids can be cloned into vectors for expression in host cells and for further cloning.

[0244] If a clone containing a nucleic acid encoding a particular antigen binding region or antibody is not available, but the sequence of the antigen binding region or antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized, or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library or isolated nucleic acid produced from any tissue or cell expressing the antibody (e.g., a hybridoma cell selected to express the antibody described herein), preferably poly A+RNA), by PCR amplification using synthetic primers that hybridize to the 3' and 5' ends of the sequence or by cloning using a specific oligonucleotide probe for a specific gene sequence to identify, for example, a cDNA clone from a cDNA library encoding the antibody. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method known in the art.

[0245] The DNA encoding the isolated anti-DDR1 antibodies described herein can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of anti-DDR1). Hybridoma cells can serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as an E. coli cell, a monkey COS cell, a Chinese hamster ovary (CHO) cell (e.g., from a CHO GS System TM (Lonza) CHO cells), or myeloma cells that do not otherwise produce immunoglobulin, to achieve synthesis of anti-DDR1 antibodies in recombinant host cells.

[0246] In order to produce a complete antibody or antigen binding region, the VH or VL sequence in the scFv clone can be amplified with PCR primers, including VH or VL nucleotide sequences, restriction sites and flanking sequences protecting the restriction sites. Using cloning techniques known to those skilled in the art, the VH domain amplified by PCR can be cloned into a vector expressing a heavy chain constant region (e.g., human γ1 or human γ4 constant region), and the VL domain amplified by PCR can be cloned into a vector expressing a light chain constant region (e.g., human κ or λ constant region). In certain embodiments, the vector for expressing the VH or VL domain includes an EF-1a promoter, a secretory signal, a cloning site for the variable region, a constant region, and a selection marker such as neomycin. The VH and VL domains can also be cloned into a vector expressing an essential constant region. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those skilled in the art to produce a stable or transient cell line expressing a full-length antibody (e.g., IgG).

[0247] The DNA may also be modified, for example, by substituting coding sequences for human heavy and light chain constant regions in place of the murine sequences, or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0248] Also provided are polynucleotides that hybridize to polynucleotides encoding antibodies described herein under high stringency, medium or low stringency hybridization conditions. In embodiments, polynucleotides described herein hybridize to polynucleotides encoding VH domains and / or VL domains provided herein under high stringency, medium or low stringency hybridization conditions.

[0249] Hybridization conditions have been described in the art and are known to those skilled in the art. For example, hybridization under stringent conditions may involve hybridization to filter-bound DNA in 6x sodium chloride / sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2x SSC / 0.1% SDS at about 50-65°C; hybridization under high stringency conditions may involve hybridization to filter-bound nucleic acids in 6x SSC at about 45°C, followed by one or more washes in 0.1x SSC / 0.2% SDS at about 68°C. Hybridization under other stringent hybridization conditions is known to those skilled in the art and has been described in, for example, Ausubel FM et al., eds., (1989) Current Protocols in Molecular Biology, Vol. I, Green Publishing Associates, Inc. and John Wiley & Sons, Inc., New York, pp. 6.3.1-6.3.6 and 2.10.3, which is incorporated herein by reference in its entirety.

[0250] In one aspect, cells (e.g., host cells) expressing (e.g., recombinantly) antibodies described herein that specifically bind to DDR1, as well as related polynucleotides and expression vectors are provided herein. Vectors (e.g., expression vectors) comprising polynucleotides are provided herein, and the polynucleotides comprise nucleotide sequences encoding anti-DDR1 antibodies or fragments for recombinant expression in host cells, preferably mammalian cells (e.g., CHO cells). Host cells are also provided herein, comprising such vectors for recombinantly expressing anti-DDR1 antibodies (e.g., human or humanized antibodies) described herein. In one aspect, methods for producing antibodies described herein are provided herein, comprising expressing the antibodies from host cells.

[0251] The recombinant expression of antibodies (e.g., full-length antigen binding regions or antibodies or heavy and / or light chains of antibodies described herein) that specifically bind to DDR1 described herein generally involves constructing an expression vector comprising a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule, a heavy chain and / or light chain of an antibody, or a fragment thereof (e.g., a heavy chain and / or light chain variable region) described herein is obtained, a vector for producing the antibody molecule can be produced by recombinant DNA technology using techniques known in the art. Therefore, a method for preparing a protein by expressing a polynucleotide containing an antibody or antibody fragment (e.g., a light chain or heavy chain) encoding nucleotide sequence is described herein. Methods known to those skilled in the art can be used to construct an expression vector containing an antibody or antibody fragment (e.g., a light chain or heavy chain) encoding sequence and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic technology, and in vivo genetic recombination. A replicable vector comprising a nucleotide sequence is also provided, the nucleotide sequence encoding comprising an antibody molecule described herein, an antibody heavy chain or light chain, an antibody heavy chain or light chain variable region or a fragment thereof, or a heavy chain or light chain CDR that is operably connected to a promoter. For example, such a vector may comprise a nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Pat. No. 5,122,464, which are incorporated herein by reference in their entirety), and the variable region of the antibody may be cloned into such a vector to express the entire heavy chain, the entire light chain, or the entire heavy and light chains.

[0252] In an embodiment, the vector comprises a polynucleotide encoding the VH, VL, heavy chain and / or light chain of an antibody described herein. In another embodiment, the vector comprises a polynucleotide encoding the VH and VL of an antibody described herein. In another embodiment, the vector comprises a polynucleotide encoding the heavy chain and light chain of an antibody described herein.

[0253] The expression vector can be transferred to a cell (e.g., a host cell) by conventional techniques, and the resulting cell can then be cultured by conventional techniques to produce an antibody or fragment thereof as described herein. Thus, provided herein are host cells containing polynucleotides encoding an antibody or fragment thereof as described herein, or a heavy chain or light chain thereof, or a fragment thereof, or a single-chain antibody as described herein, operably linked to a promoter, to express these sequences in a host cell.

[0254] In an embodiment, the host cell comprises polynucleotides encoding the VH and VL of the isolated antibody described herein. In another embodiment, the host cell comprises a vector comprising polynucleotides encoding the VH and VL of the isolated antibody described herein. In another embodiment, the host cell comprises a first polynucleotide encoding the VH of the isolated antibody described herein, and a second polynucleotide encoding the VL of the isolated antibody described herein. In another embodiment, the host cell comprises a first vector and a second vector, the first vector comprising a first polynucleotide encoding the VH of the isolated antibody described herein, and the second vector comprising a second polynucleotide encoding the VL of the isolated antibody described herein.

[0255] In embodiments, the heavy chain / heavy chain variable region expressed by the first host cell combines with the light chain / light chain variable region of the second host cell to form an anti-DDR1 antibody as described herein. In embodiments, provided herein is a host cell population comprising such a first host cell and such a second host cell.

[0256] In an embodiment, provided herein is a vector population comprising a first vector comprising a polynucleotide encoding the light chain / light chain variable region of an anti-DDR1 antibody described herein and a second vector comprising a polynucleotide encoding the heavy chain / heavy chain variable region of an anti-DDR1 antibody described herein.

[0257] Various host-expression vector systems can be used to express the antibody molecules described herein (see, e.g., U.S. Pat. No. 5,807,715, which is incorporated herein by reference in its entirety). Such host-expression systems represent vehicles by which the coding sequences of interest can be produced and subsequently purified, but also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequences, can express the antibody molecules described herein in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., Escherichia coli and Bacillus subtilis) transformed with, for example, recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces and Pichia pastoris) transformed with, for example, recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with, for example, recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., green algae, such as Chlamydomonas reinhardtii) infected with, for example, recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with, for example, recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NSO, PER.C6, VERO, CRL7030, HsS78Bst, HeLa and NIH) carrying, for example, recombinant expression constructs. 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20 and BMT10 cells), the recombinant expression construct contains a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter), or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In an embodiment, the cell used to express the antibodies described herein is a Chinese hamster ovary (CHO) cell, e.g., from a CHO GS System TM In embodiments, the heavy and / or light chains of antibodies produced by CHO cells may have N-terminal glutamine or glutamic acid residues substituted with pyroglutamate. In embodiments, cells used to express antibodies described herein are human cells, e.g., human cell lines. In embodiments, the mammalian expression vector is pOptiVEC TMOr pcDNA3.3. In an embodiment, bacterial cells such as Escherichia coli or eukaryotic cells (e.g., mammalian cells) that are particularly used to express the entire recombinant antibody molecule are used to express the recombinant antibody molecule. For example, mammalian cells, such as CHO cells, combined with vectors such as the major intermediate early gene promoter element from human cytomegalovirus, are effective expression systems for antibodies (Foecking MK & Hofstetter H (1986) Gene 45: 101-5; and Cockett MI et al., (1990) Biotechnology 8 (7): 662-7, each of which is incorporated herein by reference in its entirety). In an embodiment, the antibodies described herein are produced by CHO cells or NS0 cells. In an embodiment, the expression of the nucleotide sequence encoding the antibody specifically binding to DDR1 described herein is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

[0258] In bacterial systems, various expression vectors can be advantageously selected according to the intended use of the expressed antibody molecule. For example, when a large amount of such antibodies are to be produced in order to produce a pharmaceutical composition of an antibody molecule, it is desirable to be easy to purify a vector for the expression of a high-level fusion protein product. Such vectors include, but are not limited to, Escherichia coli expression vector pUR278 (Ruether U & Mueller-Hill B (1983) EMBO J 2: 1791-1794), wherein the coding sequence can be connected to the vector alone in frame with the lac Z coding region, thereby producing a fusion protein; pIN vector (Inouye S & Inouye M (1985) Nuc Acids Res 13: 3101-3109; Van Heeke G & Schuster SM (1989) J Biol Chem 24: 5503-5509); etc., all of which are incorporated herein by reference in their entirety. For example, pGEX vectors can also be used as fusion proteins with glutathione 5-transferase (GST) for expressing exogenous polypeptides. In general, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to matrix glutathione agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

[0259] In insect systems, for example, Autographa californica nuclear polyhedrosis virus (AcNPV) can be used as a vector for expressing foreign genes. The virus grows in fall armyworm cells. The coding sequence can be cloned separately into a non-essential region of the virus (e.g., a polyhedrin gene) and placed under the control of an AcNPV promoter (e.g., a polyhedrin promoter).

[0260] In mammalian host cells, many virus-based expression systems can be used. In the case of using adenovirus as an expression vector, the target coding sequence can be connected to an adenovirus transcription / translation control complex, for example, a late promoter and a tripartite leader sequence. Then, this chimeric gene can be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region (e.g., E1 or E3 region) of the viral genome will produce a recombinant virus that is viable and capable of expressing the molecule in an infected host (see, e.g., Logan J & Shenk T (1984) PNAS 81 (12): 3655-9, which is incorporated herein by reference in its entirety). In order to effectively translate the inserted coding sequence, specific initiation signals may also be required. These signals include the ATG initiation codon and adjacent sequences. In addition, the initiation codon must be coordinated with the reading frame of the desired coding sequence to ensure translation of the entire insertion. These exogenous translation control signals and initiation codons can have various natural and synthetic sources. The expression efficiency can be improved by adding appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bitter G et al., (1987) Methods Enzymol. 153:516-544, which is incorporated herein by reference in its entirety).

[0261] In addition, host cell strains can be selected that regulate the expression of the inserted sequences or modify and process the gene products in a desired specific manner. Such modification (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may be important for the function of the protein. Different host cells have properties and specific mechanisms for post-translational processing and modification of proteins and gene products. Suitable cell lines or host systems can be selected to ensure the correct modification and processing of the expressed foreign proteins. To this end, eukaryotic host cells can be used that have cellular machinery for appropriate processing of primary transcripts, glycosylation, and phosphorylation gene products. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NS0 (a mouse myeloma cell line without any endogenous immunoglobulin chain production), CRL7030, COS (e.g., COS1 or COS), PER.C6, VERO, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10 and HsS78Bst cells. In an embodiment, the anti-DDR1 antibodies described herein are produced in mammalian cells such as CHO cells.

[0262] In embodiments, the antibodies described herein have reduced fucose content or are fucose-free. Such antibodies can be produced using techniques known to those skilled in the art. For example, the antibodies can be expressed in cells lacking or missing fucosylation ability. In an example, a cell line knocking out both alleles of α1,6-fucosyltransferase can be used to produce antibodies with reduced fucose content. The ANTIBODYNAMIC® system (Lonza) is an example of such a system, which can be used to generate antibodies with reduced fucose content.

[0263] In order to produce recombinant proteins in a long-term and high-yield manner, stable expressing cells can be generated. For example, a cell line that stably expresses an anti-DDR1 antibody described herein can be engineered. In embodiments, the cells provided herein stably express a light chain / light chain variable region and a heavy chain / heavy chain variable region that combine to form an antigen binding region, or an antibody described herein.

[0264] In some aspects, host cells can be transformed with DNA and selectable markers controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.), rather than using expression vectors containing viral replication initiation points. After the introduction of exogenous DNA / polynucleotides, engineered cells can be grown in enriched medium for one to two days and then switched to selection medium. The selectable markers in the recombinant plasmid confer resistance to selection and allow cells to stably integrate the plasmid into their chromosomes and grow to form foci, which in turn can be cloned and amplified into cell lines. The method can be advantageously used for engineered expression of cell lines of anti-DDR1 or its fragments described herein. Such engineered cell lines may be particularly useful in screening and evaluating compositions that interact directly or indirectly with the antibody molecule.

[0265] A variety of selection systems can be used, including, but not limited to, the herpes simplex virus thymidine kinase (Wigler M et al., (1977) Cell 11(1):223-32), hypoxanthine guanine phosphoribosyltransferase (Szybalska EH & Szybalski W (1962) PNAS 48(12):2026-2034), and adenine phosphoribosyltransferase (Lowy I et al., (1980) Cell 22(3):817-23) genes in tk-, hgprt-, or aprt- cells, respectively, all of which are incorporated herein by reference in their entirety. In addition, antimetabolite resistance can serve as the basis for selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler M et al., (1980) PNAS 77(6):3567-70; O'Hare K et al., (1981) PNAS 78:1527-31); gpt, which confers resistance to mycophenolic acid (Mulligan RC & Berg P (1981) PNAS 78(4):2072-6); neo, which confers resistance to the aminoglycoside G-418 (Wu GY & Wu CH (1991) Biotherapy 3:87-95; Tolstoshev P (1993) Ann Rev Pharmacol Toxicol 32:573-596; Mulligan RC (1993) Science 260:926-932; and Morgan GY et al., (1993) Ann Rev Pharmacol Toxicol 33:573-596; Mulligan RC (1993) Science 261:574-575; and Morgan GY et al., (1993) RA & Anderson WF (1993) Ann Rev Biochem 62: 191-217; Nabel GJ & Felgner PL (1993) Trends Biotechnol 11(5):211-5); and hygro, which confers resistance to hygromycin (Santerre RF et al., (1984) Gene 30(1-3): 147-56), all of which are incorporated herein by reference in their entirety.Methods well known in the art of recombinant DNA technology can be routinely applied to select the desired recombinant clones, and these methods are described in, for example, Ausubel FM et al., (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler M, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Dracopoli NC et al., (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994), Chapters 12 and 13; Colbere-Garapin F et al., (1981) J Mol Biol 150:1-14, all of which are incorporated herein by reference in their entirety.

[0266] The expression level of the antibody molecule can be increased by vector amplification (for review, see Bebbington CR & Hentschel CCG, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3 (Academic Press, New York, 1987), which is incorporated herein by reference in its entirety). When the marker in the vector system is amplifiable, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is associated with the target gene, the production of the protein will also increase (Crouse GF et al., (1983) Mol Cell Biol 3: 257-66, which is incorporated herein by reference in its entirety).

[0267] Host cells can be co-transfected with two or more expression vectors as described herein, the first vector encoding a polypeptide from a heavy chain source, and the second vector encoding a polypeptide from a light chain source. The two vectors can contain the same selection marker, which enables equal expression of heavy chain polypeptides and light chain polypeptides. Host cells can be co-transfected with two or more expression vectors of varying amounts. For example, host cells can be transfected with the first expression vector and the second expression vector in any of the following ratios: about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:12, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45 or 1:50.

[0268] Alternatively, a single vector encoding and capable of expressing heavy and light chain polypeptides can be used. In this case, the light chain should be placed before the heavy chain to avoid excessive non-toxic heavy chain (Proudfoot NJ (1986) Nature 322: 562-565; and Kohler G (1980) PNAS 77: 2197-2199, each of which is incorporated herein by reference in its entirety). The coding sequence of heavy and light chains can include cDNA or genomic DNA. The expression vector can be a monocistronic or polycistronic. Polycistronic nucleic acid constructs can encode 2, 3, 4, 5, 6, 7, 8, 9, 10 or more genes / nucleotide sequences, or 2 to 5, 5 to 10 or 10-20 genes / nucleotide sequences. For example, a bicistronic nucleic acid construct can include a promoter, a first gene (e.g., a heavy chain of an antibody described herein) and a second gene (e.g., a light chain of an antibody described herein) in the following order. In such an expression vector, transcription of both genes can be driven by the promoter, while translation of mRNA from the first gene can occur by a cap-dependent scanning mechanism, and translation of mRNA from the second gene can occur by a cap-independent mechanism, e.g., via an IRES.

[0269] Once the antibody molecules described herein have been produced by recombinant expression, they can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly affinity for specific antigens after protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for purifying proteins. Further, the antibodies described herein can be fused to heterologous polypeptide sequences described herein or other heterologous polypeptide sequences known in the art to facilitate purification.

[0270] In embodiments, antibodies as described herein are separated or purified. In embodiments, separated antibodies are antibodies that are substantially free of other antibodies that are different from the separated antibody antigen specificity. For example, in certain embodiments, the preparations of antibodies as described herein are substantially free of cellular material and / or chemical precursors. The language of "substantially free of cellular material" includes preparations of antibodies, wherein the antibodies are separated from the cellular components of the cells or recombinantly produced cells thereof. Therefore, antibodies substantially free of cellular material include antibody preparations with less than about 30%, 20%, 10%, 5%, 2%, 1%, 0.5% or 0.1% (by dry weight) of heterologous proteins (also referred to herein as "contaminating proteins") and / or antibody variants, such as, different post-translational modified forms of antibodies or other different versions of antibodies (e.g., antibody fragments). When recombinantly producing antibodies, it is also generally substantially free of culture medium, i.e., culture medium accounts for less than about 20%, 10%, 2%, 1%, 0.5% or 0.1% of the protein preparation volume. When the antibody is produced by chemical synthesis, it is generally substantially free of chemical precursors or other chemicals, i.e., it is separated from chemical precursors and other chemicals involved in protein synthesis. Therefore, such antibody preparations contain less than about 30%, 20%, 10% or 5% (by dry weight) of chemical precursors or compounds of non-target antibodies. In embodiments, the antibodies described herein are separated or purified.

[0271] Anti-DDR1 antibodies or fragments thereof can be produced by any method known in the art for synthesizing proteins or antibodies, for example, by chemical synthesis or by recombinant expression techniques. Unless otherwise indicated, the methods described herein employ conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related art techniques. These techniques are described, for example, in the references cited herein and are fully explained in the literature. See, for example, Maniatis T et al., (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press; Sambrook J et al., (1989), Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press; Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al. al., Current Protocols in Molecular Biology, John Wiley&Sons (1987and annual updates); Current Protocols in Immunology, John Wiley&Sons (1987and annual updates); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: APractical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press, all of which are incorporated herein by reference in their entirety.

[0272] In embodiments, the antibodies described herein are prepared, expressed, formed or separated by any method involving creation, for example, by synthesis of DNA sequences, genetic engineering. In certain embodiments, such antibodies comprise sequences (e.g., DNA sequences or amino acid sequences) that are not naturally present in the antibody germline repertoire in an animal or mammal (e.g., human).

[0273] In one aspect, provided herein is a method for preparing an anti-DDR1 antibody, comprising culturing a cell or host cell as described herein. In an embodiment, the method is performed in vitro. In one aspect, provided herein is a method for preparing an anti-DDR1 antibody, comprising expressing (e.g., recombinantly expressing) an antibody using a cell or host cell as described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody as described herein). In an embodiment, the cell is an isolated cell. In an embodiment, an exogenous polynucleotide has been introduced into a cell. In an embodiment, the method further comprises a step of purifying the antibody obtained from the cell or host cell.

[0274] In an embodiment, the isolated antibody is produced by expressing polynucleotides encoding the VH and VL of the antibodies described herein in a cell under appropriate conditions, thereby expressing the polynucleotides and producing the antibody. In another embodiment, the isolated antibody is produced by expressing polynucleotides encoding the heavy chain and light chain of the antibodies described herein in a cell under appropriate conditions, thereby expressing the polynucleotides and producing the antibody. In an embodiment, the isolated antibody is produced by expressing a first polynucleotide encoding the VH of the antibodies described herein and a second polynucleotide encoding the VL of the antibodies described herein in a cell under appropriate conditions, thereby expressing the polynucleotides and producing the antibody. In an embodiment, the isolated antibody is produced by expressing a first polynucleotide encoding the heavy chain of the antibodies described herein and a second polynucleotide encoding the light chain of the antibodies described herein in a cell under appropriate conditions, thereby expressing the polynucleotides and producing the antibody.

[0275] Methods for producing polyclonal antibodies are known in the art (see, e.g., Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York, Chapter 11, which is incorporated herein by reference in its entirety).

[0276] Monoclonal antibodies can be prepared using various techniques known in the art, including the use of hybridomas, recombinant and phage display techniques, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma technology, including techniques known in the art, and, for example, Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981) The techniques taught, each of which is incorporated herein by reference in its entirety. The term "monoclonal antibody" as used herein is not limited to antibodies produced by hybridoma technology. For example, monoclonal antibodies can be recombinantly produced by host cells that exogenously express antibodies described herein or fragments thereof (such as the light chain and / or heavy chain of the antibody).

[0277] In an embodiment, a "monoclonal antibody" as used herein is an antibody produced by a single cell (e.g., a hybridoma or host cell producing a recombinant antibody), wherein the antibody specifically binds to anti-DDR1 as determined by, for example, ELISA or other antigen binding or competitive binding assays known in the art or in the examples provided herein. In an embodiment, the monoclonal antibody may be a chimeric antibody or a humanized antibody. In an embodiment, the monoclonal antibody is a monovalent antibody or a multivalent (e.g., bivalent) antibody. In an embodiment, the monoclonal antibody is a monospecific or multispecific antibody (e.g., a bispecific antibody). The monoclonal antibodies described herein can be prepared, for example, by the hybridoma method as described in Kohler G&Milstein C (1975) Nature 256:495, which is incorporated herein by reference in its entirety, or can be separated from a phage library, for example, using the techniques described herein. Other methods for preparing clonal cell lines and monoclonal antibodies expressed thereby are well known in the art (see, e.g., Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., Chapter 11, supra).

[0278] As used herein, when an antibody comprises at least two (e.g., two or more) monovalent binding regions, the antibody is multivalent (e.g., bivalent) binding antigen, and each monovalent binding region is capable of binding an epitope on the antigen. Each monovalent binding region can bind to the same or different epitopes on the antigen.

[0279] The method of using hybridoma technology to produce and screen specific antibodies is conventional and well known in the art. For example, in the hybridoma method, mice or other suitable host animals, such as sheep, goats, rabbits, rats, hamsters or macaques, are immunized to induce lymphocytes that produce or can produce antibodies, and the antibodies will specifically bind to the protein (e.g., DDR1) used for immunity. Alternatively, lymphocytes can be immunized in vitro. Then, lymphocytes are fused with myeloma cells using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells (Goding JW (ed.), Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986), which is incorporated herein by reference in its entirety). In addition, RIMMS (multi-site repeated immunization) technology can be used for immunizing animals (Kilpatrick KE et al., (1997) Hybridoma 16: 381-9, which is incorporated herein by reference in its entirety).

[0280] In an embodiment, a mouse (or other animal, such as a rat, monkey, donkey, pig, sheep, hamster or dog) can be immunized with an antigen (e.g., DDR1), and once an immune response is detected, such as detection of antibodies specific for the antigen in mouse serum, the mouse spleen is harvested and splenocytes are isolated. The splenocytes are then fused to any suitable myeloma cells by known techniques, such as those available from the American Type Culture Collection. (Manassas, VA) to form hybridomas. Hybridomas are selected and cloned by limiting dilution. In an embodiment, lymph nodes of immunized mice are harvested and fused with NS0 myeloma cells.

[0281] The hybridoma cells thus prepared are seeded and grown in an appropriate culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of cells lacking HGPRT.

[0282] In embodiments, effective fusion, support selected antibody production cells to stably produce antibodies at high levels, and myeloma cells sensitive to culture media such as HAT culture media are used. In these myeloma cell lines, there are mouse myeloma lines, such as NS0 cell lines or cell lines derived from MOPC-21 and MPC-11 mouse tumors that can be obtained from the Salk Institute Cell Distribution Center (Salk Institute Cell Distribution Center) in San Diego, California, USA, and SP-2 or X63-Ag8.653 cells that can be obtained from the American Type Culture Collection (American Type Culture Collection) in Rockville, Maryland, USA. It is also described that human myeloma and mouse-human heteromyeloma cell lines are used to produce human monoclonal antibodies (Kozbor D (1984) J Immunol 133: 3001-5; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987, each of which is incorporated herein by reference in its entirety).

[0283] The culture medium in which the hybridoma cells are grown is assayed for production of monoclonal antibodies directed against DDR1. The binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by methods known in the art, for example, immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0284] After identifying hybridoma cells that produce antibodies of the desired specificity, affinity and / or activity, the clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding JW (ed.), Monoclonal Antibodies: Principles and Practice, supra). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 culture media. In addition, the hybridoma cells can be grown in animals as ascites tumors.

[0285] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0286] The antibodies described herein include, for example, antibody fragments that recognize DDR1 and can be produced by any technique known to those skilled in the art. For example, the Fab and F(ab')2 fragments described herein can be produced by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (producing Fab fragments) or pepsin (producing F(ab')2 fragments). The Fab fragment corresponds to one of the two identical arms of the antibody molecule and contains a complete light chain paired with the VH and CH1 domains of the heavy chain. The F(ab')2 fragment contains two antigen binding arms of the antibody molecule connected by a disulfide bond in the hinge region.

[0287] Further, antibodies described herein can also be produced using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles carrying polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., cDNA libraries of affected tissues of humans or mice). DNA encoding VH and VL domains are recombined with scFv linkers by PCR and cloned into phagemid vectors. The vector enters Escherichia coli by electroporation, and the Escherichia coli is infected by helper phage. The phage used in these methods is generally filamentous phage, including fd and M13, and VH and VL domains are generally recombinantly fused with phage gene III or gene VIII. Phages expressing antigen binding regions that bind to specific antigens can be selected or identified with antigens, for example, using labeled antigens or antigens that are bound or captured to solid surfaces or beads. Examples of phage display methods that can be used to prepare the antibodies described herein include those disclosed in Brinkman U et al., (1995) J Immunol Methods 182:41-50; Ames RS et al., (1995) J Immunol Methods 184:177-186; Kettleborough CA et al., (1994) Eur J Immunol 24:952-958; Persic L et al., (1997) Gene 187:9-18; Burton DR & Barbas CF (1994) Advan Immunol 57:191-280; WO 90 / 02809; WO 91 / 10737; WO 92 / 01047; WO 92 / 18619; WO 93 / 11236; WO 95 / 15982; WO 95 / 20401; and WO 97 / 13844; and U.S. Patents 5,698,426; 5,223,409; 5,403,484; 5,427,908; 5,516,637; 5,571,698; 5,580,717; 5,658,727; 5,733,743; 5,780,225; 5,821,047; and 5,969,108, all of which are incorporated herein by reference in their entirety.

[0288] As described in the above references, after phage selection, the antibody coding regions from the phage can be isolated and used to produce complete antibodies, including human antibodies or any other desired antigen-binding fragments, and expressed in any desired host, including, for example, mammalian cells, insect cells, plant cells, yeast and bacteria, as described below. Techniques for recombinant production of antibody fragments such as Fab, Fab' and F(ab')2 fragments can also use methods known in the art, such as those disclosed in PCT Publication No. WO 92 / 22324; Mullinax RL et al., (1992) BioTechniques 12(6):864-9; Sawai H et al., (1995) Am J Reprod Immunol 34:26-34; and Better M et al., (1988) Science 240:1041-1043, all of which are incorporated herein by reference in their entirety.

[0289] In certain embodiments, to produce complete antibodies, PCR primers, including VH or VL nucleotide sequences, restriction sites, and flanking sequences protecting the restriction sites can be used to amplify VH or VL sequences from templates such as scFv clones. Using cloning techniques known to those skilled in the art, PCR-amplified VH domains can be cloned into vectors expressing VH constant regions, and PCR-amplified VL domains can be cloned into vectors expressing VL constant regions such as human kappa or lambda constant regions. VH and VL domains can also be cloned into a vector expressing the necessary constant regions. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those skilled in the art to produce a stable or transient cell line expressing a full-length antibody such as IgG.

[0290] A chimeric antibody is a molecule in which different parts of an antibody are derived from different immunoglobulin molecules. For example, a chimeric antibody may contain a variable region of a non-human mammalian (e.g., mouse, rat, rabbit, etc.) monoclonal antibody fused to a constant region of a human antibody. Methods for producing chimeric antibodies are known in the art. See, e.g., Morrison SL (1985) Science 229: 1202-7; Oi VT & Morrison SL (1986) Bio Techniques 4: 214-221; Gillies SD et al., (1989) J Immunol Methods 125: 191-202; and U.S. Pat. Nos. 4,816,397, 4,816,567, 5,807,715 and 6,331,415, all of which are incorporated herein by reference in their entirety.

[0291] Humanized antibodies can bind to a predetermined antigen, and they include a framework region that has substantially the amino acid sequence of a human immunoglobulin, and a CDR that has substantially the amino acid sequence of a non-human immunoglobulin (e.g., mouse immunoglobulin). In certain embodiments, humanized antibodies also include at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin constant region. The antibody may also include CH1, hinge, CH2, CH3, and CH4 regions of a heavy chain. Humanized antibodies may be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including IgG1, IgG2, IgG3, and IgG4.Humanized antibodies can be produced using various techniques known in the art, including, but not limited to, CDR grafting (European Patent No. EP239400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5225539, 5530101, and 5585089), veneering or resurfacing (European Patent Nos. EP592106 and EP519596; Padlan EA (1991) Mol Immunol 28(4 / 5):489-498; Studnicka GM et al., (1994) Prot Engineering 7(6):805-814; and Roguska MA et al., (1994) PNAS 91:969-973), chain displacement (U.S. Pat. No. 5565332), and the techniques disclosed in, for example, U.S. Pat. Nos. 576686 and 6407213, WO International Publication No. 93 / 17105; Tan P et al., (2002) J lmmunol 169:1119-25; Caldas C et al., (2000) Protein Eng.13(5):353-60; MoreaV et al., (2000) Methods20(3):267-79; Baca M et al. al., (1997) J Biol Chem 272(16):10678-84; Roguska MA et al., (1996) Protein Eng 9(10):895 904; Couto JR et al., (1995) Cancer Res. 55(23Supp):5973s-5977s; Couto JR et al. al., (1995) Cancer Res55(8):1717-22; Sandhu JS (1994) Gene 150 (2): 409-10; and Pedersen JT et al., (1994) J Mol Biol 235 (3): 959-73, all of which are incorporated herein by reference in their entirety. See also, U.S. Patent Application Publication No. US2005 / 0042664A1, which is incorporated herein by reference in its entirety.

[0292] Methods for making multispecific antibodies (e.g., bispecific antibodies) have been described, see, e.g., U.S. Pat. Nos. 5,837,242; 5,869,620; 5,989,830; 6,132,992; 7,183,076; 7,951,917; 8,227,577, all of which are incorporated herein by reference in their entirety.

[0293] Bispecific, bivalent antibodies and methods for their preparation are described in, for example, U.S. Pat. Nos. 5,731,168, 5,807,706, and 5,821,333, and U.S. Patent Application Publication No. 2002 / 0155537, each of which is incorporated herein by reference in its entirety. Bispecific tetravalent antibodies and methods for their preparation are described in, for example, International Publications Nos. WO 02 / 096948 and WO 00 / 44788, the disclosures of which are incorporated herein by reference in their entirety. See generally, International Publication Nos. WO 91 / 00360; WO 92 / 08802; WO 92 / 05793; and WO 93 / 17715; Tutte et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; and 5,601,819; and Kostelny et al., J. Immunol. 148:1547-1553 (1992); each of which is incorporated herein by reference in its entirety.

[0294] The bispecific antibodies described herein can be produced according to the DuoBody technology platform (Genmab A / S), described in, for example, WO 2008 / 119353; WO 2011 / 131746; WO 2011 / 147986; and International Publication No. WO 2013 / 060867; and Labrijn AF et al., (2013) PNAS 110(13):5145-5150. DuoBody technology can be used to combine a first monospecific antibody or half of a first antigen-binding region containing two heavy chains and two light chains with a second monospecific antibody or half of a second antigen-binding region containing two heavy chains and two light chains. The resulting heterodimer contains one heavy chain and one light chain from the first antibody or first antigen-binding region, paired with one heavy chain and one light chain from the second antibody or second antigen-binding region. When two monospecific antibodies or antigen-binding regions recognize different epitopes on different antigens, the resulting heterodimer is a bispecific antibody.

[0295] DuoBody technology requires that each monospecific antibody or antigen-binding region comprises a heavy chain constant region with a single point mutation in the CH3 domain. The point mutation allows for stronger interactions between the CH3 domains in the resulting bispecific antibody than between the CH3 domains in any one of the monospecific antibodies or antigen-binding regions. The single point mutation in each monospecific antibody or antigen-binding region is located at residues 366, 368, 370, 399, 405, 407 or 409 numbered according to the EU numbering system in the CH3 domain of the heavy chain constant region, such as described in International Publication No. WO 2011 / 131746. In addition, the single point mutation is located at a different residue in one monospecific antibody or antigen-binding region compared to another monospecific antibody or antigen-binding region. For example, one monospecific antibody or antigen-binding region may comprise a mutation F405L (i.e., a mutation of phenylalanine to leucine at residue 405), while another monospecific antibody or antigen-binding region may comprise a mutation K409R (i.e., a mutation of lysine to arginine at residue 409), according to the EU numbering system. The heavy chain constant region or antigen-binding region of the monospecific antibody may be of the IgG1, IgG2, IgG3, or IgG4 isotype (e.g., human IgG1 isotype), and the bispecific antibodies produced by the DuoBody technology may retain Fc-mediated effector functions.

[0296] Another method for producing bispecific antibodies is called the "knobs-into-holes" strategy (see, e.g., International Publication No. WO 2006 / 028936). This technique reduces mispairing of Ig heavy chains by mutating selected amino acids that form the interface of the CH3 domain in IgG. At the position where the two heavy chains directly interact within the CH3 domain, amino acids with small side chains (holes) are introduced into the sequence of one heavy chain, and amino acids with large side chains (knobs) are introduced into the corresponding interacting residue positions on the other heavy chain. In some embodiments, the compositions of the present invention have immunoglobulin chains in which the CH3 domain is modified by mutating selected amino acids that interact at the interface between the two polypeptides, thereby preferentially forming bispecific antibodies. The bispecific antibodies can be composed of immunoglobulin chains of the same subclass (e.g., IgG1 or IgG3) or different subclasses (e.g., IgG1 and IgG3, or IgG3 and IgG4).

[0297] In some cases, the bispecific antibody may comprise IgG4 and IgG1, IgG4 and IgG2, IgG4 and IgG2, IgG4 and IgG3, or IgG1 and IgG3 chain heterodimers. These heterodimeric heavy chain antibodies can generally be engineered by, for example, modifying selected amino acids that form the interface of the CH3 domains in human IgG4 and IgG1 or IgG3 to facilitate the formation of heterodimeric heavy chains.

[0298] In an embodiment, the antibodies described herein that bind to the same DDR1 epitope as the anti-DDR1 antibodies described herein are human antibodies. In an embodiment, the antibodies described herein that competitively block (e.g., in a dose-dependent manner) any of the antibodies described herein that bind to DDR1 are human antibodies. Human antibodies can be produced using any method known in the art. For example, transgenic mice that cannot express functional endogenous immunoglobulins but can express human immunoglobulin genes can be used. In particular, human heavy chain and light chain immunoglobulin gene complexes can be introduced into mouse embryonic stem cells randomly or by homologous recombination. Alternatively, in addition to human heavy chain and light chain genes, human variable regions, constant regions, and diversity regions can be introduced into mouse embryonic stem cells. Mouse heavy chain and light chain immunoglobulin genes can lose function alone or at the same time as the introduction of human immunoglobulin sites by homologous recombination. In particular, J HHomozygous deletion of the region prevents the production of endogenous antibodies. Modified embryonic stem cells are amplified and microinjected into blastocysts to produce chimeric mice. Chimeric mice are then cultivated to produce homozygous offspring expressing human antibodies. Transgenic mice are immunized with selected antigens in a normal manner, such as all or part of an antigen (e.g., DDR1). Using conventional hybridoma technology, monoclonal antibodies against the antigen can be obtained from immunized transgenic mice. The human immunoglobulin transgene carried by transgenic mice is rearranged during B cell differentiation, and class switching and somatic mutations occur subsequently. Therefore, it is possible to use this technology to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg N & Huszar D (1995) Int Rev Immunol 13: 65-93, which is incorporated herein by reference in its entirety. For a detailed discussion of the use of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing these antibodies, see, e.g., International Publication Nos. WO 96 / 33735; WO 96 / 34096; and WO 98 / 24893; and U.S. Pat. Nos. 5,413,923; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016; 5,814,318; and 5,939,598, all of which are incorporated herein by reference in their entirety. Examples of mice capable of producing human antibodies include the XenoMouse. TM (Abgenix, Inc.; U.S. Patent Nos. 6,075,181 and 6,150,184), HuAb-Mouse TM (Medarex, Inc. / GenPharm; U.S. Patents 5,545,806 and 5,569,825), TransChromo Mouse TM (Kirin) and KM Mouse TM (Medarex / Kirin), all of which are incorporated herein by reference in their entirety.

[0299] Human antibodies that specifically bind to DDR1 can be prepared by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences as described above. See also, U.S. Pat. Nos. 4,444,887; 4,716,111; and 5,885,793; and WO 91 / 10741; WO 96 / 34096; WO 96 / 33735; WO 98 / 16654; WO 98 / 24893; WO 98 / 46645; and WO 98 / 50433, all of which are incorporated herein by reference in their entirety.

[0300] In certain embodiments, mouse-human hybridomas can be used to produce human antibodies. For example, human peripheral blood lymphocytes transformed with Epstein-Barr virus (EBY) can be fused with mouse myeloma cells to produce mouse-human hybridomas that secrete human monoclonal antibodies, and these mouse-human hybridomas can be screened to determine the hybridomas that secrete human monoclonal antibodies that specifically bind to target antigens (e.g., DDR1). These methods are known and described in the art, see, e.g., Shinmoto H et al., (2004) Cytotechnology 46: 19-23; Naganawa Y et al., (2005) Human Antibodies 14: 27-31, each of which is incorporated herein by reference in its entirety.

[0301] 7.5 Usage

[0302] In one aspect, the present disclosure provides a method for monitoring the effectiveness of an anti-DDR1 antibody in a subject in need thereof. In one embodiment, the method comprises administering an effective amount of the anti-DDR1 antibody to the subject, and detecting the level of DDR1 phosphorylation in a sample from the subject. In some embodiments, the anti-DDR1 antibody comprises an anti-DDR1 antibody disclosed herein or a nucleic acid encoding an anti-DDR1 antibody. In some embodiments, the anti-DDR1 antibody is administered by a suitable route. Non-limiting examples of suitable routes of administration include intravenous, oral, parenteral, ocular, pulmonary, and topical administration.

[0303] In some embodiments, the DDR1 phosphorylation level comprises the proportion of the total DDR1 in the sample that is phosphorylated at one or more phosphorylation sites. In one embodiment, the one or more phosphorylation sites comprise a tyrosine residue. In one embodiment, the tyrosine residue is a tyrosine residue that is known to be autophosphorylated in response to stimulation of the extracellular portion of DDR1. In one embodiment, the tyrosine residue is a tyrosine residue that is known to be autophosphorylated by one or more collagens in response to stimulation of the extracellular portion of DDR1. In some embodiments, the DDR1 phosphorylation level comprises the absolute amount of phosphorylated DDR1 protein in the sample. In some embodiments, the DDR1 phosphorylation level comprises the absolute amount of phosphorylated DDR1 sites in the sample.

[0304] In some embodiments, the DDR1 phosphorylation level includes the ratio of the sheared form of DDR1 phosphorylated on one or more phosphorylation sites in the sample. In some embodiments, the sheared form of DDR1 has a lower molecular weight relative to the unsheared form. In some embodiments, the sheared form has a molecular weight of less than 125kDa (e.g., less than 120kDa, less than 115kDa, less than 110kDa, less than 105kDa, less than 100kDa, less than 95kDa, less than 90kDa, less than 85kDa, less than 80kDa, less than 75kDa, less than 70kDa, less than 65kDa, less than 60kDa, less than 55kDa, less than 50kDa, less than 45kDa, less than 40kDa, less than 35kDa, less than 30kDa, less than 25kDa, less than 20kDa, less than 15kDa, less than 10kDa, or less than 5kDa). In some embodiments, the sheared form has a molecular weight of about 60kDa.

[0305] Technicians will recognize that any method for detecting DDR1 phosphorylation can be used together with the method of the present invention. Various methods for detecting phosphorylation are known in the art, including but not limited to radioisotope labeling, mass spectrometry, immunoassays (e.g., immunoblotting, enzyme-linked immunosorbent assay, intracellular flow cytometry, etc.) using phosphorylation-specific antibodies. Various antibodies specific to phosphorylated DDR1 are readily available and known in the art. Exemplary phosphorylation-specific DDR1 antibodies include but are not limited to phosphorylated DDR1 (Tyr513) (E1N8F) rabbit mAb #14531 (Cell Signaling Technology), phosphorylated DDR1 (Tyr792) antibody #11994 (Cell Signaling Technology), phosphorylated DDR1 (Tyr796) polyclonal antibody phosphorylated DDR1 (Tyr796) polyclonal antibody PA5-106123 (Thermo Fisher Scientific) and anti-phosphorylated DDR1 (pTyr513) SAB4504671 (MilliporeSigma).

[0306] The skilled person will further recognize that any method of preparing a sample for detecting DDR1 phosphorylation can be used in the method of the present invention. As a non-limiting example, cells or tissues can be lysed by physical (e.g., ultrasound) and / or chemical (e.g., surfactant) means and treated to remove cell debris (e.g., centrifugation). In some embodiments, one or more phosphatase inhibitors are included in the sample to prevent premature dephosphorylation of DDR1. In some embodiments, one or more protease inhibitors are included in the sample to prevent premature degradation of DDR1.

[0307] In some embodiments, the subject in need has an elevated DDR1 phosphorylation level compared to a reference sample. In some embodiments, the DDR1 phosphorylation level is elevated by at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 300%, or 400% compared to a reference sample. In some embodiments, the DDR1 phosphorylation level is elevated by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times compared to a reference sample. In some embodiments, the elevated DDR1 level is associated with a disease or disorder. In some embodiments, the elevated DDR1 level is a direct result of a disease or disorder. In some embodiments, compared with a reference sample, an increase in DDR1 phosphorylation level of at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 300% or 400% indicates a disease or disorder. In some embodiments, compared with a reference sample, an increase in DDR1 phosphorylation level of at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times or 15 times indicates a disease or disorder. In some embodiments, the disease or disorder is associated with an increased expression level of DDR1 (e.g., cancer, fibrosis, etc.). In some embodiments, the disease or disorder is associated with an increased binding of DDR1 to collagen. In some embodiments, the disease or disorder is associated with an increased phosphorylation level of DDR1. In some embodiments, the disease or disorder is associated with an increased expression level of DDR1, binding to collagen and / or an increased phosphorylation level of DDR1.

[0308] In one embodiment, a decrease in DDR1 phosphorylation in a sample from the subject compared to a reference sample indicates that the administration of the anti-DDR1 antibody is effective. In one embodiment, a decrease in DDR1 phosphorylation in a sample from the subject compared to a reference sample indicates that the administration of the anti-DDR1 antibody is effective for treating a disease or disorder associated with elevated levels of DDR1 phosphorylation. In one embodiment, a decrease in DDR1 phosphorylation in a sample from the subject compared to a reference sample of at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75% or 100% indicates that the administration of the anti-DDR1 antibody is effective for treating a disease or disorder associated with elevated levels of DDR1 phosphorylation. In one embodiment, a decrease in DDR1 phosphorylation in a sample from the subject of at least 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold or 0.1-fold indicates that the administration of the anti-DDR1 antibody is effective for treating a disease or disorder associated with elevated levels of DDR1 phosphorylation.

[0309] In one aspect, the present disclosure provides a method for treating a DDR1-related disorder in a subject. In one embodiment, the method comprises administering an effective amount of an anti-DDR1 antibody to the subject, and detecting the level of DDR1 phosphorylation in a sample from the subject and a reference sample. In some embodiments, a reduction in DDR1 phosphorylation in a sample from the subject, as compared to the reference sample, indicates that treatment is effective. In one embodiment, a reduction in DDR1 phosphorylation in a sample from the subject, as compared to the reference sample, of at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75% or 100%, indicates that administration of the anti-DDR1 antibody is effective for treating the DDR1-related disease or disorder. In one embodiment, a reduction in DDR1 phosphorylation in a sample from the subject, as compared to the reference sample, of at least 0.9-fold, 0.8-fold, 0.7-fold, 0.6-fold, 0.5-fold, 0.4-fold, 0.3-fold, 0.2-fold or 0.1-fold indicates that administration of the anti-DDR1 antibody is effective for treating the DDR1-related disease or disorder.

[0310] In one embodiment, the method comprises detecting the level of DDR1 phosphorylation in a sample from a subject, and administering an effective amount of an anti-DDR1 antibody to the subject if DDR1 phosphorylation is higher in the sample from the subject compared to a reference sample. In one embodiment, an effective amount of an anti-DDR1 antibody is administered if DDR1 phosphorylation in the subject sample is at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 300%, or 400% higher than a reference sample. In one embodiment, an effective amount of an anti-DDR1 antibody is administered if DDR1 phosphorylation in the subject sample is at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, or 15-fold higher than a reference sample.

[0311] In one aspect, the present disclosure provides a method for screening an object with a DDR1-related disorder that may be effectively treated with an anti-DDR1 antibody. In one embodiment, the method comprises detecting the level of DDR1 phosphorylation in a sample from the object, wherein if the DDR1 phosphorylation in the sample from the object is higher than that in a reference sample, the DDR1-related disorder may be effectively treated with an anti-DDR1 antibody. In some embodiments, if the DDR1 phosphorylation in the object sample is at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 75%, 100%, 150%, 200%, 300% or 400% higher than the reference sample, the DDR1-related disorder may be effectively treated by anti-DDR1. In some embodiments, if the DDR1 phosphorylation in the object sample is at least 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times or 15 times higher than the reference sample, the DDR1-related disorder may be effectively treated by anti-DDR1. In some embodiments, the likelihood of effectively treating a DDR1-related disorder comprising elevated levels of DDR1 phosphorylation by an anti-DDR1 antibody is 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times greater than a therapeutic agent that non-specifically targets DDR1. In some embodiments, the likelihood of effectively treating a DDR1-related disorder comprising elevated levels of DDR1 phosphorylation by an anti-DDR1 antibody is 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or 10 times greater than an antibody that non-specifically targets DDR1.

[0312] In various embodiments of the methods described herein, the object sample or the reference sample includes object cells, tissues, biological fluids or derivatives thereof. In some embodiments, the cells include blood cells, skin cells, cancer cells or cells derived from fibrotic tissue. In some embodiments, the blood cells include red blood cells, white blood cells or platelets. In some embodiments, the white blood cells include monocytes, lymphocytes, neutrophils, eosinophils, basophils or macrophages. In some embodiments, the tissue includes skin tissue, cancer tissue or fibrotic tissue. In some embodiments, skin tissue is collected by skin puncture biopsy. In some embodiments, the biological fluid includes blood (e.g., whole blood, plasma, serum, etc.), saliva or sputum. In some embodiments, the object or the derivative of the reference cell or tissue is a lysate. In some embodiments, the object or the derivative of the reference biological fluid is an isolate.

[0313] In various embodiments of the methods described herein, the phosphorylation level of DDR1 in the sample of the object is compared with the phosphorylation level of DDR1 in the reference sample. Non-limiting examples of reference samples include, but are not limited to, negative controls, positive controls, standard controls, standard values, expected normal background values ​​of the object, historical normal background values ​​of the object, reference standards, reference levels, expected normal background values ​​of the population of which the object is a member, or historical normal background values ​​of the population of which the object is a member. In some embodiments, the reference sample includes a sample of a healthy individual. In some embodiments, the reference sample includes a sample of a healthy individual obtained after successful treatment of a DDR1-related disease or disorder. In some embodiments, the reference sample includes a sample of a healthy individual with no known history of a DDR1-related disease or disorder. In some embodiments, the reference sample includes a sample of an object. In some embodiments, the reference sample includes a sample of an object obtained before suffering from a DDR1-related disease or disorder. In some embodiments, the reference sample includes a sample of an object obtained after suffering from a DDR1-related disease or disorder.

[0314] In one aspect, the present disclosure provides a method for screening anti-DDR1 antibodies. In some embodiments, the method comprises administering an effective amount of an anti-DDR1 antibody to a cell, and detecting the level of DDR1 phosphorylation in the cell. In one embodiment, the method comprises screening an anti-DDR1 antibody that is effective in treating a DDR1-related disorder, wherein a decrease in DDR1 phosphorylation in the cell compared to a reference cell indicates that the anti-DDR1 antibody is effective in treating cancer. In another embodiment, the method comprises screening an anti-DDR1 antibody that is effective in reducing the interaction of collagen with a cell, wherein a decrease in DDR1 phosphorylation in the cell compared to a reference cell indicates that the anti-DDR1 antibody is effective in reducing the interaction of collagen with the cell. Exemplary collagen types include, but are not limited to, collagen I, collagen II, collagen III, collagen IV, collagen V, collagen VI, collagen VII, collagen VIII, collagen IX, collagen X, collagen XI, collagen XII, collagen XIII, collagen XIV, collagen XV, collagen XVI, collagen XVII, collagen XVIII, collagen XIX, collagen XX, collagen XXI, collagen XXII, collagen XXIII, collagen XXIV, collagen XXV, collagen XXVI, collagen XXVII, and collagen XXVIII. In some embodiments, the collagen type includes collagen I, collagen II, collagen III, or collagen V.

[0315] In various embodiments of the methods described herein, the disease or disorder associated with increased DDR1 phosphorylation (i.e., the DDR1-associated disease or disorder) includes cancer. Exemplary cancer tissues that may be associated with increased DDR1 phosphorylation include, but are not limited to, cancer or cancer cells of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, intestine, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, pancreas, testicles, tongue, cervix, or uterus.

[0316] Exemplary histological types of cancers that may be associated with elevated DDR1 phosphorylation include, but are not limited to, malignant tumors; carcinomas; undifferentiated carcinomas; giant and spindle cell carcinomas; small cell carcinomas; papillary carcinomas; squamous cell carcinomas; epithelial lymphocytic carcinomas; basal cell carcinomas; pilomatricomas; transitional cell carcinomas; papillary transitional cell carcinomas; adenocarcinomas; malignant gastrinomas; cholangiocarcinomas; hepatocellular carcinomas; combined hepatocellular and cholangiocarcinomas; trabecular adenocarcinomas; adenoid cystic carcinomas; adenocarcinomas in adenomatous polyps; adenocarcinomas in familial polyposis; solid carcinoma; malignant carcinoid tumor; bronchioloalveolar adenocarcinoma; papillary adenocarcinoma; chromophobe cell carcinoma; oncocytic carcinoma; oncocytic adenocarcinoma; basophilic cell carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulated sclerosing carcinoma; adrenocortical carcinoma; endometrial carcinoma; skin appendage carcinoma; sweat gland adenocarcinoma; sebaceous gland carcinoma; cerumen gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; bookmark ring cell carcinoma; invasive ductal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the breast; acinar cell carcinoma; adenosquamous cell carcinoma; adenocarcinoma with squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; malignant testicular blastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; glomus sarcoma; malignant melanoma Pigmented tumor; Amelanotic melanoma; Superficial spreading melanoma; Malignant melanoma in giant pigmented nevus; Epithelioid cell melanoma; Malignant blue nevus; Sarcoma; Fibrosarcoma; Malignant fibrous histiocytoma; Myxosarcoma; Liposarcoma; Leiomyosarcoma; Rhabdomyosarcoma; Embryonic rhabdomyosarcoma; Alveolar rhabdomyosarcoma; Stromal sarcoma; Malignant mixed tumor; Mullerian mixed tumor; Wilms tumor; Hepatoblastoma; Carcinosarcoma; Malignant mesenchymal tumor; Malignant Br enner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant ovarian thyroid tumor; choriocarcinoma; malignant mesonephric duct tumor; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangiopericytoma; lymphangiosarcoma; osteosarcoma; paracortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma; ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroglioma; primitive neuroectodermal tumor; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor;Malignant lymphoma; Hodgkin's disease; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specific non-Hodgkin's lymphoma; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative enteropathy; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocyte leukemia; myeloid sarcoma; and hairy cell leukemia. In some aspects, the tumor can include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing's sarcoma, glioblastoma, neuroblastoma, or leukemia. ;

[0317] In various embodiments of the methods described herein, the disease or disorder associated with increased DDR1 phosphorylation (i.e., the DDR1-associated disease or disorder) comprises a fibrotic condition. In some embodiments, the fibrotic condition comprises organ fibrosis. In some embodiments, the fibrotic condition comprises fibrosis of the skin, kidney, liver, lung, or heart. In some embodiments, the fibrotic condition comprises hypertrophic scarring of the skin, scleroderma, lung scarring, interstitial lung disease, idiopathic pulmonary fibrosis, cirrhotic liver fibrosis, or renal fibrosis.

[0318] 7.6 Kit

[0319] Also provided is a kit including one or more antibodies described herein or its pharmaceutical composition or conjugate. In an embodiment, provided herein is a drug package or a kit, which includes one or more containers containing one or more components of a pharmaceutical composition described herein, such as one or more antibodies provided herein. In an embodiment, the kit includes a pharmaceutical composition as described herein and any preventive or therapeutic agent, such as those described herein. In an embodiment, the kit may include a T cell mitogen, such as, for example, phytohemagglutinin (PHA) and / or phorbol ester (PMA), or a TCR complex stimulating antibody, such as an anti-CD3 antibody and an anti-CD28 antibody. Alternatively, associated with such containers may be a statement in a form prescribed by a government agency regulating the manufacture, use or sale of drugs or biological products for humans, which reflects approval by a government agency regulating manufacture, use or sale.

[0320] Also provided are kits that can be used for the above methods. In an embodiment, the kit includes an antibody described herein, preferably a purified antibody, in one or more containers. In an embodiment, the kit described herein includes a substantially isolated DDR1 antigen as a control. In an embodiment, the kit described herein further includes a control antibody that does not react with the DDR1 antigen. In an embodiment, the kit described herein includes one or more elements for detecting the binding of an antibody to a DDR1 antigen (e.g., the antibody can be coupled to a detectable substrate, such as a fluorescent compound, an enzymatic substrate, a radioactive compound, or a luminescent compound, or a second antibody that recognizes the first antibody can be coupled to a detectable substrate). In an embodiment, the kit provided herein may include a recombinantly produced or chemically synthesized DDR1 antigen. The DDR1 antigen provided in the kit may also be attached to a solid support. In an embodiment, the detection tool of the above kit includes a solid support to which the DDR1 antigen is attached. Such a kit may also include a non-attached reporter-labeled anti-human antibody or anti-mouse / rat antibody. In this embodiment, the binding of the antibody to the DDR1 antigen can be detected by the binding of the reporter-labeled antibody. In certain embodiments, the present invention relates to the use of the kit of the present invention for in vitro determination and / or detection of DDR1 antigens in biological samples. In embodiments, the kit provided herein includes one or more antibodies capable of specifically detecting one or more phosphorylation sites of DDR1. In embodiments, the kit provided herein includes one or more biomolecules (e.g., collagen) capable of binding and / or stimulating DDR1 phosphorylation in cells. In embodiments, the kit provided herein includes reference samples and / or cells as described herein. 8. Examples

[0321] The examples in this section (ie, Section 8) are provided by way of illustration and not limitation.

[0322] 8.1 Example 1: In vitro characterization of anti-DDR1 monoclonal antibodies (mAbs)

[0323] Anti-DDR1 mAbs were tested for their ability to inhibit collagen-induced DDR1 phosphorylation, their effects on cell proliferation and cell death in cultured cancer cells, and their effects on collagen-dependent adhesion of DDR1-overexpressing cells.

[0324] A. Inhibition of collagen-induced DDR1 phosphorylation in T47D cells

[0325] Cultured T47D breast cancer cells were serum starved for 16 hours, pretreated with increasing concentrations of 9H1-WT or IgG1-WT (as a negative control) monoclonal antibody for 2 hours, and then treated with 50 μg / ml human or rat collagen I for 90 minutes. Cells treated with 200 nM 2.45-IN [2-(4-bromo-2-oxo-l'-(1H-pyrazolo[4,3-b]pyridine-5-carbonyl)spiro[indole-3,4'-piperidin]-1-yl)-N-(2,2,2-trifluoroethyl)acetamide] were used as a positive control for inhibition of DDR1 phosphorylation. Cell lysates were then analyzed by Jess immunoassay (ProteinSimple). As shown in Figure 1, 9H1-WT was able to inhibit human collagen I ( Figure 1A and Figure 1C ) and rat collagen I ( Figure 1B and Figure 1D )-induced DDR1 phosphorylation.

[0326] B. Effects of 9H1-WT and Ab#33 mAb on cell proliferation and cell death

[0327] To evaluate the potential cell growth inhibition or cytotoxic effects of 9H1-WT and Ab#33 mAbs, serum-starved T47D cells were pretreated with 9H1-WT or Ab#33, their corresponding IgG controls, or the known chemotherapeutic agent paclitaxel (as a positive control for cytotoxicity) and chased for 96 hours. To measure proliferation, Hoechst staining was used to stain cell nuclei, and CellMask TM (Thermo Fisher Scientific) was used to stain cell membranes. To measure cell death, Annexin V was used to stain cell surface phosphatidylserine (a marker of apoptosis), and Cytoxin Green (Sartorius) was used to stain cells with compromised membrane integrity (a marker of cell death). As shown in Figure 2, 9H1-WT had an effect on cell proliferation ( Figure 2A and Figure 2C to Figure 2D ) or cell death ( Figure 2A and FIG. 2E to FIG. 2F ) had no measurable effect, while paclitaxel reduced proliferation and increased cell death, as expected. Similarly, Ab #33 had no measurable effect on cell proliferation ( Figure 2B and Figure 2G to Figure 2H ) or cell death ( Figure 2B and Figures 2I to 2J ) had no measurable effect, indicating that 9H1-WT and Ab#33 had no significant cell growth inhibitory or cytotoxic effects.

[0328] C. Specific collagen types induce DDR1 phosphorylation

[0329] To determine which type of collagen effectively induces DDR1 phosphorylation, serum-starved T47D cells were treated with 25 μg / ml or 50 μg / ml of human or rat collagen I, human collagen IV, or human collagen V. Figure 3A and Figure 3B As shown, rat or human collagen I and human collagen V induced measurable amounts of DDR1 phosphorylation, whereas human collagen IV did not, indicating that DDR1 primarily responded to collagen I and collagen V stimulation.

[0330] D. Inhibition of collagen I and V-induced DDR1 phosphorylation

[0331] Serum-starved T47D cells were pretreated with increasing concentrations of 9H1-WT, IgG1-WT as a negative control, or 2.45-IN as a positive control for two hours and then stimulated with 50 μg / ml human collagen I or V for 90 minutes. Figure 5A As shown, 9H1-WT was able to inhibit collagen I and collagen V-induced DDR1 phosphorylation at a concentration as low as 0.1 μg / ml. Figure 5B It was demonstrated that total DDR1 protein levels were largely unaffected by the treatments.

[0332] E. Calculation of IC50 for 9H1-WT inhibition of collagen I-induced pDDR1

[0333] To determine the IC50 of 9H1-WT inhibition of DDR1 phosphorylation, serum-starved T47D cells were pretreated with increasing concentrations (logarithmic scale) of 9H1-WT, IgG1-WT as a negative control, or 2.45-IN as a positive control for two hours and then stimulated with 50 μg / ml human collagen I for 90 minutes. FIG. 6A to FIG. 6D As shown, 9H1-WT (PRTH-101) inhibited collagen I-induced DDR1 phosphorylation with an average IC50 of 0.05-0.06 μg / ml.

[0334] F. Inhibition of adhesion of DDR1 overexpressing (DDR1 OE) cells to collagen I

[0335] To determine whether 9H1-WT could inhibit DDR1-mediated cell adhesion, HEK293 cells overexpressing DDR1 and WT HEK293 cells were pretreated with increasing concentrations of PRTH-101 or control IgG1-WT and cultured on plates coated with 0.5 μg / cm 2 Incubate on collagen I plate for 30 min and stain nuclei with Hoechst. 7A to 7CAs shown, DDR1 overexpression resulted in increased adhesion to collagen I, which was inhibited by 9H1-WT (PRTH-101).

[0336] G. Calculation of IC50 of 9H1-WT inhibition of DDR1 OE cell adhesion

[0337] To determine the IC50 of 9H1-WT inhibition of DDR1 OE cell adhesion, HEK293-DDR1 OE cells were pretreated with increasing concentrations (logarithmic scale) of 9H1-WT or control IgG1-WT for 1 h and plated with 0.5 μg / cm 2 Incubate the plates with collagen I for 30 min and stain the nuclei with Hoechst. Figure 8 As shown, the calculated IC50 of 9H1-WT (PRTH-101) was about 0.065 μg / ml, which was comparable to the calculated IC50 of collagen I-induced DDR1 phosphorylation shown in FIG6 .

[0338] H. Inhibition of collagen II and III-induced DDR1 phosphorylation

[0339] Serum-starved T47D cells were pretreated with increasing concentrations of 9H1-WT or IgG1-WT as a negative control for two hours and then stimulated with 50 μg / ml of human collagen I, II, or III for 90 minutes. Fig.9A As shown, collagen II and, to a lesser extent, collagen III induced DDR1 phosphorylation in T47D, and both were inhibited by 9H1-WT (PRTH-101). Fig. 9B Confirmed Fig.9A and showed a similar pattern for collagen I.

[0340] I. Rabbit and chimeric mAb #33-mediated inhibition of pDDR1

[0341] Serum-starved T47D cells were pretreated with increasing concentrations of mAbs or IgG control for two hours and then stimulated with 50 μg / ml human collagen I for 90 minutes. Fig. 10A As shown, rabbit mAb #33 was able to inhibit collagen I-induced DDR1 phosphorylation in a similar pattern and with comparable potency to 9H1-WT (PRTH-101). Fig. 10B As shown, chimeric rabbit / human mAb#33, comprising the rabbit mAb#33 heavy and light chain variable domains fused to human IgG1 heavy and light chain constant domains (see Table 4), produced results similar to those of 9H1-WT and rabbit mAb#33.

[0342] 8.2 Example 2: In vivo characterization of anti-DDR1 mAbs

[0343] To evaluate the pharmacokinetic characteristics of anti-DDR1 mAb, female C57B16JrJ mice were intraperitoneally administered with a 10 mg / kg dose of mAb, and serial blood sampling was performed over time ( Figure 4A ), and ELISA was used to measure circulating free and partially bound antibodies. FIG. 4B to FIG. 4D As shown, exposure to humanized mAb #9H1 ( Figure 4B ), humanized mAb #9H1 with WT IgG1 Fc ( Figure 4C ) and chimeric rabbit / human mAb #33 with an inert IgG1 Fc ( Figure 4D ) is comparable to the expected exposure of IgG1. Figure 4E As shown in Table S1 below, the maximum circulating (free and partially bound) concentration of each mAb was higher than the concentration required for binding to the target (as shown by the surface plasmon resonance results of mAb binding to the mouse DDR1 extracellular domain; see Table S2 below). In addition, the maximum circulating (free and partially bound) concentration of mAb #9H1 WT IgG1 was higher than the in vitro concentration required to inhibit collagen I-induced DDR1 phosphorylation in T47D cells (see Figure 1).

[0344] Table S1. Pharmacokinetic parameters of anti-DDR1 mAbs

[0345]

[0346] Table S2. SPR binding characterization of mAbs to mouse DDR1 extracellular domain

[0347] mAbs kon(1 / Ms) Koff(1 / s) KD(nM) mAb#33 Inert IgG1 Fc l,05E+06 l,06E-03 1.01 mAb#9H1 Inert IgG1 Fc l,97E+05 3,75E-03 19.0 mAb#9H1 WT IgG1 Fc 2,14E+05 3,77E-03 17.6

[0348] 8.3 Example 3: Characterization of DDR1 phosphorylation in skin samples

[0349] Methods for detecting pDDR1 in skin samples were developed. Initially, pDDR1 was detected in reference human skin samples from healthy subjects, as described below. Strikingly, the pDDR1 detected in these reference samples was almost entirely the spliced ​​form of DDR1.

[0350] Healthy human subject 4 mm fresh frozen skin punch biopsy samples were obtained from Discovery Life Sciences. Sample 1 was a normal skin sample from a 36-year-old Caucasian female, collected via abdominoplasty on July 13, 2022 (patient ID: 122299014). Sample 2 was a normal skin sample from a 51-year-old Caucasian female, collected via abdominoplasty on July 20, 2022 (patient ID: 122305452).

[0351] For protein extraction, samples were taken out of the -80°C freezer and placed in 2 ml Pierce RIPA buffer (Thermo) + 1 x Halt in GentleMacs M tubes (Miltenyi). TM Suspended in protease / phosphatase inhibitor cocktail (Thermo) + 5mM EDTA + 5nM Batimastat (R&D Systems). The sample was cycled twice on the Protein 01_01 cycle of the GentleMacs Disassociator (Miltenyi) at 4°C and spun at 220Ref for 1 minute and 30 seconds at 4°C to dissipate the foam and collect the sample. 300 μL of whole skin lysate was removed (2-cycle skin lysate sample). 200 μL of 10% SDS was added to 300 μL of whole skin lysate and the mixture was boiled at 95°C for 5 minutes (2-cycle skin lysate 4% SDS sample). The remaining lysate was centrifuged at 220Ref for 4 minutes at 4°C and the supernatant was removed (2-cycle skin supernatant sample). The remaining lysate was run a third time on the Protein01_01 cycle and centrifuged at 220Ref for 4 minutes at 4°C. The supernatant was removed (3-cycle skin supernatant sample). The remaining lysate was resuspended and 200 μL 10% SDS was added to 300 μL lysate and the mixture was boiled as described above (3 cycle lysate 4% SDS sample). Finally, about 300 μL of the remaining lysate was recovered (3 cycle skin lysate sample).

[0352] All lysates were boiled at 95°C for 20 minutes to redissolve the proteins, and 200 μg / mL samples were prepared with 0.1x sample buffer. Phosphorylated DDR1 in the samples was detected using the Jess Total Protein Detection Chemiluminescent Assay in combination with a 12-230 kDa Fluorescence Separation Module, Replex Module, Anti-Rabbit Detection Module, and Total Protein Detection Module (all from Protein Simple) and a phospho-DDR1 (Tyr513) (E1N8F) antibody (Cell Signaling; mAb Cat. #5583).

[0353] like Fig.11A As shown, pDDR1 was detected in all preparations of skin lysates from two healthy subjects. Interestingly, only cleaved intracellular pDDR1 (approximately 60 kDa) was detected in the skin lysate samples ( Fig.11A , indicated by white arrows). In contrast, T47D breast cancer cells stimulated with collagen (e.g., as described above) only expressed full-length pDDR1 (approximately 125 kDa; Fig.11A , indicated by the black arrow).

[0354] The above results do not indicate that full-length DDR1 is not present in the samples assayed, as the antibody used is specific for pDDR1. Using a DDR1 (D1G6) antibody (Cell Signaling) that is nonspecific for pDDR1, it was shown that both full-length and cleaved DDR1 could be detected in skin lysate samples. Fig. 11B , see lane indicated by black arrow). However, only the sheared form is phosphorylated ( Fig. 11B , see lane indicated by white arrow).

[0355] The scope of the present invention is not limited by the specific embodiments described herein. In fact, various modifications of the present invention, in addition to those described, will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0356] All references (e.g., publications or patents or patent applications) cited herein are incorporated by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0357] Other embodiments are within the following claims.

Claims

1. A method for monitoring the effectiveness of an anti-discoidin domain receptor tyrosine kinase 1 (DDR1) antibody or an antigen-binding fragment thereof in a subject in need thereof, comprising: a) administering an effective amount of an anti-DDR1 antibody to the subject; as well as b) detecting the level of DDR1 phosphorylation in the sample from the subject, wherein a decrease in DDR1 phosphorylation in the sample from the subject compared to a positive reference sample indicates that administration of the anti-DDR1 antibody is effective.

2. The method of claim 1, wherein the subject suffers from cancer.

3. The method of claim 2, wherein the cancer is selected from the group consisting of pancreatic cancer; lung cancer, including small cell lung cancer and non-small cell lung cancer; colon cancer and colorectal cancer; head and neck cancer; stomach (gastric) cancer; ovarian cancer; breast cancer; kidney cancer; liver cancer; prostate cancer; cervical cancer; brain cancer; skin cancer, including melanoma; sarcoma; bile duct cancer; and bone cancer.

4. The method of claim 1, wherein the subject has a fibrotic disorder.

5. The method of claim 4, wherein the fibrotic disorder is selected from the group consisting of hypertrophic scarring of the skin, scleroderma, lung scarring, idiopathic pulmonary fibrosis, cirrhotic liver fibrosis, renal fibrosis, and interstitial lung disease.

6. A method for treating a DDR1-related disorder in a subject in need thereof, comprising: a) administering to the subject an effective amount of an anti-DDR1 antibody or an antigen-binding fragment thereof; as well as b) detecting the level of DDR1 phosphorylation in a sample from the subject, wherein a decrease in DDR1 phosphorylation in the sample from the subject compared to a positive reference sample indicates that the treatment is effective.

7. A method for screening a subject for a DDR1-related disorder that may be effectively treated with an anti-DDR1 antibody, comprising detecting the level of DDR1 phosphorylation in a sample from the subject, wherein if DDR1 phosphorylation is higher in the sample from the subject compared to a negative reference sample, then the DDR1-related disorder may be effectively treated with the anti-DDR1 antibody.

8. A method for treating a DDR1-related disorder in a subject in need thereof, comprising: a) detecting the level of DDR1 phosphorylation in a sample from the subject; as well as b) if DDR1 phosphorylation is higher in the sample from the subject compared to the negative reference sample, administering to the subject an effective amount of an anti-DDR1 antibody or antigen-binding fragment thereof.

9. The method according to any one of claims 6 to 8, wherein the DDR1-related disorder is cancer.

10. The method of claim 9, wherein the cancer is selected from the group consisting of pancreatic cancer; lung cancer, including small cell lung cancer and non-small cell lung cancer; colon cancer and colorectal cancer; head and neck cancer; stomach (gastric) cancer; ovarian cancer; breast cancer; kidney cancer; liver cancer; prostate cancer; cervical cancer; brain cancer; skin cancer, including melanoma; sarcoma; bile duct cancer; and bone cancer.

11. The method of any one of claims 6 to 8, wherein the DDR1-related disorder is a fibrotic disorder.

12. The method of claim 11, wherein the fibrotic disorder is selected from the group consisting of hypertrophic scarring of the skin, scleroderma, lung scarring, idiopathic pulmonary fibrosis, cirrhotic liver fibrosis, renal fibrosis, and interstitial lung disease.

13. The method of any one of claims 1 to 3 or 6 to 10, wherein the sample comprises tumor tissue.

14. The method according to any one of claims 1 to 12, wherein the sample comprises one or more selected from blood cells, skin tissue, lung tissue, kidney tissue and liver tissue.

15. The method of any one of claims 1 to 12, wherein the sample comprises a skin punch biopsy sample.

16. A method for screening an anti-DDR1 antibody or an antigen-binding fragment thereof that is effective in treating a DDR1-related disorder, comprising: a) administering an effective amount of an anti-DDR1 antibody or an antigen-binding fragment thereof to a cell; and b) detecting the phosphorylation level of DDR1 in the cell, Wherein a reduction in DDR1 phosphorylation in the cells compared to positive reference cells indicates that the anti-DDR1 antibody or antigen-binding fragment thereof is effective in treating the DDR1-related disorder.

17. A method for screening an anti-DDR1 antibody or an antigen-binding fragment thereof that is effective in reducing the interaction between collagen and cells, comprising: a) administering an effective amount of an anti-DDR antibody or an antigen-binding fragment thereof to the cell; as well as b) detecting the phosphorylation level of DDR1 in the cell, Wherein a reduction in DDR1 phosphorylation in the cell compared to a positive reference cell indicates that the anti-DDR1 antibody or antigen-binding fragment thereof is effective in reducing the interaction of collagen with the cell.

18. The method of claim 16 or 17, wherein the cell is a cancer cell.

19. The method of claim 18, wherein the cancer cells are derived from a cancer selected from the group consisting of pancreatic cancer; lung cancer, including small cell lung cancer and non-small cell lung cancer; colon cancer and colorectal cancer; head and neck cancer; stomach (gastric) cancer; ovarian cancer; breast cancer; kidney cancer; liver cancer; prostate cancer; cervical cancer; brain cancer; skin cancer, including melanoma; sarcoma; bile duct cancer; and bone cancer.

20. The method according to claim 16 or 17, wherein the cells are one or more selected from skin cells, lung cells, kidney cells and liver cells.

21. The method according to any one of claims 1 to 20, wherein the anti-DDR1 antibody or antigen-binding fragment thereof comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), the VH comprising the CDRH1, CDRH2 and CDRH3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 4 or 13, and the VL comprising the CDRL1, CDRL2 and CDRL3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 3, 11 or 12.

22. The method of claim 21, wherein: a) the CDRL1 comprises the amino acid sequence of SEQ ID NO: 5; b) the CDRL2 comprises the amino acid sequence of QAS; c) the CDRL3 comprises the amino acid sequence of SEQ ID NO: 7; d) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 8; e) the CDRH2 comprises the amino acid sequence of SEQ ID NO: 9; and f) the CDRH3 comprises the amino acid sequence of SEQ ID NO:

10.

23. The method of claim 21, wherein: a) the CDRL1 comprises the amino acid sequence of SEQ ID NO: 17; b) the CDRL2 comprises the amino acid sequence of GVF; c) the CDRL3 comprises the amino acid sequence of SEQ ID NO: 19; d) the CDRH1 comprises the amino acid sequence of SEQ ID NO: 20; e) the CDRH2 comprises the amino acid sequence of SEQ ID NO: 21; and f) the CDRH3 comprises the amino acid sequence of SEQ ID NO:

22.

24. The method of claim 22 or 23, wherein the anti-DDR1 antibody comprises: a) a VL domain comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11 and 12; and b) a VH domain comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 13.

25. The method of claim 22 or 23, wherein the anti-DDR1 antibody comprises: a) a VL domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3, 11 and 12; and b) a VH domain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 13.

26. The method of claim 25, wherein the anti-DDR1 antibody comprises a VL domain and a VH domain selected from: a) SEQ ID NO: 3 and 4, respectively; b) SEQ ID NOs: 11 and 13, respectively; and c) SEQ ID NOs: 12 and 13, respectively.

27. The method of claim 26, wherein the anti-DDR1 antibody comprises a VL domain and a VH domain, and the VL domain and the VH domain comprise the amino acid sequences of SEQ ID NOs: 3 and 4, respectively.

28. The method of claim 26, wherein the anti-DDR1 antibody comprises a VL domain and a VH domain, and the VL domain and the VH domain comprise the amino acid sequences of SEQ ID NOs: 11 and 13, respectively.

29. The method of claim 26, wherein the anti-DDR1 antibody comprises a VL domain and a VH domain, and the VL domain and the VH domain comprise the amino acid sequences of SEQ ID NOs: 12 and 13, respectively.

30. The method of any one of claims 1 to 29, wherein detecting the phosphorylation level of DDR1 comprises detecting the phosphorylation level of a spliced ​​form of DDR1.

31. The method of claim 30, wherein the spliced ​​form of DDR1 has a molecular weight of about 65 kDa.

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