Novel DDR1 antibody and its use

Abstract

To provide antibodies binding to tumor discoidin domain receptor 1 (DDR1) and the uses of the antibodies in detecting and treating cancer.SOLUTION: In one aspect, the present disclosure provides an isolated monoclonal antibody or an antigen-binding fragment thereof that binds specifically to DDR1. In certain embodiments, the antibody or antigen-binding fragment, when bound to DDR1, modulates the activity of DDR1, i.e., suppresses DDR1.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 949,300, filed on 17 December 2019, the entire contents of which are incorporated herein by reference.

[0002] Statement regarding government-sponsored research This invention was made possible with government support under grant number CA220578, awarded by the National Institutes of Health. The U.S. Government reserves certain rights in this invention.

[0003] Sequence listing reference The sequence listing was submitted along with the specification as an ASCII text file named P0362WO_ST25.txt, created on December 17, 2020, and measuring 127 kilobytes. The electronic information of the sequence listing is part of the specification and is incorporated herein by reference in its entirety.

[0004] 1. Field This disclosure generally relates to the fields of medicine, oncology, and immunology. More specifically, this disclosure relates to an antibody that binds to DDR1 and can treat cancer, including breast cancer. [Background technology]

[0005] 2. Description of related technologies Abnormal expression of tumor discoidin domain receptors 1 (DDR1) and 2 (DDR2) proteins has been associated with the progression of several solid tumor types, including breast cancer (Valiathan et al., 2012; Rammal et al., 2016; Gao et al., 2016; Bayer et al., 2019). Published data support the role of DDR proteins in promoting tumor progression and metastatic potential (Gao et al., 2016; Bayer et al., 2019; Hidalgo-Carcedo et al., 2011; Marcotte et al., 2012). However, gene excision of Ddr1 in MMTV-PyMT mice promotes spontaneous tumorigenesis (Takai et al., 2018), suggesting a stage-dependent tumor DDR1 function in cancer development and progression. [Overview of the Initiative]

[0006] overview Accordingly, in one embodiment, the present disclosure provides an isolated monoclonal antibody or an antigen-binding fragment thereof that specifically binds to DDR1. In certain embodiments, the antibody or antigen-binding fragment, upon binding to DDR1, modulates the activity of DDR1, i.e., inhibits DDR1. In certain embodiments, the antibody or antigen-binding fragment, upon binding to DDR1, specifically blocks the binding of a ligand to DDR1.

[0007] In one embodiment, the isolated monoclonal antibody or its antigen-binding fragment each comprises a light chain (LC) variable region (VL) and a heavy chain (HC) variable region (VH) containing the CDR amino acid sequences of the clone pair described in Tables 1 and 2, as well as variants thereof, each comprising one or more LC-CDRs and / or one or more HC-CDRs having one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment is a mouse antibody, rodent antibody, rabbit antibody, chimeric antibody, humanized antibody, or human antibody. In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment may each have VL and VH chains having amino acid sequences at least 90% or 95% identical to the sequences of the clone pair described in Tables 3 and 4. In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment may have VL and VH chains, respectively, encoded by nucleic acid sequences that are at least 80% or 90% identical to the sequences of the clone pairs in Tables 8 and 9. In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment may have VL and VH chains, respectively, having amino acid sequences identical to the sequences of the clone pairs in Tables 3 and 4. In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment may have VL and VH chains, respectively, encoded by nucleic acid sequences identical to the sequences of the clone pairs in Tables 8 and 9.

[0008] A variant may be one in which one or more HC-CDRs or LC-CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. In certain embodiments, each CDR is defined according to the Kabat definition, Chothia definition, IMGT definition, a combination of the Kabat and Chothia definitions, the AbM definition, or a contact definition of the CDR.

[0009] In another embodiment, the disclosure provides isolated monoclonal antibodies or their antigen-binding fragments that compete for the same epitopes as antibodies having the light chain and heavy chain CDR amino acid sequences of clone pairs from Tables 1 and 2, respectively.

[0010] In certain embodiments, the isolated monoclonal antibody described herein is a chimeric antibody, a humanized antibody, or a human antibody. In certain embodiments, the isolated monoclonal antibody described herein is of type IgG1, IgG2, IgG3, or IgG4. In certain embodiments, the antigen-binding fragment described herein is a recombinant ScFv (single-chain variable region fragment) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment.

[0011] In another embodiment, a pharmaceutical composition is provided comprising an isolated monoclonal antibody or an antigen-binding fragment thereof provided herein, and at least one pharmaceutically acceptable carrier.

[0012] In another embodiment, an isolated nucleic acid is provided which encodes an isolated monoclonal antibody or an antigen-binding fragment thereof.

[0013] In another embodiment, a vector comprising isolated nucleic acids provided herein is provided.

[0014] In another embodiment, a host cell containing a vector provided herein is provided. The host cell may be a mammalian cell. The host cell may be a CHO cell.

[0015] In another embodiment, a hybridoma is provided that encodes or produces an isolated monoclonal antibody provided herein.

[0016] In another embodiment, a method for producing antibodies is provided. This method may include culturing host cells provided herein under conditions suitable for expressing antibodies and recovering them.

[0017] In another embodiment, a chimeric antigen receptor (CAR) protein comprising an antigen-binding fragment provided herein is provided.

[0018] In another embodiment, isolated nucleic acids encoding CAR proteins provided herein are provided.

[0019] In another embodiment, engineered cells comprising isolated nucleic acids provided herein are provided. In certain embodiments, the cells are T cells, NK cells, or myeloid cells. In another embodiment, a method is provided for treating or mitigating the effects of cancer in a subject, or for treating or mitigating fibrosis (e.g., organofibrosis), the method comprising administering to the subject a therapeutically effective amount of an antibody or antigen-binding fragment as defined herein. In the treatment of cancer, the method can reduce or eradicate the tumor burden in the subject, reduce the number of tumor cells, reduce the tumor size, or eradicate the tumor in the subject. In some embodiments, the cancer to be treated is breast cancer.

[0020] Antibodies or their antigen-binding fragments may be administered topically, intravenously, intraarterially, intratumorally, or subcutaneously. In some embodiments, the method may target topoisomerase inhibitors, anthracycline topoisomerase inhibitors, anthracyclines, daunorubicin, nucleoside metabolism inhibitors, cytarabine, hypomethylating agents, low-dose cytarabine (LDAC), combinations of daunorubicin and cytarabine, daunorubicin and cytarabine liposomes for injection, Vyxeos®, azacitidine, Vidaza®, decitabine, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine dihydrochloride, Ceplene®, interleukin-2, aldesleukin, Proleukin®, getumuzumab Ozogamicin, Mylotarg®, FLT-3 inhibitor, Midostaurin, Rydapt®, Clofarabine, Farnesyltransferase inhibitor, Decitabine, IDH1 inhibitor, Ivosidenib, Tibsovo®, IDH2 inhibitor, Enasidenib, Idhifa®, Smoothened (SMO) inhibitor, Grasdegib, Arginase inhibitor, IDO inhibitor, Epacadostat, BCL-2 inhibitor, Venetoclax, Venclexta®, Platinum complex derivative, Oxaliplatin, Kinase inhibitor, Tyrosine kinase inhibitor, PI3 kinase inhibitor, BTK inhibitor, Ibrutinib, IMBRUVICA® The process further comprises administering one or more drugs selected from the group consisting of registered trademarks, acalabrutinib, CALQUENCE®, zanubrutinib, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG3 antibody, ICOS antibody, TIGIT antibody, TIM3 antibody, CD40 antibody, 4-1BB antibody, CD47 antibody, SIRP1α antibody or fusion protein, E-selectin antagonists, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to bone marrow cells or NK cell surface markers, alkylating agents, nitrosourea agents, antimetabolites, antitumor antibiotics, plant-derived alkaloids, hormonal therapy agents, hormone antagonists, aromatase inhibitors, and P-glycoprotein inhibitors.

[0021] In some embodiments, the isolated monoclonal antibody or its antigen-binding fragment may include an antitumor drug linked thereto. The antitumor drug may be linked to the antibody via a photo-unstable linker. The antitumor drug may be linked to the antibody via an enzymatically cleavable linker. The antitumor drug may be a toxin, a radioisotope, a cytokine, or an enzyme.

[0022] In another embodiment, a method is provided for detecting cancer cells or fibrous tissue in a sample or subject, comprising: (a) contacting a subject or a sample from a subject with an antibody or its antigen-binding fragment as defined herein; and (b) detecting the binding of the aforementioned antibody to cancer cells or fibrous tissue in the aforementioned subject or sample. The sample may be a body fluid or biopsy, or blood, bone marrow, sputum, tears, saliva, mucous membrane, serum, urine, or feces. Detection may include immunohistochemistry, flow cytometry, FACS, ELISA, RIA, or Western blotting. In some embodiments, steps (a) and (b) may further be performed again, and the change in detection level compared to the first time may be determined. The isolated monoclonal antibody or its antigen-binding fragment may further include labeling such as a peptide tag, enzyme, magnetic particle, chromophore, fluorescent molecule, chemiluminescent molecule, or dye. The isolated monoclonal antibody or its antigen-binding fragment may be conjugated into liposomes or nanoparticles.

[0023] The use of the words "a" or "an," when used in conjunction with the term "including" in the claims and / or specification, may mean "one," but may also correspond to the meanings of "one or more," "at least one," and "one or more than one." The word "about" means plus or minus 5% of the stated number.

[0024] [Invention 1001] An isolated monoclonal antibody or its antigen-binding fragment that specifically binds to the discoidine domain receptor 1 (DDR1) protein, A light chain variable region having LC-LC-CDR1, LC-CDR2, and LC-CDR3 within a light chain variable region selected from sequence numbers 108 to 128, and Sequence ID: A heavy chain variable region having HC-CDR1, HC-CDR2, and HC-CDR3 within a heavy chain variable region selected from Sequence IDs 129 to 149. Includes, or The variants thereof, wherein one or more of the LC-CDR and / or the HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. including, The isolated monoclonal antibody or its antigen-binding fragment. [Invention 1002] LC-LC-CDR1, LC-CDR2, and LC-CDR3 of the clone pairs selected from Table 1, and Select HC-CDR1, HC-CDR2, and HC-CDR3 from Table 2. Includes, or The variants thereof, wherein one or more of the LC-CDR and / or the HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. including, An isolated monoclonal antibody or its antigen-binding fragment according to the present invention 1001. [Invention 1003] A light chain variable region having LC-CDR1 containing the amino acid sequence QSIGSV (SEQ ID NO: 11), LC-CDR2 containing the amino acid sequence GVF, and LC-CDR3 containing the amino acid sequence QYIPYGSSP (SEQ ID NO: 12), and Heavy chain variable region having HC-CDR1 containing amino acid sequence GFSLNRYY (SEQ ID NO: 57), HC-CDR2 containing amino acid sequence ISYGDTT (SEQ ID NO: 58), and HC-CDR3 containing amino acid sequence ARADTGDNGYLGLQL (SEQ ID NO: 59). Includes (DDR1-9), or The variants thereof, wherein one or more of the LC-CDR and / or the HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. including, An isolated monoclonal antibody or its antigen-binding fragment according to the present invention 1001. [Invention 1004] A light chain variable region having LC-CDR1 containing the amino acid sequence ESINSW (SEQ ID NO: 41), LC-CDR2 containing the amino acid sequence DAS, and LC-CDR3 containing the amino acid sequence QSYYIINRSNYGNS (SEQ ID NO: 42), and Heavy chain variable region containing HC-CDR1 with amino acid sequence GFSLSSYY (SEQ ID NO: 102), HC-CDR2 with amino acid sequence ITTAGPL (SEQ ID NO: 103), and HC-CDR3 with amino acid sequence ARGHAGSIYYSYFDL (SEQ ID NO: 104). Includes (DDR1-32), or The variants thereof, wherein one or more of the LC-CDR and / or the HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof. including, An isolated monoclonal antibody or its antigen-binding fragment according to the present invention 1001. [Invention 1005] Light chain variable region having an amino acid sequence selected from SEQ ID NOs: 108-128, and heavy chain variable region having an amino acid sequence selected from SEQ ID NOs: 129-149 An isolated monoclonal antibody or its antigen-binding fragment according to the present invention 1001, comprising the above. [Invention 1006] (a) Light chain variable region (DDR1-1K) having the amino acid sequence of SEQ ID NO: 108, and heavy chain variable region (DDR1-1H) having the amino acid sequence of SEQ ID NO: 129, (b) Light chain variable region (DDR1-3K) having the amino acid sequence of SEQ ID NO: 109, and heavy chain variable region (DDR1-3H) having the amino acid sequence of SEQ ID NO: 130, (c) Light chain variable region (DDR1-5K) having the amino acid sequence of SEQ ID NO: 110, and heavy chain variable region (DDR1-5H) having the amino acid sequence of SEQ ID NO: 131, (d) Light chain variable region (DDR1-6K) having the amino acid sequence of SEQ ID NO: 111, and heavy chain variable region (DDR1-6H) having the amino acid sequence of SEQ ID NO: 132, (e) Light chain variable region (DDR1-9K) having the amino acid sequence of SEQ ID NO: 112, and heavy chain variable region (DDR1-9H) having the amino acid sequence of SEQ ID NO: 133, (f) Light chain variable region (DDR1-11K) having the amino acid sequence of SEQ ID NO: 113, and heavy chain variable region (DDR1-11H) having the amino acid sequence of SEQ ID NO: 134, (g) Light chain variable region (DDR1-12K) having the amino acid sequence of SEQ ID NO: 114, and heavy chain variable region (DDR1-12H) having the amino acid sequence of SEQ ID NO: 135, (h) Light chain variable region (DDR1-13K) having the amino acid sequence of SEQ ID NO: 115, and heavy chain variable region (DDR1-13H) having the amino acid sequence of SEQ ID NO: 136, (i) Light chain variable region (DDR1-14K) having the amino acid sequence of SEQ ID NO: 116, and heavy chain variable region (DDR1-14H) having the amino acid sequence of SEQ ID NO: 137, (j) Light chain variable region (DDR1-15K) having the amino acid sequence of SEQ ID NO: 117, and heavy chain variable region (DDR1-15H) having the amino acid sequence of SEQ ID NO: 138, (k) Light chain variable region (DDR1-17K) having the amino acid sequence of SEQ ID NO: 118, and heavy chain variable region (DDR1-17H) having the amino acid sequence of SEQ ID NO: 139, (l) Light chain variable region (DDR1-20K) having the amino acid sequence of SEQ ID NO: 119, and heavy chain variable region (DDR1-20H) having the amino acid sequence of SEQ ID NO: 140, (m) Light chain variable region (DDR1-21K) having the amino acid sequence of SEQ ID NO: 120, and heavy chain variable region (DDR1-21H) having the amino acid sequence of SEQ ID NO: 141, (n) Light chain variable region (DDR1-22K) having the amino acid sequence of SEQ ID NO: 121, and heavy chain variable region (DDR1-22H) having the amino acid sequence of SEQ ID NO: 142, (o) Light chain variable region (DDR1-23K) having the amino acid sequence of SEQ ID NO: 122, and heavy chain variable region (DDR1-23H) having the amino acid sequence of SEQ ID NO: 143, (p) Light chain variable region (DDR1-26K) having the amino acid sequence of SEQ ID NO: 123, and heavy chain variable region (DDR1-26H) having the amino acid sequence of SEQ ID NO: 144, (q) Light chain variable region (DDR1-28K) having the amino acid sequence of SEQ ID NO: 124, and heavy chain variable region (DDR1-28H) having the amino acid sequence of SEQ ID NO: 145, (r) Light chain variable region (DDR1-29K) having the amino acid sequence of SEQ ID NO: 125, and heavy chain variable region (DDR1-29H) having the amino acid sequence of SEQ ID NO: 146, (s) Light chain variable region (DDR1-32K) having the amino acid sequence of SEQ ID NO: 126, and heavy chain variable region (DDR1-32H) having the amino acid sequence of SEQ ID NO: 147, (t) Light chain variable region (DDR1-33K) having the amino acid sequence of SEQ ID NO: 127, and heavy chain variable region (DDR1-33H) having the amino acid sequence of SEQ ID NO: 148, or (u) Light chain variable region (DDR1-34K) having the amino acid sequence of SEQ ID NO: 128, and heavy chain variable region (DDR1-34H) having the amino acid sequence of SEQ ID NO: 149 Includes, or The variants wherein the light chain variable region has 80% or more amino acid sequence identity with the amino acid sequence of the parent light chain variable region, and the heavy chain variable region has 80% or more sequence identity with the amino acid sequence of the parent heavy chain variable region, and the variants that specifically bind to the DDR1 protein. including, An isolated monoclonal antibody or its antigen-binding fragment according to the present invention 1001. [Invention 1007] Light chain variable region (DDR1-9K) having the amino acid sequence of SEQ ID NO: 112, and heavy chain variable region (DDR1-9H) having the amino acid sequence of SEQ ID NO: 133. An isolated monoclonal antibody or its antigen-binding fragment according to the present invention 1001, comprising the above. [Invention 1008] Light chain variable region (DDR1-32K) having the amino acid sequence of SEQ ID NO: 126, and heavy chain variable region (DDR1-32H) having the amino acid sequence of SEQ ID NO: 147. An isolated monoclonal antibody or its antigen-binding fragment according to the present invention 1001, comprising the above. [Invention 1009] The isolated monoclonal antibody or its antigen-binding fragment according to Invention 1001 or 1002, wherein the isolated monoclonal antibody is a mouse antibody, rodent antibody, rabbit antibody, chimeric antibody, humanized antibody, or human antibody. [Invention 1010] The isolated monoclonal antibody according to Invention 1009, wherein the isolated monoclonal antibody is a rabbit antibody or a chimeric antibody, or an isolated monoclonal antibody or its antigen-binding fragment. [Invention 1011] The isolated monoclonal antibody according to Invention 1009, wherein the isolated monoclonal antibody is a rabbit antibody or a chimeric antibody, or an isolated monoclonal antibody or its antigen-binding fragment. [Invention 1012] The isolated monoclonal antibody is a humanized antibody, and is an isolated monoclonal antibody or its antigen-binding fragment according to any of the present invention 1001 to 1004. [Invention 1013] SEQ ID NO: A light chain variable region having amino acid sequence 150 or 151 (DDR1-9hu_Lv1 or DDR1-9hu_Lc2), and Sequence ID: 152, a heavy chain variable region (DDR1-9hu_Hv) containing the amino acid sequence. Includes, or The variants wherein the light chain variable region has 80% or more amino acid sequence identity with the amino acid sequence of the parent light chain variable region, and the heavy chain variable region has 80% or more sequence identity with the amino acid sequence of the parent heavy chain variable region, and the variants that specifically bind to the DDR1 protein. An isolated monoclonal antibody or its antigen-binding fragment according to the present invention 1001, comprising the above. [Invention 1014] An isolated monoclonal antibody or its antigen-binding fragment according to any of the present invention 1001 to 1013, wherein the antigen-binding fragment is an ScFv (single-chain variable region fragment) antibody, a Fab fragment, an F(ab')2 fragment, or an Fv fragment. [Invention 1015] An isolated monoclonal antibody or its antigen-binding fragment that specifically binds to an epitope or to all or part of the same epitope, as specified in any of invention 1001 to 1014. [Invention 1016] An isolated monoclonal antibody or its antigen-binding fragment that competes for the same epitope as any of the isolated monoclonal antibody or its antigen-binding fragments of the present invention 1001 to 1014. [Invention 1017] An antibody-drug conjugate comprising an isolated monoclonal antibody or its antigen-binding fragment according to any of invention 1001 to 1016, and an antitumor drug linked to the antibody or its antigen-binding fragment. [Invention 1018] The antibody-drug conjugate of the present invention 1017, wherein the antitumor drug is linked to the antibody via a photo-unstable linker or an enzymatically cleavable linker. [Invention 1019] The antibody-drug conjugate of the present invention 1017 or 1018, wherein the antitumor drug is a toxin, radioisotope, cytokine, or enzyme. [Invention 1020] A pharmaceutical composition comprising an isolated monoclonal antibody or its antigen-binding fragment according to any of the present invention 1001 to 1014, and a pharmaceutically acceptable carrier. [Invention 1021] An isolated polynucleotide encoding any isolated monoclonal antibody or its antigen-binding fragment according to any of invention 1001 to 1014. [Invention 1022] A vector comprising the isolated nucleic acid of the present invention 1021. [Invention 1023] The vector of the present invention 1022, comprising an expression vector capable of expressing the encoded antibody or its antigen-binding fragment. [Invention 1024] A host cell containing the polynucleotide of Invention 1021 or the vector of Invention 1022 or 1023. [Invention 1025] A mammalian cell, which is the host cell of the present invention 1024. [Invention 1026] The host cell of the present invention 1025, which is a CHO cell. [Invention 1027] Hybridomas or engineered cells that encode and / or produce any isolated monoclonal antibody of the present invention 1001 to 1014. [Invention 1028] A host cell capable of expressing the monoclonal antibody or its antigenic fragment according to any of the present invention 1024 to 1026. A method for producing antibodies, comprising the step of culturing, The method comprising the steps of culturing the host cells under conditions suitable for expressing the antibody or its antigen-binding fragment, and isolating the antibody or its antigen-binding fragment. [Invention 1029] A chimeric antigen receptor (CAR) protein comprising any antibody or antigen-binding fragment thereof according to invention 1001 to 1014. [Invention 1030] An isolated nucleic acid encoding the CAR protein of the present invention 1029. [Invention 1031] A vector comprising the isolated nucleic acid of the present invention 1030. [Invention 1032] A vector according to the present invention 1031 that can express the aforementioned CAR protein. [Invention 1033] Engineered cells comprising isolated nucleic acids of Invention 1030 or vectors of Invention 1032. [Invention 1034] The manipulated cells of the present invention 1033, which are T cells, NK cells, or macrophages. [Invention 1035] A step of administering to a target an effective amount of any antibody according to invention 1001 to 1014 or its antigen-binding fragment, or manipulated cells according to invention 1033 or 1034. Methods for treating or mitigating the effects of cancer in a subject, including those mentioned above. [Invention 1036] A method according to the present invention 1035 for reducing or eradicating tumor burden in the subject. [Invention 1037] A method according to the present invention 1036 for reducing the number of tumor cells or reducing tumor size. [Invention 1038] The method of the present invention 1035, wherein the cancer is a solid tumor or a tumor. [Invention 1039] The method of the present invention 1038, wherein the cancer is selected from pancreatic cancer; lung cancer including small cell lung cancer and non-small cell lung cancer; colon 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; and bone cancer. [Invention 1040] The method of the present invention 1038, wherein the cancer is breast cancer. [Invention 1041] The method of the present invention 1040, wherein the breast cancer is a breast cancer subtype selected from (a) luminal A (ER+ and / or PR+, HER2-), (b) luminal B (ER+ and / or PR+, HER2-), (c) triple-negative (ER-, PR-, HER2-), (d) HER2+ / ER- (ER-, PR-, and HER2+), and (e) unclassified (ER-, PR-, HER2-, cytokeratin 5 / 6-, and HER1-). [Invention 1042] A method according to any one of items 1035 to 1041 of the present invention, wherein the cancer cells of the cancer are identified as expressing secreted DDR1 protein and / or DDR1 protein on the surface of the cancer cells, or are determined to express it. [Invention 1043] The method according to any one of items 1035 to 1042 of the present invention, wherein the antibody or its antigen-binding fragment is administered intravenously, intraarterially, intratumorally, or subcutaneously. [Invention 1044] The aforementioned targets include topoisomerase inhibitors, anthracycline topoisomerase inhibitors, anthracyclines, daunorubicin, nucleoside metabolism inhibitors, cytarabine, hypomethylating agents, low-dose cytarabine (LDAC), combinations of daunorubicin and cytarabine, daunorubicin and cytarabine liposomes for injection, Vyxeos®, azacitidine, Vidaza®, decitabine, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine dihydrochloride, Ceplene®, interleukin-2, aldesleukin, Proleukin®, and getumuzumab. Ozogamicin, Mylotarg®, FLT-3 inhibitor, Midostaurin, Rydapt®, Clofarabine, Farnesyltransferase inhibitor, Decitabine, IDH1 inhibitor, Ivosidenib, Tibsovo®, IDH2 inhibitor, Enasidenib, Idhifa®, Smoothened (SMO) inhibitor, Glasdegib, Arginase inhibitor, IDO inhibitor, Epacadostat, BCL-2 inhibitor, Venetoclax, Venclexta®, Platinum complex derivative, Oxaliplatin, Kinase inhibitor, Tyrosine kinase inhibitor, PI3 kinase inhibitor, BTK inhibitor, Ibrutinib, IMBRUVICA®, A The method of the present invention 1035 further comprises the step of administering one or more drugs selected from the group consisting of calabrutinib, CALQUENCE®, zanubrutinib, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG3 antibody, ICOS antibody, TIGIT antibody, TIM3 antibody, CD40 antibody, 4-1BB antibody, CD47 antibody, SIRP1α antibody or fusion protein, E-selectin antagonists, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to bone marrow cells or NK cell surface markers, alkylating agents, nitrosourea agents, antimetabolites, antitumor antibiotics, plant-derived alkaloids, hormonal therapy agents, hormone antagonists, aromatase inhibitors, and P-glycoprotein inhibitors. [Invention 1045] Any method of the present invention 1035 to 1044, wherein the isolated monoclonal antibody or its antigen-binding fragment further comprises an antitumor drug linked thereto. [Invention 1046] The method of the present invention 1045, wherein the antitumor drug is a toxin, a radioisotope, a cytokine, or an enzyme. [Invention 1047] The process of administering a therapeutically effective amount of any antibody according to Invention 1001 to 1014 or its antigen-binding fragment, or manipulated cells according to Invention 1033 or 1034, to the subject. Methods for treating or alleviating the effects of fibrosis in a subject, including those mentioned above. [Invention 1048] The method of the present invention 1047, wherein the subject has organ fibrosis. [Invention 1049] The method of the present invention 1048, wherein the subject has fibrosis of the kidney, liver, lung, or heart. [Invention 1050] The method of the present invention 1049, wherein the subject has pulmonary fibrosis. [Invention 1051] The method of the present invention 1050, wherein the subject has interstitial lung disease. [Invention 1052] The method of the present invention 1050, wherein the subject has idiopathic pulmonary fibrosis (IPF) or pulmonary scarring. [Invention 1053] A method for detecting cancer cells or cancer stem cells in a sample or subject, (a) A step of contacting a subject or a sample from a subject with any antibody or antigen-binding fragment of the present invention 1001 to 1014, and (b) A step of detecting the binding of the antibody to cancer cells or cancer stem cells in the subject or sample. The method, including the method described above. [Invention 1054] The method of the present invention 1053, wherein the sample is a body fluid or a biopsy. [Invention 1055] The method of the present invention 1054, wherein the sample is blood, bone marrow, sputum, tears, saliva, mucus, serum, urine, or feces. [Invention 1056] The detection is performed by any of the methods 1053 to 1055 of the present invention, comprising immunohistochemistry, flow cytometry, FACS, ELISA, RIA, or Western blotting. [Invention 1057] A process of repeating process (a) and process (b), and A process for determining the change in detection level compared to the first measurement. The method of the present invention 1053, further comprising: [Invention 1058] The method according to any one of the present invention 1053 to 1057, wherein the isolated monoclonal antibody or its antigen-binding fragment further comprises labeling. [Invention 1059] The method of the present invention 1058, wherein the label is a peptide tag, an enzyme, magnetic particles, a chromophore, a fluorescent molecule, a chemiluminescent molecule, or a dye. [Invention 1060] The method according to any of items 1035 to 1059 of the present invention, wherein the isolated monoclonal antibody or its antigen-binding fragment is conjugated to liposomes or nanoparticles. Any method or composition described herein is intended to be implementable in relation to any other method or composition described herein. Other purposes, features, and advantages of this disclosure will become apparent from the following detailed description. However, various changes and modifications within the spirit and scope of this disclosure will become apparent to those skilled in the art from this detailed description, so while specific embodiments of the invention are shown, it should be understood that the detailed description and specific examples are given for illustrative purposes only. [Brief explanation of the drawing]

[0025] The drawings form part of this specification and are included to further illustrate specific aspects of the invention. The invention may be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein. [Figure 1] DDR1 confers mammary tumor growth in immune hosts. (Figure 1a) Immunoblot of DDR1, DDR2, and loading control GAPDH in DDR1 WT / KO mammary tumor E0771 cells. (Figure 1b) Cell proliferation of WT / KO E0771 cells as measured by MTT assay (n=6). (Figures 1c-d) Quantification of DDR1 WT / KO M-Wnt cell migration (c) and invasion (d) (n=6 random field). (Figure 1e) Growth of DDR1 WT / KO E0771 tumors in Rag1- / - (n=6, e) hosts. (Figures 1f-h) Growth of WT / KO E0771 (n=6, f), M-Wnt (n=7, g), and AT-3 (n=7, h) tumors in C57BL / 6 mice. (Figure 1i-j) Tumor growth dynamics (i) and weight (j) at the endpoint of E0771 DDR1 WT / KO tumors transplanted from Rag1- / - to C57BL / 6 hosts (n=8). (Figure 1k) Figure for tumor rechallenge. In the first round of inoculation, mice were challenged with saline buffer or DDR1 KO E0771 cells in one side of the inguinal mammary gland. After 30 days, mice were rechallenged with DDR1 WT E0771 tumor cells in both sides of the inguinal mammary gland. L, left; R, right. (Figure 1l-m) Tumor curves (Figure 1l) and weight (Figure 1m) from rechallenged mice (n=6). Values ​​represent mean ± SEM, p-value, as shown. [Figure 2]DDR1 excludes anti-tumor immune cell infiltration. (Figure 2a) Representative images (n=3) of CD8+ and CD4+ T cell staining in the tumor margin (upper panel, indicated by red dashed line) and tumor center (lower panel). Scale bar: 50 μm. (Figures 2b-g) Number of tumor-infiltrating lymphocytes (TILs) normalized by tumor weight per gram. Cell counts of CD8+ (Figure 2b) and CD4+ (Figure 2c) T cells, cytokine IFN-γ+ CD8+ (Figure 2d) and CD4+ (Figure 2e) T cells, as well as CD44-high CD62L-low-activation CD8+ (Figure 2f) and CD4+ (Figure 2g) T cells. (Figures 2h-j) WT / KO E0771 tumor growth in C57BL / 6 hosts with prior treatment of either anti-IgG or anti-CD8 antibody (n=5). The tumor volume (Figure 2h), image (Figure 2i), and weight (Figure 2j) at the endpoint are shown. Scale bar: 1 cm. (Figures 2k~m) Adoptive transfer of CD8+ T cells or culture medium (sham) into Rag1- / - (n=6) mice loaded with DDR1 WT / KO E0771 tumors. Arrows (Figure 2k) indicate CD8+ T cell transfer at day 17. Image (Figure 2l) and weight (Figure 2m) of the tumor are shown. Scale bar: 1 cm. Values ​​represent mean ± SEM, p-value, as shown. [Figure 3]DDR1-dependent ECM remodeling inhibits antitumor immune infiltration. (Figure 3a) Figures (top) and tumor curves of full-length DDR1 in DDR1 KO E0771 tumor cells with various DDR1 expression vectors: wild-type (WT), empty vector (EV), kinase domain deletion (ΔKD), and DDR1 with only the extracellular domain (ECD). All p-values ​​compared to the KO group. TM: transmembrane domain. (Figure 3b) Figure (top) of ECD consisting of DS and DS-like (DSL) domains, and crystal structure (bottom) of the mouse DDR1 DS domain generated by Jmol software. Shows targeted amino acid residues in mutation analysis. (Figure 3c) Collagen binding of WT and point mutation ECD expressed in KO E0771 cells, evaluated by ELISA (n=4). (Figures 3d-k) Individual tumor growth curves of KO E0771 tumor cells with ectopically expressed ECD WT or point mutation. Tumor incidence is shown in parentheses for each panel. (Figure 3l) Representative SEM image of WT / KO E0771 tumor cells cultured in vitro. (Figure 3m) Decellularized ECM from E0771 cells inhibits T cell migration in an ECD-dependent manner. The figure for the Transwell migration assay is shown on the left. (Figure 3n) WT / KO E0771 tumors transplanted from Rag1- / - to C57BL / 6 hosts were analyzed by SHG (gray), CD3 staining (green), To-pro-3 staining (red), and collagen fiber individualization (far right panel). Block arrows indicate tumor margins. Scale bar: 50 μm. (Figures 3o-p) Quantification of tumor fiber alignment (o) and fiber length (p) by CT Fire software. Values ​​represent mean ± SEM, p-value, as shown. [Figure 4]DDR1 is a potential therapeutic target for enhancing antitumor immunity. (Figure 4a) Immunoblots of ectopic human (hu)DDR1 and endogenous mouse DDR1 in cell lysates and culture medium of E0771-derived cells. (Figures 4b-c) Tumor growth curves (Figure 4b) and weights (Figure 4c) of E0771-derived DDR1 WT, KO, and KO+huDDR1 cells (n=6). Images of tumors are shown above. (Figures 4d-e) Growth curves of individual KO+huDDR1 tumors in C57BL / 6 hosts treated with isotype IgG (Figure 4d) or anti-huDDR1 antibody #9 (Figure 4e). Arrows indicate the start date of antibody administration. (Figure 4f) Survival curves of isotype IgG and anti-huDDR1 treatment groups. (Figure 4g) KO+huDDR1 tumors were analyzed by SHG (gray), CD3 staining (green), To-pro-3 staining (red), and collagen fiber individualization (far right panel). Block arrows indicate tumor margins. Scale bar: 50 μm. (Figures 4h-i) Quantification of tumor fiber parameters. Fiber alignment (angle of coefficient variation, Figure 4h), fiber length (Figure 4i). (Figure 4j) Correlation between huDDR1 mRNA and the abundance of tumor-invasive CD8+T in 1,093 breast cancer patient samples (TIMER). (Figures 4k-m) Correlation between DDR1 mRNA levels and anti-tumor immune markers IFNG (Figure 4k), CD8A (Figure 4l), and GZMB (Figure 4m) in 37 TNBC patient samples (GSE88847). (Figure 4n) Model diagram of ECD (red circle) reforming collagen fibers (curve) forming a barrier to prevent immune cell infiltration. Values ​​represent mean ± SEM, p-value, as shown. [Figure 5]Tumor DDR1 excision inhibits tumor growth in immune hosts. (Figure 5a) DDR1 expression in normal (N) tissue and mammary tumor (T) samples in the TCGA database. P values ​​were analyzed using the Wilcoxon test. (Figure 5b) DDR1 immunoblots of WT / KO cells derived from mouse mammary tumor cells M-Wnt and AT-3. (Figures 5c-d) Genomic DNA sequencing of sgRNA target sites at the mouse DDR1 locus. Both AT-3 and E0771 KO clones contain a single base pair insertion (Figure 5c) (SEQ ID NO: 312; SEQ ID NO: 313; SEQ ID NO: 314). The M-Wnt DDR1 KO clone has an 8 bp deletion (Figure 5d). The excision was performed upstream of the PAM sequence. (Figure 5e) Representative images of migration and invasion of DDR1 WT / KO M-Wnt and AT-3 cells. (SEQ ID NO: 315, SEQ ID NO: 316). (Figure 5f) In vitro cell proliferation of WT / KO M-Wnt tumor cells. (Figure 5g) M-Wnt tumor growth in nude mice (n=4). (Figure 5h) Individual tumor growth curves of DDR1 KO E0771 cells inoculated in various numbers into C57BL / 6 mice (0.5, 5, 10, and 20 × 10⁶ per mouse, n=4). (Figures 5i~k) Tumor volume (Figure 5i), image (Figure 5j), and weight (Figure 5k) (n=8) of M-Wnt DDR1 WT, KO, and 1:1 mixture at inoculation. Values ​​represent mean ± SEM, p-value, as shown. [Figure 6]Immunodeficiency and adoptive transfer of CD8+ cells. (Figures 6a-d) Percentage of T cells from KO and parental controls that are positive for Ki67 (CD4+ in Figure 6a and CD8+ in Figure 6b), IFNγ (CD8+ in Figure 6c), or Gzmb (CD8+ in Figure 6d). (Figures 6e-f) Flow cytometry of CD4+ and CD8+ T cells from splenocytes of anti-IgG2b treated mice or anti-CD8 antibody treated C57BL / 6 mice (e), as well as the percentage of CD8+ among CD3+ T cells in the blood (f, n=5). (Figure 6g) Number of CD8+ T cells in TILs normalized by tumor weight in Rag1- / - mice (n=6). (Figures 6h-i) RNA-seq heatmaps of T cell homing genes and chemokine genes derived from DDR1 WT / KO E0771 tumors transplanted from Rag1- / - to C57BL / 6 hosts. ***, p<0.001. The values ​​represent the mean ± SEM, p-value, as shown. [Figure 7] DDR1-mediated collagen remodeling is required for immune exclusion. (Figures 7a-b) Growth curves (Figure 7a) and tumor weights (Figure 7b) of E0771 DDR1 KO+ECD, KO, KO+DS, and KO+DSL tumors in C57BL / 6 hosts (n=10). (Figure 7c) Co-IP of type I collagen and Flag-tagged ECD WT and mutants. (Figure 7d) Immunoblots of intracellular full-length DDR1 and soluble ECD in conditional medium from mouse (left) and various human (right) breast cancer cell lines. (Figure 7e) Top 10 biological processes (BP) based on gene ontology analysis of RNA-seq data from E0771 DDR1 WT and KO tumors transplanted from Rag1- / - to C57BL / 6 hosts. Values ​​represent mean ± SEM, p-value, as shown. [Figure 8]Screening of huDDR1 neutralizing antibodies. (Figure 8a) Transwell migration assay of purified CD8+ T cells in the presence of conditional medium from endogenous DDR1 WT, DDR1 KO, and DDR1 KO-containing E0771 cells, as well as ectopic expression of huDDR1. (Figure 8b) Representative neutralizing antibody screening by CD8+ T cell migration assay using conditional medium from KO or KO+huDDR1 E0771 cells. Control: Isotyped IgG; Anti-huDDR1 antibodies: #3, #9, #14, and #33. (Figure 8c) Body weight measurement of mice treated with control (α-IgG) and anti-huDDR1 antibody #9 (n=5). (Figures 8d-e) KO+huDDR1 E0771 tumors in C57BL / 6 (Figure 8d) and Rag1- / - hosts (Figure 8e) treated with either isotyped IgG or anti-huDDR1 #33 antibody. (Figure 8f) The central region of KO+huDDR1 tumors was analyzed by SHG (gray), CD3 staining (green), To-pro-3 staining (red), and collagen fiber individualization (far right panel). Scale bar: 50 μm. (Figures 8g~i) Correlation between huDDR1 mRNA levels and immunocytotoxic marker genes IFNG (Figure 8g), GZMB (Figure 8h), and PRF1 (Figure 8i) in 1,093 breast cancer tumors in the TIMER database. Values ​​represent mean ± SEM, p-value, as shown. [Figure 9] Binding of DDR1-mAb to DDR1, as analyzed by ELISA. [Figure 10] Determination of antibody binding affinity to human and mouse DDR1 using titration ELISA. [Figure 11] The kinetic coupling curve of an anti-DDR1 antibody measured using an Octet instrument. [Figure 12]Anti-hECD antibodies inhibit spontaneous tumor growth. To demonstrate the efficacy of anti-DDR1 antibody therapy against breast cancer development at various stages, MMTV-PyMT mice (C57BL / 6 strain background) were treated with control IgG or anti-DDR1 antibody for 2 weeks when the mean tumor size reached 100 mm3 ("post-tumor"). (Figure 12A-B) Tumor growth dynamics (per mouse, Figure 12A) and tumor incidence (per mouse, Figure 12B) in a C57BL / 6 genetic background MMTV-PyMT spontaneous mammary tumor model treated with Ctrl (n=7) or humanized anti-DDR1#9 antibody (n=8) in the "post-tumor" scheme. (Figure 12C) Representative images of tumors from the post-tumor treated group, analyzed by SHG (gray), CD3 staining (green), To-pro-3 staining (red), and collagen fiber individualization (far right panel). Scale bar: 50 μm. [Figure 13] Comparison of anti-hECD antibodies and DDR1 kinase inhibitors. E0771 mammary gland tumors were treated with anti-hECD antibodies, and tumor growth (Figure 13A) and host body weight (Figure 13B) were evaluated. The previously published small molecule DDR1 kinase inhibitor 7rh did not reduce tumor growth (Figure 13C). This is consistent with the assertion that DDR1-dependent elimination of antitumor immunity is independent of its kinase activity (Figure 13D). 7rh did not affect host body weight (Figure 13E). Tumors treated with 7rh had substantially lower levels of DDR1 autophosphorylation, a marker of DDR1 tyrosine kinase activity (Gao et al., J Med Chem 2013). [Figure 14] DDR1 is required for the growth of several tumor types. (Figure 14A) CRISPR-based gene resection of tumor Ddr1 significantly increased the survival of immune hosts with ID8agg ovarian tumors. (Figures 14B-C) Ddr1 knockout in B16 melanoma or MC38 colorectal tumors did not affect tumor growth in syngeneic immune hosts. [Figure 15A]Correlation between DDR1 and antitumor immune markers. (Figure 15A) DDR1 mRNA levels in multiple cancers were negatively correlated with cytotoxic immune markers such as granzyme B (GZMB), suggesting that DDR1 may antagonize antitumor immunity in many cancer types. [Figure 15B] Correlation between DDR1 and antitumor immune markers. (Figure 15B) Analysis of the TCGA breast cancer proteome dataset (NCI CPTAC) showed that DDR1 protein levels also had a negative correlation with CD8 and proteins in the cytolytic effector pathway. [Figure 15C] Correlation between DDR1 and antitumor immune markers. (Figure 15C) Analysis of the TCGA breast cancer proteome dataset (NCI CPTAC) showed that DDR1 protein levels also had a negative correlation with CD8 and proteins in the cytolytic effector pathway. [Figure 16] High tumor DDR1 protein levels correlate with immunity in TNBCs. (Left) Images of multiplexed IHC for DDR1, CD8, and tumor-specific panCK using treatment-naive DDR1-high (n=7) and DDR1-low (n=5) TNBC tumor samples. Scale bar: 200 mm. Using treatment-naive TNBC cohorts in multiplexed IHC, DDR1-high tumors were shown to have a lower percentage of CD8+ T cells within the tumor and a higher percentage of CD8+ T cells at the tumor margin compared to DDR1-low tumors, where the percentage of CD8+ T cells distributed throughout the tumor was higher (right). [Figure 17] Schematic diagram of recombinant constructs of the DDR1 extracellular (ECD) protein domain for expression in HEK293 cells. DS: N-terminal discoidine domain; DSL: DS-like domain; JM: near-membrane domain. [Figure 18A]Determination of DDR1 antibody binding to ECD and DS or DSL domains using ELISA. Recombinant DDR1 extracellular (ECD) protein and domain protein were coated onto high-binding 96-well plates at a concentration of 2 μg / ml (Figure 18A). Binding curves and EC50 were determined using titration with DDR1-9Hu antibody. Deletion of the DS or DSL domain of DDR1 ECD resulted in reduced binding by humanized DDR1-9Hu antibody. Deletion of the DS domain alone reduced binding at an EC50 of 168 ng / ml vs. 83 ng / ml, while deletion of the DSL domain completely abolished binding with DDR1-9 (Figure 18B) and DDR1-14 antibodies (Figure 18C). [Figure 18B] See the explanation in Figure 18A. [Figure 18C] See the explanation in Figure 18A. [Figure 19] Determination of cross-reactivity of a panel of monoclonal antibodies against human DDR2 using ELISA. DDR1 or DDR2 ECD proteins were coated onto high-binding plates, and each monoclonal antibody was added at a concentration of 1 ug / ml for binding detection with HRP-conjugated anti-rabbit antibody (Jackson Immune Research, PA). [Modes for carrying out the invention]

[0026] Description of Exemplary Embodiments The inventors determined that tumor dyscoidine domain receptor 1 (DDR1) plays a crucial role in regulating host immunity. DDR1 is a collagen receptor with tyrosine kinase activity. The inventors found that DDR1 induces tumor defense, preventing host immune cells from infiltrating tumor tissue and attacking the tumor itself in a kinase-independent manner. The inventors have isolated a panel of novel monoclonal antibodies that recognize the DDR1 protein, which can be used for cancer treatment. Anti-human DDR1 antibodies are antagonists of DDR1 function and prevent the induction of DDR1-mediated tumor defense.

[0027] The following descriptions in this disclosure are intended merely to illustrate various embodiments of this disclosure. Therefore, any particular modifications considered should not be construed as limitations on the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure. Such equivalent embodiments are understood to be included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0028] I. Definition It should be understood that both the general description above and the detailed description below are illustrative and descriptive only and do not limit the claimed invention. In this application, unless otherwise specified, the use of the singular includes the plural. In this application, the use of “or” means “and / or” unless otherwise specified. Furthermore, the use of the term “including,” as well as other forms such as “includes” and “included,” is not limiting. Also, terms such as “element” or “component,” unless otherwise specified, include both elements and components containing one unit, and elements and components containing one or more subunits. Also, the use of the term “part” may include a portion of a part or the whole of a part.

[0029] As used herein, the singular forms "a," "an," and "the" include references to the plural unless the context clearly indicates otherwise.

[0030] When referring to measurable values ​​such as quantity or duration, the term “approximately” as used herein means that a variation of up to ±10% from the specified value is included. Unless otherwise indicated, all figures representing quantities such as components, properties such as molecular weight, and reaction conditions used herein and in the claims should be understood in all cases to be modified by the term “approximately.” Thus, unless otherwise indicated, the numerical parameters described in the following specification and the appended claims are approximations that may vary depending on the desired properties to be obtained by the disclosed subject matter. At the very least, and without attempting to limit the application of the equivalent view to the claims, each numerical parameter should be interpreted by applying the usual rounding technique in light of at least the number of significant digits reported. Although the numerical ranges and parameters describing the broad scope of the present invention are approximations, the numerical values ​​described in particular examples are reported as accurately as possible. However, every numerical value inherently contains a certain error that inevitably arises from the standard deviation found in their respective test measurements.

[0031] The term “antibody” refers to an intact immunoglobulin of any isotype, or their fragments that can compete with an intact antibody for specific binding to a target antigen, including, for example, chimeric, humanized, fully human, and bispecific antibodies. “Antibodies” are a class of antigen-binding proteins. Intact antibodies generally contain at least two full-length heavy chains and two full-length light chains, but in some cases, they may contain fewer chains, such as antibodies naturally occurring in camelids that may contain only heavy chains. Antibodies may originate from only a single source or they may be “chimeric,” i.e., different parts of an antibody may originate from two different antibodies, as further described below. Antigen-binding proteins, antibodies, or binding fragments may be produced in hybridomas by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. Unless otherwise indicated, the term “antibody” includes antibodies containing two full-length heavy chains and two full-length light chains, as well as their derivatives, variants, fragments, and mutaines, examples of which are listed below. Furthermore, unless explicitly excluded, antibodies include monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimes”), chimeric antibodies, humanized antibodies, human antibodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), and fragments thereof. In some embodiments, this term also includes peptide bodies.

[0032] Naturally occurring antibody structural units typically contain tetramers. Each such tetramer typically consists of two identical polypeptide chain pairs, each pair having one full-length "light" chain (about 25 kDa in certain embodiments) and one full-length "heavy" chain (about 50–70 kDa in certain embodiments). The amino-terminal portion of each chain typically contains a variable region of about 100–110 or more amino acids involved in antigen recognition. The carboxyl-terminal portion of each chain typically defines a constant region that may be involved in effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, defining antibody isotypes as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has several subclasses, including but not limited to IgG1, IgG2, IgG3, and IgG4. IgM has subclasses including, but not limited to, IgM1 and IgM2. IgA is similarly subdivided into subclasses including, but not limited to, IgA1 and IgA2. Within the full-length light and heavy chains, the variable and constant regions are typically linked by a "J" region of about 12 or more amino acids, and the heavy chain also includes a "D" region of about 10 or more amino acids. See, for example, Fundamental Immunology, Ch.7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989) (all of which are incorporated for all purposes by reference)). The variable region of each light / heavy chain pair typically forms the antigen-binding site.

[0033] The term "variable region" or "variable domain" refers to a portion of the light and / or heavy chain of an antibody, typically containing approximately 120–130 amino acids at the amino-terminus of the heavy chain and approximately 100–110 amino acids at the amino-terminus of the light chain. In certain embodiments, the variable regions of different antibodies can vary widely in amino acid sequence, even among antibodies of the same species. The variable region of an antibody typically determines the specificity of a particular antibody against its target.

[0034] The variable regions typically exhibit the same general structure as a relatively conserved framework region (FR) linked by three hypervariable regions, also called complementarity-determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework region, which allows binding to specific epitopes. From the N-terminus to the C-terminus, the variable regions of both the light and heavy chains typically contain the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The amino acid assignments to each domain typically follow the definitions in Kabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)), Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987) or Chothia et al., Nature, 342:878-883 (1989).

[0035] In certain embodiments, the antibody heavy chain binds to the antigen in the absence of the antibody light chain. In certain embodiments, the antibody light chain binds to the antigen in the absence of the antibody heavy chain. In certain embodiments, the antibody binding region binds to the antigen in the absence of the antibody light chain. In certain embodiments, the antibody binding region binds to the antigen in the absence of the antibody heavy chain. In certain embodiments, individual variable regions specifically bind to the antigen in the absence of other variable regions.

[0036] In certain embodiments, a definitive description of the CDR and identification of residues containing the antibody binding site are achieved by elucidating the structure of the antibody and / or the structure of the antibody-ligand complex. In certain embodiments, this can be achieved by any of the various techniques known to those skilled in the art, such as X-ray crystallography. In certain embodiments, the CDR region can be identified or approximated using various analytical methods. Examples of such methods include, but are not limited to, the Kabat definition, Chothia definition, AbM definition, IMGT definition, and contact definition.

[0037] The Kabat definition is a standard for numbering residues in antibodies and is typically used to identify CDR regions. See, for example, Johnson & Wu, Nucleic Acids Res., 28:214-8 (2000). The Chothia definition is similar to the Kabat definition, but takes into account the location of specific structural loop regions. See, for example, Chothia et al., J. Mol. Biol., 196:901-17 (1986) and Chothia et al., Nature, 342:877-83 (1989). The AbM definition uses an integrated set of computer programs developed by the Oxford Molecular Group to model antibody structures. For example, see Martin et al., Proc Natl Acad Sci (USA), 86:9268-9272 (1989), “AbM (trademark), A Computer Program for Modeling Variable Regions of Antibodies,” Oxford, UK, Oxford Molecular, Ltd. The AbM definition models the tertiary structure of an antibody from primary sequence using a combination of knowledge databases and ab initio methods, such as those described by Samudrala et al., “Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach,” in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198 (1999). The definition of contact is based on the analysis of available complex crystal structures. For example, see MacCallum et al., J. Mol. Biol., 5:732-45 (1996).The IMGT definition utilizes a unique numbering system that combines framework (FR) and CDR region definitions, structural data from X-ray diffraction studies, and hypervariable loop characterization, as described by Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol 27:55-77 (2003). In one preferred embodiment, the CDR sequence is based on the IMGT definition.

[0038] By convention, CDR regions in the heavy chain are typically designated H1, H2, and H3, and are sequentially numbered from the amino terminus to the carboxyl terminus. CDR regions in the light chain are typically designated L1, L2, and L3, and are sequentially numbered from the amino terminus to the carboxyl terminus. In this disclosure, CDR regions in the light chain variable region are also referred to as LC-CDR1, LC-CDR2, and LC-CDR3, and CDR regions in the heavy chain variable region are referred to as HC-CDR1, HC-CDR2, and HC-CDR3.

[0039] The term "light chain" includes full-length light chains and their fragments that have a variable region sequence sufficient to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of a light chain is located at the amino terminus of the polypeptide. Examples of light chains include kappa chains and lambda chains.

[0040] The term "heavy chain" includes full-length heavy chains and their fragments that have a variable region sequence sufficient to confer binding specificity. A full-length heavy chain comprises a variable region domain, VH, and three constant region domains, CH1, CH2, and CH3. The VH domain is located at the amino terminus of the polypeptide, the CH domain is at the carboxyl terminus, and CH3 is closest to the carboxyl terminus of the polypeptide. The heavy chain can be any isotype, including IgG (including IgG1, IgG2, IgG3, and IgG4 subtypes), IgA (including IgA1 and IgA2 subtypes), IgM, and IgE.

[0041] Bispecific or bifunctional antibodies are typically artificial hybrid antibodies that have two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including but not limited to hybridoma fusion or Fab' fragment conjugation. See, for example, Songsivilai et al., Clin. Exp. Immunol., 79:315-321 (1990) and Kostelny et al., J. Immunol., 148:1547-1553 (1992).

[0042] The term "antigen" refers to a substance that can induce an adaptive immune response. Specifically, an antigen is a substance that is targeted by a receptor for an adaptive immune response. Typically, an antigen is a molecule that binds to an antigen-specific receptor but cannot induce an immune response in the body on its own. Antigens are usually proteins or polysaccharides, but less frequently they can also be lipids. Preferred antigens include, but are not limited to, parts of bacteria (coat, capsule, cell wall, flagella, cilia, and toxins), viruses, and other microorganisms. Antigens also include tumor antigens, such as antigens produced by mutations in tumors. As used herein, antigens also include immunogens and haptens.

[0043] The term “antigen-binding protein” (“ABP” as used herein means any protein that binds to a particular target antigen. In this application, the particular target antigen is the DDR1 protein or a fragment thereof. “Antigen-binding protein” includes, but is not limited to, antibodies and their antigen-binding fragments. A peptide body is another example of an antigen-binding protein.

[0044] The term “antigen-binding fragment,” as used herein, refers to a portion of a protein that can specifically bind to an antigen. In certain embodiments, the antigen-binding fragment is derived from an antibody containing one or more CDRs, or from any other antibody fragment that binds to an antigen but does not contain an intact native antibody structure. In certain embodiments, the antigen-binding fragment is derived not from an antibody, but rather from a receptor. Examples of antigen-binding fragments include, but are not limited to, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, single-domain antibodies (sdAb), camel antibodies, or nanobodies, domain antibodies, and bivalent domain antibodies. In certain embodiments, the antigen-binding fragment can bind to the same antigen to which the parent antibody binds. In certain embodiments, the antigen-binding fragment may comprise one or more CDRs from a specific human antibody grafted onto a framework region from one or more different human antibodies. In certain embodiments, the antigen-binding fragment is derived from a receptor and contains one or more mutations. In certain embodiments, the antigen-binding fragment does not bind to the native ligand of the receptor from which the antigen-binding fragment originates.

[0045] The term "Fab fragment" includes one light chain and one heavy chain's CH1 and variable region. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule.

[0046] The term "Fab' fragment" comprises one light chain and a portion of one heavy chain containing a VH domain, a CH1 domain, and a region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains of two Fab' fragments to form an F(ab')2 molecule.

[0047] The "F(ab')2 fragment" contains two light chains and two heavy chains containing a portion of the constant region between the CH1 domain and the CH2 domain, such that an interchain disulfide bond is formed between the two heavy chains. Thus, the F(ab')2 fragment consists of two Fab' fragments held together by a disulfide bond between the two heavy chains.

[0048] The "Fc" region contains two heavy chain fragments, each containing the CH1 and CH2 domains of the antibody. These two heavy chain fragments are held together by two or more disulfide bonds and hydrophobic interactions of the CH3 domain.

[0049] The "Fv region" includes variable regions from both the heavy and light chains, but lacks a steady region.

[0050] A "single-chain antibody" is an Fv molecule in which the heavy chain and light chain variable regions are linked by a flexible linker to form a single polypeptide chain that forms an antigen-binding region. Single-chain antibodies are described in detail in International Patent Application Publication No. 88 / 01649, and U.S. Patents No. 4,946,778 and No. 5,260,203, which are incorporated by reference.

[0051] A "domain antibody" is an immunofunctional immunoglobulin fragment that contains only the variable region of the heavy chain or the variable region of the light chain. In some cases, two or more VH regions covalently bind to a peptide linker to create a bivalent domain antibody. The two VH regions of a bivalent domain antibody can target the same or different antigens.

[0052] A "bivalent antigen-binding protein" or "bivalent antibody" contains two antigen-binding sites. In some cases, the two binding sites have the same antigen specificity. Bivalent antigen-binding proteins and bivalent antibodies can be bispecific (see below). In certain embodiments, bivalent antibodies other than "multispecific" or "multifunctional" antibodies are typically understood to have identical binding sites.

[0053] A "multispecific antigen-binding protein" or "multispecific antibody" targets multiple antigens or epitopes.

[0054] "Bispecific," "dual-specific," or "bifunctional" antigen-binding proteins or antibodies are hybrid antigen-binding proteins or antibodies that have two different antigen-binding sites. Bispecific antigen-binding proteins and antibodies are a type of multispecific antigen-binding protein-antibody and can be produced by various methods, including but not limited to hybridoma fusion or Fab' fragment binding. See, for example, Songsivilai and Lachmann, 1990, Clin. Exp. Immunol. 79:315-321 and Kostelny et al., 1992, J. Immunol. 148:1547-1553. The two binding sites of a bispecific antigen-binding protein or antibody bind to two different epitopes that may be present on the same or different protein targets.

[0055] "Binding affinity" generally refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X for its partner Y can generally be expressed by a dissociation constant (Kd). Affinity can be measured by common methods known in the art, including those described herein. Low-affinity antibodies generally tend to bind slowly to antigens and dissociate easily, while high-affinity antibodies generally tend to bind more quickly to antigens and remain bound for longer. Various methods for measuring binding affinity are known in the art, and any of them can be used for the purposes of the present invention. Specific illustrative and exemplary embodiments for measuring binding affinity are described below.

[0056] An antibody that "specifically binds" to or "is specific to" a particular polypeptide or epitope on a particular polypeptide is an antibody that binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. For example, the DDR1-specific antibody of the present invention is specific to DDR1. In some embodiments, the antibody that binds to DDR1 has a concentration of 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example, 10 -9 M~10 -13 It has a dissociation constant (Kd) of M.

[0057] When used in the context of competing antigen-binding proteins (e.g., antibodies or their antigen-binding fragments) for the same epitope, the term “competing” means competition between antigen-binding proteins, as determined by an assay, where the antigen-binding protein being tested (e.g., an antibody or its antigen-binding fragment) prevents or inhibits (e.g., reduces) the specific binding of a reference antigen-binding protein (e.g., a ligand or reference antibody) to a common antigen (e.g., DDR1 or its fragment). Various types of competitive binding assays can be used to determine whether one antigen-binding protein is competing with others, for example, solid-phase direct or indirect radioimmunoassay (RIA), solid-phase direct or indirect enzyme immunoassay (EIA), sandwich competition assays (see, e.g., Stahli et al., 1983, Methods in Enzymology 9:242-253), solid-phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J.Immunol. 137:3614-3619), solid-phase direct labeling assays, solid-phase direct labeling sandwich assays (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press), and solid-phase direct labeling RIA using 1-125 labeling (e.g., Morel et al. Examples include solid-phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176:546-552), and directly labeled RIA (see, Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). Typically, such assays involve the use of purified antigens bound to a solid surface or cell having either an unlabeled test antigen-binding protein or a labeled reference antigen-binding protein. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cell in the presence of the test antigen-binding protein. Usually, the test antigen-binding protein is present in excess.Antigen-binding proteins identified by competitive assays (competitive antigen-binding proteins) include antigen-binding proteins that bind to the same epitope as the reference antigen-binding protein, and antigen-binding proteins that bind to adjacent epitopes that are sufficiently close to the epitope bound by the reference antigen-binding protein due to steric hindrance. Further details on methods for determining competitive binding are provided in the examples herein. Typically, when competitive antigen-binding proteins are present in excess, they inhibit (e.g., reduce) the specific binding of the reference antigen-binding protein to the common antigen by at least 40–45%, 45–50%, 50–55%, 55–60%, 60–65%, 65–70%, 70–75%, or more than 75%. In some cases, binding is inhibited by at least 80–85%, 85–90%, 90–95%, 95–97%, or more than 97%.

[0058] As used herein, the term "epitope" refers to a specific atom or amino acid group on an antigen to which an antibody binds. An epitope can be either a linear epitope or a structural epitope. Linear epitopes are formed by a continuous sequence of amino acids derived from the antigen and interact with the antibody based on their primary structure. Structural epitopes, on the other hand, consist of discontinuous fragments of the amino acid sequence of the antigen and interact with the antibody based on the 3D structure of the antigen. Generally, epitopes are approximately 5 or 6 amino acid long. Two antibodies may bind to the same epitope within an antigen if they exhibit competitive binding to that antigen.

[0059] As used herein, "cell" may refer to either a prokaryote or a eukaryote. Prokaryotic cells include, for example, bacteria. Eukaryotic cells include, for example, fungi, plant cells, and animal cells.Examples of animal cell types (e.g., mammalian cells or human cells) include, for example, cells from the circulatory / immune system or organs, e.g., B cells, T cells (cytotoxic T cells, natural killer T cells, regulatory T cells, helper T cells), natural killer cells, granulocytes (e.g., basophils, eosinophils, neutrophils, and hypersegmented neutrophils), monocytes or macrophages, erythrocytes (e.g., reticulocytes), mast cells, platelets or megakaryocytes, and dendritic cells; cells from the endocrine system or organs, e.g., thyroid cells (e.g., thyroid epithelial cells, parafollicular cells), parathyroid cells (e.g., For example, chief parathyroid cells (eosinophilic cells), adrenal cells (e.g., chromaffin cells), and pineal gland cells (e.g., pineal gland cells); cells from the nervous system or organs, for example, glioblasts (e.g., astrocytes and oligodendrocytes), microglia, large neuroendocrine cells, astrocytes, Betschel cells, and pituitary cells (e.g., gonadotropin-secreting cells, adrenocorticotropin-secreting cells, thyroid-stimulating hormone-secreting cells, growth hormone-secreting cells, and mammary gland-stimulating hormone-secreting cells); cells from the respiratory system or organs, for example, lung cells (Type I). Lung cells and type II lung cells), Clara cells, goblet cells, and alveolar macrophages; cells from the circulatory system or organs (e.g., cardiomyocytes and peripheral cells); cells from the digestive system or organs, e.g., chief cells, parietal cells, goblet cells, Paneth cells, G cells, D cells, ECL cells, I cells, K cells, S cells, enteroendocrine cells, enterochromaffin cells, APUD cells, and hepatocytes (e.g., hepatocytes and Kupffer cells); cells from the cortical system or organs, e.g., osteocytes (e.g., osteoblasts, osteocytes, and osteoclasts), tooth cells (e.g., cementum Examples include cells of the genital system or organs (e.g., blast cells and ameloblasts), chondrocytes (e.g., chondrocytes and chondrocytes), skin / hair cells (e.g., hair matrix cells, keratinocytes, and melanocytes (nevus cells)), muscle cells (e.g., myocytes), adipocytes, fibroblasts, and tendinocytes; cells from the urinary system or organs (e.g., podocytes, juxtaglomerular cells, intraglomerular mesangial cells, extraglomerular mesangial cells, renal proximal tubular brush margin cells, and macula densa cells), as well as cells from the reproductive system or organs (e.g., sperm, Sertoli cells, Leydig cells, oocytes, and oocytes).Cells may be normal and healthy cells, or diseased or unhealthy cells (e.g., cancer cells). Cells may further include mammalian juvenile cells, or stem cells, including embryonic stem cells, fetal stem cells, induced pluripotent stem cells, and adult stem cells. Stem cells are cells that can differentiate into specific cell types through a cycle of cell division while maintaining an undifferentiated state. Stem cells may be totipotent stem cells, pluripotent stem cells, multipotent stem cells, oligopotent stem cells, and unipotent stem cells, all of which can be induced from somatic cells. Stem cells may also include cancer stem cells. Mammalian cells may be rodent cells, e.g., mouse, rat, or hamster cells. Mammalian cells may be lagomorph cells, e.g., rabbit cells. Mammalian cells may also be primate cells, e.g., human cells.

[0060] As used herein, the terms “chimeric antigen receptor” or “CAR” refer to an artificially constructed hybrid protein or polypeptide containing an antigen-binding site (e.g., a single-chain variable region fragment (scFv)) of an antibody bound to a domain or signaling pathway that activates immune cells, e.g., T cells or NK cells (see, e.g., Kershaw et al., above; Eshhar et al., Proc. Natl. Acad. Sci. USA, 90(2):720-724 (1993); and Sadelain et al., Curr. Opin. Immunol. 21(2):215-223 (2009)). CARs can leverage the antigen-binding properties of monoclonal antibodies to redirect immune cell specificity and reactivity to selected targets in a non-MHC-restricted manner. Non-MHC-restricted antigen recognition confers the ability of immune cells expressing CARs to recognize antigens independently of antigen processing, thus bypassing the primary mechanism of tumor escape. In addition, when expressed on T cells, CARs do not dimerize favorably with the endogenous T cell receptor (TCR) alpha and beta chains.

[0061] As used herein, “essentially not present” with respect to a particular component means that none of the particular component is intentionally formulated in the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of any particular component resulting from any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferably, the composition is one in which the presence of the particular component cannot be detected using standard analytical methods.

[0062] The term "host cell" refers to a cell that has been transformed, or can be transformed, by a nucleic acid sequence to express the gene of interest. This term includes offspring of a parent cell, regardless of whether the offspring are morphologically or genetically identical to the original parent cell, as long as the gene of interest is present.

[0063] The term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, determined by aligning and comparing their sequences. "Identity percentage" means the percentage of identical residues between amino acids or nucleotides in the molecules being compared, calculated based on the smallest size of the molecules being compared. For these calculations, the alignment gap (if any) is preferably addressed by a specific mathematical model or computer program (i.e., an "algorithm"). Methods that can be used to calculate the identity of aligned nucleic acids or polypeptides include those described in Computational Molecular Biology, (Lesk, AM, ed.), 1988, New York: Oxford University Press; Biocomputing Informatics and Genome Projects, (Smith, DW, ed.), 1993, New York: Academic Press; Computer Analysis of Sequence Data, Part I, (Griffin, AM, and Griffin, HG, eds.), 1994, New Jersey: Humana Press; von Heinje, G., 1987, Sequence Analysis in Molecular Biology, New York: Academic Press; Sequence Analysis Primer, (Gribskov, M. and Devereux, J., eds.), 1991, New York: M. Stockton Press; and Carillo et al., 1988, SIAM J. Applied Math. 48:1073.

[0064] When calculating the percentage of identity, the sequences being compared are typically aligned in a way that yields the greatest match between them. One example of a computer program that can be used to determine the percentage of identity is the GCG program package, which includes GAP (Devereux et al., 1984, Nucl. Acid Res. 12:387, Genetics Computer Group, University of Wisconsin, Madison, Wis.). The GAP computer algorithm is used to align two polypeptides or polynucleotides for which the percentage of sequence identity is determined. The sequences are aligned for the best possible matching of each amino acid or nucleotide (the "matched span" determined by the algorithm). A gap-start penalty (calculated as 3 times the mean diagonal, where the "mean diagonal" is the average of the diagonals of the comparison matrix used, and the "diagonal" is the score or number assigned to each perfect amino acid match by a particular comparison matrix), and a gap-extension penalty (usually 1 / 10 of the gap-start penalty), as well as comparison matrices such as PAM250 or BLOSUM62, are used in conjunction with the algorithm. In certain embodiments, standard comparison matrices are also used by the algorithm (see Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix, and Henikoff et al., 1992, Proc. Natl. Acad. Sci. USA 89:10915-10919 for the BLOSUM 62 comparison matrix).

[0065] Examples of parameters that can be used to determine the identity percentage of polypeptides or nucleotide sequences using the GAP program can be found in Needleman et al., 1970, J.Mol.Biol.48:443-453.

[0066] A particular alignment scheme for aligning two amino acid sequences may result in matching only short regions of the two sequences, and this aligned small region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Therefore, the selected alignment method (GAP program) can be adjusted if it is desired to thus result in alignment over at least 50 or more consecutive amino acids of the target polypeptide.

[0067] As used herein, the term “bonding” refers to intramolecular interactions, such as covalent bonds, metallic bonds, and / or ionic bonds, or associations via intermolecular interactions, such as hydrogen bonds or non-covalent bonds.

[0068] The term "operatably linked" refers to the arrangement of elements configured so that the components described in this way perform their normal functions. Thus, a given signal peptide operatably linked to a polypeptide directs the secretion of the polypeptide from the cell. In the case of a promoter, a promoter operatably linked to a coding sequence directs the expression of the coding sequence. Promoters or other regulatory elements do not need to be adjacent to the coding sequence insofar as they function to direct its expression. For example, an intervening untranslated but transcribed sequence can exist between the promoter sequence and the coding sequence, and the promoter sequence can still be considered "operatably linked" to the coding sequence.

[0069] The use of the term “or” in the claims is used to mean “and / or” unless expressly indicated to mean only the alternatives, or unless the alternatives are mutually exclusive; however, this disclosure supports the definition that refers only to the alternatives and “and / or.” As used herein, “another” may mean at least a second one, or more.

[0070] The terms "polynucleotide" or "nucleic acid" include both single-chain and double-chain nucleotide polymers. Nucleotides containing polynucleotides can be ribonucleotides or deoxyribonucleotides, or modified forms of either type of nucleotide. The aforementioned modifications include base modifications such as bromouridine and inosine derivatives, ribose modifications such as 2',3'-dideoxyribose, and internucleotide ligation modifications such as phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphoranilothioates, phosphoraniradiates, and phosphoramidates.

[0071] The terms “polypeptide” or “protein” mean macromolecules having the amino acid sequence of a natural protein, i.e., proteins produced by naturally occurring non-recombinant cells, or molecules produced by genetically engineered or recombinant cells having the amino acid sequence of a natural protein, or molecules having one or more amino acid deletions, additions, and / or substitutions of the natural sequence. The term also includes amino acid polymers, in which one or more amino acids are chemical analogs of the corresponding naturally occurring amino acids and polymers. The terms “polypeptide” and “protein” specifically encompass DDR1 antigen-binding proteins, antibodies, or sequences having one or more amino acid deletions, additions, and / or substitutions of an antigen-binding protein. The term “polypeptide fragment” refers to polypeptides having amino-terminal deletions, carboxyl-terminal deletions, and / or internal deletions compared to the full-length natural protein. Such fragments may also contain modified amino acids compared to the natural protein. In certain embodiments, the fragments are about 5 to 500 amino acid long. For example, a fragment may be at least 5, 6, 8, 10, 14, 20, 50, 70, 100, 110, 150, 200, 250, 300, 350, 400, or 450 amino acids long. Useful polypeptide fragments include immunofunctional fragments of an antibody containing a binding domain. In the case of DDR1-binding antibodies, useful fragments include, but are not limited to, the CDR region, variable domains of the heavy and / or light chain, a portion of the antibody chain, or just the variable region containing two CDRs.

[0072] The pharmaceutically acceptable carriers useful in the present invention are conventional ones. Remington's Pharmaceutical Sciences, by EW Martin, Mack Publishing Co., Easton, PA, 15th Edition (1975) describes compositions and formulations suitable for the pharmaceutically acceptable delivery of the fusion proteins disclosed herein. Generally, the properties of the carrier will depend on the specific mode of administration used. For example, parenteral formulations typically contain an injectable fluid as a vehicle, which includes a pharmaceutically and physiologically acceptable fluid such as water, saline, equilibrium salt solution, aqueous dextrose, or glycerol. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the administered pharmaceutically acceptable composition may contain small amounts of non-toxic adjuncts, such as wetting or emulsifying agents, preservatives, and pH buffers, such as sodium acetate or sorbitan monolaurate.

[0073] As used herein, the term “subject” refers to a human or any non-human animal (e.g., a mouse, rat, rabbit, dog, cat, cattle, pig, sheep, horse, or primate). Human includes prenatal and postnatal forms. In many embodiments, the subject is a human. The subject may be a patient, referring to a human being presented to a healthcare provider for the diagnosis or treatment of a disease. The term “subject” is used herein as synonymous with “individual” or “patient.” The subject may have a disease or disorder, or may be susceptible to a disease or disorder, and may or may not exhibit symptoms thereof.

[0074] As used herein, the terms “therapeutic dose” or “effective dose” refer to the dosage or concentration of a drug that is effective in treating a disease or condition. For example, with respect to the use of a monoclonal antibody or its antigen-binding fragment disclosed herein for the treatment of cancer, the therapeutic dose is the dosage or concentration of a monoclonal antibody or its antigen-binding fragment that enables a reduction in tumor volume, eradication of all or part of a tumor, inhibition or delay of tumor growth or invasion of cancer cells into other organs, inhibition of the growth or proliferation of cells mediating the cancerous condition, inhibition or delay of tumor cell metastasis, improvement of any symptoms or markers associated with a tumor or cancerous condition, prevention or delay of the onset of a tumor or cancerous condition, or a combination thereof.

[0075] As used herein, “treatment” or “procedure” for a condition includes preventing or alleviating a condition, slowing the onset or rate of onset of a condition, reducing the risk of developing a condition, preventing or delaying the onset of symptoms associated with a condition, reducing or terminating symptoms associated with a condition, producing complete or partial regression of a condition, curing a condition, or any combination thereof.

[0076] As used herein, “vector” refers to a nucleic acid molecule that is introduced into a host cell to produce a transformed host cell. A vector may contain nucleic acid sequences that enable replication in the host cell, such as an origin of replication. A vector may also contain one or more therapeutic genes and / or selectable marker genes and other genetic elements known in the art. A vector can transduce, transform, or infect a cell, thereby causing the cell to express nucleic acids and / or proteins that are not native to the cell. A vector may optionally contain materials that assist in achieving entry of nucleic acids into the cell, such as viral particles, liposomes, or protein coatings.

[0077] II. DDR1 and DDR1 Antibodies A.DDR1 Receptor tyrosine kinases (RTKs) play a crucial role in communication between cells and their microenvironment. These molecules are involved in regulating cell growth, differentiation, and metabolism. The DDR1 protein, encoded by the DDR1 gene, is an RTK 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 that has a region homologous to the Dictyostelium discoideum protein discoidin I within its extracellular domain. Its autophosphorylation is achieved by all collagens (types I-VI) tested to date. A closely related family member is the DDR2 protein. In situ studies and Northern blot analyses have shown that the expression of the DDR1-encoded protein is limited 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. This gene is located on chromosome 6p21.3, close to several HLA class I genes. Alternative splicing of this gene results in multiple transcriptional variants. The representative mRNA sequence of DDR1 is NM_001202521 (SEQ ID NO: 1), and the representative amino acid sequence is NP_001189450 (SEQ ID NO: 2).

[0078] Antibody against B.DDR1 protein The antibodies or antigen-binding fragments provided in this disclosure can first be defined by their binding specificity to DDR1, in this case. A person skilled in the art can determine whether such an antibody falls within the scope of the claims by evaluating the binding specificity / affinity of a given antibody using techniques well known to those skilled in the art.

[0079] In one aspect, antibodies and antigen-binding fragments that specifically bind to DDR1 are provided. In some embodiments, such antibodies modulate the activation of DDR1 when bound to DDR1. In certain embodiments, the antibody or antigen-binding fragment activates DDR1 when bound to DDR1. In certain embodiments, the antibody or antigen-binding fragment inhibits the activation of DDR1 when bound to DDR1. In certain embodiments, the antibody or antigen-binding fragment can specifically interfere with, block, or reduce the interaction between DDR1 and its binding partner when bound to DDR1. In certain embodiments, the antibodies or antigen-binding fragments provided herein specifically or selectively bind to human DDR1.

[0080] In some embodiments, the antibody or antigen-binding fragment specifically binds to human DDR1 and / or substantially inhibits the binding of human DDR1 to its binding partner by at least about 20% - 40%, 40 - 60%, 60 - 80%, 80 - 85%, or more (e.g., by the assays disclosed in the examples). In some embodiments, the antibody or antigen-binding fragment has a Kd less than (more tightly binding) 10 -7 、10 -8 、10 -9 、10 -10 、10 -11 、10 -12 、10 -13 M.

[0081] The antibodies described herein are produced as IgG, but it may be useful to modify their constant regions to alter their function. The constant region of an antibody typically mediates the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Thus, the term “antibody” includes intact immunoglobulins of the types IgA, IgG, IgE, IgD, and IgM (and their subtypes), and the light chain of an immunoglobulin may be of the kappa or lambda type. Within the light and heavy chains, the variable and constant regions are linked by “J” regions of about 12 or more amino acids, and the heavy chain also includes “D” regions of about 10 or more amino acids. Generally, see Fundamental Immunology Ch.7 (Paul, W., ed., 2 nd See ed. Raven Press, NY (1989).

[0082] In some embodiments, the Disclosure provides an antibody or antigen-binding fragment that specifically binds to the DDR1 protein, wherein the antibody comprises LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region sequences shown in Table 3, and HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region sequences shown in Table 4, or variants thereof, wherein one or more of the LC-CDR and / or HC-CDRs have one, two, or three amino acid substitutions, additions, deletions, or combinations.

[0083] In some embodiments, the isolated antibody or its antigen-binding fragment includes LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region sequences shown in Table 3, and HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region sequences shown in Table 4, or variants thereof, in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations, where the light chain variable region sequences and heavy chain variable region sequences are, respectively, the light chain variable region sequences and heavy chain variable region sequences of a clone pair (e.g., of the same mAb designation) shown in Tables 3 and 4. Exemplary embodiments of the "clone pairs" of light chain variable region sequences LC-CDR1, LC-CDR2, and LC-CDR3, as well as the heavy chain variable region sequences HC-CDR1, HC-CDR2, and HC-CDR3, are the LC-CDR and HC-CDR of the light chain variable region sequence and heavy chain variable region sequence of mAb DDR1-1, respectively.

[0084] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1, LC-CDR2, and LC-CDR3 selected from the LC-CDR1, LC-CDR2, and LC-CDR3 sequences for each mAb shown in Table 1, and a heavy chain variable region having HC-CDR1, HC-CDR2, and HC-CDR3 selected from the HC-CDR1, HC-CDR2, and HC-CDR3 sequences for each mAb shown in Table 2, or variants thereof, which include variants having one, two, or three amino acid substitutions, additions, deletions, or combinations of one or more LC-CDRs and / or HC-CDRs. In some embodiments, the isolated antibody or its antigen-binding fragment includes the light chain variable regions having LC-CDR1, LC-CDR2, and LC-CDR3, and the heavy chain variable regions having HC-CDR1, HC-CDR2, and HC-CDR3, respectively, of the LC-CDR and HC-CDR of the clone pairs shown in Tables 1 and 2, or variants thereof, which include variants having one, two, or three amino acid substitutions, additions, deletions, or combinations of one or more of the LC-CDR and / or HC-CDR.

[0085] (Table 1) CDR of the light chain amino acid variable region sequence of DDR1 antibodies TIFF0007883266000001.tif190145

[0086] (Table 2) CDR of the heavy chain amino acid variable region sequence of DDR1 antibodies TIFF0007883266000002.tif190157

[0087] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QNIYSN (SEQ ID NO: 3), LC-CDR2 containing the amino acid sequence GAS, and LC-CDR3 containing the amino acid sequence QSGYYSSSTDIA (SEQ ID NO: 4), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFSLSRYA (SEQ ID NO: 45), HC-CDR2 containing the amino acid sequence IGSSGLT (SEQ ID NO: 46), and HC-CDR3 containing the amino acid sequence ARGMWYDDSDDYEDYFNL (SEQ ID NO: 47) (DDR1-1), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0088] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QTISSW (SEQ ID NO: 5), LC-CDR2 containing the amino acid sequence YAF, and LC-CDR3 containing the amino acid sequence QQGISSSNVDNV (SEQ ID NO: 6), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GIDLSSYA (SEQ ID NO: 48), HC-CDR2 containing the amino acid sequence INIGGGT (SEQ ID NO: 49), and HC-CDR3 containing the amino acid sequence ARDVDAHTLTYFTL (SEQ ID NO: 50) (DDR1-3), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0089] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QTISSW (SEQ ID NO: 7), LC-CDR2 containing the amino acid sequence YAF, and LC-CDR3 containing the amino acid sequence QCTYGSGSSSSYGCA (SEQ ID NO: 8), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFTLSNNA (SEQ ID NO: 51), HC-CDR2 containing the amino acid sequence IYASGRT (SEQ ID NO: 52), and HC-CDR3 containing the amino acid sequence ARGDTETDYGIPYFDL (SEQ ID NO: 53) (DDR1-5), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0090] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QSVYSNY (SEQ ID NO: 9), LC-CDR2 containing the amino acid sequence ETS, and LC-CDR3 containing the amino acid sequence QGGYSEIIENT (SEQ ID NO: 10), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFSFSSSYY (SEQ ID NO: 54), HC-CDR2 containing the amino acid sequence IYASSGST (SEQ ID NO: 55), and HC-CDR3 containing the amino acid sequence AILGADYRLTRLDL (SEQ ID NO: 56) (DDR1-6), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0091] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QSIGSV (SEQ ID NO: 11), LC-CDR2 containing the amino acid sequence GVF, and LC-CDR3 containing the amino acid sequence QYIPYGSSP (SEQ ID NO: 12), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFSLNRYY (SEQ ID NO: 57), HC-CDR2 containing the amino acid sequence ISYGDTT (SEQ ID NO: 58), and HC-CDR3 containing the amino acid sequence ARADTGDNGYLGLQL (SEQ ID NO: 59) (DDR1-9), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations.

[0092] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QSIGSTY (SEQ ID NO: 13), LC-CDR2 containing the amino acid sequence KAS, and LC-CDR3 containing the amino acid sequence LYGGFGSSTGDA (SEQ ID NO: 14), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFSFSSGYY (SEQ ID NO: 60), HC-CDR2 containing the amino acid sequence IYTGRTDFT (SEQ ID NO: 61), and HC-CDR3 containing the amino acid sequence ARGDYSGGVGGNYWLDL (SEQ ID NO: 62) (DDR1-11), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0093] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QTIYSN (SEQ ID NO: 15), LC-CDR2 containing the amino acid sequence QAS, and LC-CDR3 containing the amino acid sequence QSYYGADDYT (SEQ ID NO: 16), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GIDLSNTW (SEQ ID NO: 63), HC-CDR2 containing the amino acid sequence ITDSGTT (SEQ ID NO: 64), and HC-CDR3 containing the amino acid sequence GRDPGDITSGTNDL (SEQ ID NO: 65) (DDR1-12), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0094] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence KSVYNNNA (SEQ ID NO: 17), LC-CDR2 containing the amino acid sequence GVS, and LC-CDR3 containing the amino acid sequence AGDYSDISDNN (SEQ ID NO: 18), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence SGFSLNNY (SEQ ID NO: 66), HC-CDR2 containing the amino acid sequence IFNNGDI (SEQ ID NO: 67), and HC-CDR3 containing the amino acid sequence ARTGYRTGGWL (SEQ ID NO: 68) (DDR1-13), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0095] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QSISSY (SEQ ID NO: 19), LC-CDR2 containing the amino acid sequence EAS, and LC-CDR3 containing the amino acid sequence QNNNGFSGSNFNN (SEQ ID NO: 20), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GIDLSYYA (SEQ ID NO: 69), HC-CDR2 containing the amino acid sequence INGRGDT (SEQ ID NO: 70), and HC-CDR3 containing the amino acid sequence AREDSAIPFIVGNYYGMDL (SEQ ID NO: 71) (DDR1-14), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0096] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QTIYSS (SEQ ID NO: 21), LC-CDR2 containing the amino acid sequence KAS, and LC-CDR3 containing the amino acid sequence QQGSSISNVDKNA (SEQ ID NO: 22), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence TFSFNSRYW (SEQ ID NO: 72), HC-CDR2 containing the amino acid sequence INNGIS (SEQ ID NO: 73), and HC-CDR3 containing the amino acid sequence AKGGNLAGDCYGL (SEQ ID NO: 74) (DDR1-15), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0097] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QSIGSY (SEQ ID NO: 23), LC-CDR2 containing the amino acid sequence EAS, and LC-CDR3 containing the amino acid sequence QNNNGMTVSDFNA (SEQ ID NO: 24), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFSLNRYA (SEQ ID NO: 75), HC-CDR2 containing the amino acid sequence IGSSGST (SEQ ID NO: 76), and HC-CDR3 containing the amino acid sequence ARDLDDSYGYTYATGMDIRLDL (SEQ ID NO: 77) (DDR1-17), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0098] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QIIDHDH (SEQ ID NO: 25), LC-CDR2 containing the amino acid sequence RAS, and LC-CDR3 containing the amino acid sequence QNNNGMTVSDFNA (SEQ ID NO: 26), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFSLSDYA (SEQ ID NO: 78), HC-CDR2 containing the amino acid sequence INSRDDT (SEQ ID NO: 79), and HC-CDR3 containing the amino acid sequence AREDSSIPFIVGNYYGMDL (SEQ ID NO: 80) (DDR1-20), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0099] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QSVVDKNW (SEQ ID NO: 27), LC-CDR2 containing the amino acid sequence EAS, and LC-CDR3 containing the amino acid sequence AGDFESGVSG (SEQ ID NO: 28), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFSLSSYG (SEQ ID NO: 81), HC-CDR2 containing the amino acid sequence IYPSGSI (SEQ ID NO: 82), and HC-CDR3 containing the amino acid sequence VRYLTGSSDLHL (SEQ ID NO: 83) (DDR1-21), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0100] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence KNIYNNNA (SEQ ID NO: 29), LC-CDR2 containing the amino acid sequence GAS, and LC-CDR3 containing the amino acid sequence AADYSDISDNN (SEQ ID NO: 30), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFSLSDYA (SEQ ID NO: 84), HC-CDR2 containing the amino acid sequence INGDIY (SEQ ID NO: 85), and HC-CDR3 containing the amino acid sequence ARPGYRTGIWL (SEQ ID NO: 86) (DDR1-22), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0101] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QSVYSNNY (SEQ ID NO: 31), LC-CDR2 containing the amino acid sequence AAS, and LC-CDR3 containing the amino acid sequence LGGYNDDAN (SEQ ID NO: 32), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFDLRSYYY (SEQ ID NO: 87), HC-CDR2 containing the amino acid sequence IHGGEGNT (SEQ ID NO: 88), and HC-CDR3 containing the amino acid sequence RGGWTNYF (SEQ ID NO: 89) (DDR1-23), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0102] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence ESVYSNNH (SEQ ID NO: 33), LC-CDR2 containing the amino acid sequence AAS, and LC-CDR3 containing the amino acid sequence LGGYNDDAN (SEQ ID NO: 34), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFDLSSNYY (SEQ ID NO: 90), HC-CDR2 containing the amino acid sequence IYSSNTRT (SEQ ID NO: 91), and HC-CDR3 containing the amino acid sequence RGGWTNYL (SEQ ID NO: 92) (DDR1-26), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0103] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QSIDNND (SEQ ID NO: 35), LC-CDR2 containing the amino acid sequence RTS, and LC-CDR3 containing the amino acid sequence QSYCVNTYGYT (SEQ ID NO: 36), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFSLSSHD (SEQ ID NO: 93), HC-CDR2 containing the amino acid sequence IISSGNT (SEQ ID NO: 94), and HC-CDR3 containing the amino acid sequence ARDVYSGASP (SEQ ID NO: 95) (DDR1-28), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0104] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence QSISNH (SEQ ID NO: 37), LC-CDR2 containing the amino acid sequence RAS, and LC-CDR3 containing the amino acid sequence QSYYIINRSNYANS (SEQ ID NO: 38), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence TFSFNSRYW (SEQ ID NO: 96), HC-CDR2 containing the amino acid sequence INNGSDIT (SEQ ID NO: 97), and HC-CDR3 containing the amino acid sequence AKGGNLAGDCYGL (SEQ ID NO: 98) (DDR1-29), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0105] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence ESINSW (SEQ ID NO: 39), LC-CDR2 containing the amino acid sequence DAS, and LC-CDR3 containing the amino acid sequence QSYYIINRSNYGNS (SEQ ID NO: 40), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFSLSSYY (SEQ ID NO: 99), HC-CDR2 containing the amino acid sequence ITTAGPL (SEQ ID NO: 100), and HC-CDR3 containing the amino acid sequence ARGHAGSIYYSYFDL (SEQ ID NO: 101) (DDR1-32), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0106] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence ETISSR (SEQ ID NO: 41), LC-CDR2 containing the amino acid sequence QAS, and LC-CDR3 containing the amino acid sequence QGCYYGGGSFYDSA (SEQ ID NO: 42), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFSLSSYD (SEQ ID NO: 102), HC-CDR2 containing the amino acid sequence SWNSGFV (SEQ ID NO: 103), and HC-CDR3 containing the amino acid sequence ARLGADDIYYFNL (SEQ ID NO: 104) (DDR1-33), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0107] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence ETISSR (SEQ ID NO: 41), LC-CDR2 containing the amino acid sequence QAS, and LC-CDR3 containing the amino acid sequence QGCYYGGGSFYDSA (SEQ ID NO: 42), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFSLSSYD (SEQ ID NO: 102), HC-CDR2 containing the amino acid sequence SWNSGFV (SEQ ID NO: 103), and HC-CDR3 containing the amino acid sequence ARLGADDIYYFNL (SEQ ID NO: 104) (DDR1-33), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0108] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1 containing the amino acid sequence ENLYKDNY (SEQ ID NO: 43), LC-CDR2 containing the amino acid sequence GAS, and LC-CDR3 containing the amino acid sequence AGGYDSVVD (SEQ ID NO: 44), and a heavy chain variable region having HC-CDR1 containing the amino acid sequence GFDLSSYYY (SEQ ID NO: 105), HC-CDR2 containing the amino acid sequence SIYTSSGAT (SEQ ID NO: 106), and HC-CDR3 containing the amino acid sequence RGGWCDFNL (SEQ ID NO: 107) (DDR1-34), or variants thereof, including variants in which one or more of the LC-CDR and / or HC-CDR have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof.

[0109] In some embodiments, the LC-CDRs in Table 1 and the HC-CDRs in Table 2 are encoded by the polynucleotides in Tables 6 and 7, respectively, as will be further discussed below.

[0110] In some embodiments, the LC-CDR and HC-CDR are placed in a suitable context of the framework region (FR) sequence to form light chain variable regions and heavy chain variable regions that define the antibody binding specificity. In some embodiments, the framework region sequence of the antibody having the identified LC-CDR and HC-CDR is the framework sequence of the original antibody isolate. In some embodiments, the LC-CDR and HC-CDR are used in conjunction with framework region sequences derived from different mammalian species, e.g., primates. In some embodiments, the framework sequence is a humanized or human framework sequence for forming an antibody that specifically binds to the DDR1 protein. In some embodiments, the light chain variable region is, for example, the FR LC 1. FR LC 2. FR LC 3, and FR LC It includes four light chain framework regions designated as 4, and LC-CDR, and follows the following configuration in the direction from NH2 to COOH: FR LC 1--LC-CDR1--FR LC 2-LC-CDR2-FR LC 3,-LC-CDR3-FR LC 4. In some embodiments, the heavy chain variable region is, for example, FR HC 1. FR HC 2. FR HC 3, and FR HC It includes four heavy chain framework regions designated as 4, and HC-CDR, and follows the following configuration in the direction from NH2 to COOH: FR HC 1--HC-CDR1--FR HC 2-HC-CDR2-FR HC 3,-HC-CDR3-FR HC 4. In some embodiments, the framework region of the parent light chain variable region is replaced with the framework region of the human light chain variable region to form a humanized light chain variable region. In some embodiments, the framework region of the parent heavy chain variable region is replaced with the framework region of the human heavy chain variable region to form a humanized heavy chain variable region.

[0111] In some embodiments, an isolated antibody or antigen-binding fragment that specifically binds to the DDR1 protein includes a light chain variable region having the sequence shown in Table 3, i.e., a light chain variable region amino acid sequence selected from SEQ ID NOs: 108-128. In some embodiments, an isolated antibody or antigen-binding fragment that specifically binds to the DDR1 protein includes a heavy chain variable region having the sequence shown in Table 4, i.e., a heavy chain variable region amino acid sequence selected from SEQ ID NOs: 129-149.

[0112] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having a light chain variable region amino acid sequence selected from SEQ ID NOs: 108-128 and a heavy chain variable region having a heavy chain variable region amino acid sequence selected from SEQ ID NOs: 129-149. In various embodiments, any one of the variable light chain amino acid sequences corresponding to SEQ ID NOs: 108-128 can be used together with any one of the variable heavy chain amino acid sequences corresponding to SEQ ID NOs: 129-149.

[0113] In some embodiments, the isolated antibody or its antigen-binding fragment includes a light chain variable region having a light chain variable region amino acid sequence selected from SEQ ID NOs: 108-128 and a heavy chain variable region having a heavy chain variable region amino acid sequence selected from SEQ ID NOs: 129-149. In various embodiments, any one of the variable light chain amino acid sequences corresponding to SEQ ID NOs: 108-128 can be used together with any one of the variable heavy chain amino acid sequences corresponding to SEQ ID NOs: 129-149. In some embodiments, the antibody or its antigen-binding fragment that specifically binds to the DDR1 protein includes the light chain variable region and heavy chain variable region of the light chain variable region amino acid sequence and heavy chain variable region amino acid sequence of the clone pair shown in Tables 3 and 4, respectively.

[0114] (Table 3) Amino acid sequence of the light chain variable region of anti-DDR1 antibody TIFF0007883266000003.tif245157TIFF0007883266000004.tif26157

[0115] (Table 4) Amino acid sequence of the heavy chain variable region of anti-DDR1 antibody TIFF0007883266000005.tif185157TIFF0007883266000006.tif89157

[0116] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-1K) having the amino acid sequence of SEQ ID NO: 108 and a heavy chain variable region (DDR1-1H) having the amino acid sequence of SEQ ID NO: 129.

[0117] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-3K) having the amino acid sequence of SEQ ID NO: 109 and a heavy chain variable region (DDR1-3H) having the amino acid sequence of SEQ ID NO: 130.

[0118] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-5K) having the amino acid sequence of SEQ ID NO: 110 and a heavy chain variable region (DDR1-5H) having the amino acid sequence of SEQ ID NO: 131.

[0119] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-6K) having the amino acid sequence of SEQ ID NO: 111 and a heavy chain variable region (DDR1-6H) having the amino acid sequence of SEQ ID NO: 132.

[0120] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-9K) having the amino acid sequence of SEQ ID NO: 112 and a heavy chain variable region (DDR1-9H) having the amino acid sequence of SEQ ID NO: 133.

[0121] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-11K) having the amino acid sequence of SEQ ID NO: 113 and a heavy chain variable region (DDR1-11H) having the amino acid sequence of SEQ ID NO: 134.

[0122] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-12K) having the amino acid sequence of SEQ ID NO: 114 and a heavy chain variable region (DDR1-12H) having the amino acid sequence of SEQ ID NO: 135.

[0123] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-13K) having the amino acid sequence of SEQ ID NO: 115 and a heavy chain variable region (DDR1-13H) having the amino acid sequence of SEQ ID NO: 136.

[0124] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-14K) having the amino acid sequence of SEQ ID NO: 116 and a heavy chain variable region (DDR1-14H) having the amino acid sequence of SEQ ID NO: 137.

[0125] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-15K) having the amino acid sequence of SEQ ID NO: 117 and a heavy chain variable region (DDR1-15H) having the amino acid sequence of SEQ ID NO: 138.

[0126] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-17K) having the amino acid sequence of SEQ ID NO: 118 and a heavy chain variable region (DDR1-17H) having the amino acid sequence of SEQ ID NO: 139.

[0127] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-20K) having the amino acid sequence of SEQ ID NO: 119 and a heavy chain variable region (DDR1-20H) having the amino acid sequence of SEQ ID NO: 140.

[0128] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-21K) having the amino acid sequence of SEQ ID NO: 120 and a heavy chain variable region (DDR1-21H) having the amino acid sequence of SEQ ID NO: 141.

[0129] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-22K) having the amino acid sequence of SEQ ID NO: 121 and a heavy chain variable region (DDR1-22H) having the amino acid sequence of SEQ ID NO: 142.

[0130] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-23K) having the amino acid sequence of SEQ ID NO: 122 and a heavy chain variable region (DDR1-23H) having the amino acid sequence of SEQ ID NO: 143.

[0131] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-26K) having the amino acid sequence of SEQ ID NO: 123 and a heavy chain variable region (DDR1-26H) having the amino acid sequence of SEQ ID NO: 144.

[0132] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-28K) having the amino acid sequence of SEQ ID NO: 124 and a heavy chain variable region (DDR1-28H) having the amino acid sequence of SEQ ID NO: 145.

[0133] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-29K) having the amino acid sequence of SEQ ID NO: 125 and a heavy chain variable region (DDR1-29H) having the amino acid sequence of SEQ ID NO: 146.

[0134] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-32K) having the amino acid sequence of SEQ ID NO: 126 and a heavy chain variable region (DDR1-32H) having the amino acid sequence of SEQ ID NO: 147.

[0135] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-33K) having the amino acid sequence of SEQ ID NO: 127 and a heavy chain variable region (DDR1-33H) having the amino acid sequence of SEQ ID NO: 148.

[0136] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region (DDR1-34K) having the amino acid sequence of SEQ ID NO: 128 and a heavy chain variable region (DDR1-34H) having the amino acid sequence of SEQ ID NO: 149.

[0137] In some embodiments, the antibody or its antigen-binding fragment is a humanized antibody of the parent antibody DDR1-9 described in the examples. The light chain variable region and heavy chain variable region are shown in Table 5. The polynucleotide sequences encoding the humanized variable region are shown in Table 10 below.

[0138] (Table 5) Amino acid sequences of humanized DDR1-9hu antibodies TIFF0007883266000007.tif47156

[0139] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1, LC-CDR2, and LC-CDR3 within the light chain variable region amino acid sequence of SEQ ID NO: 150 (DDR1-9hu_Lv1). In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1, LC-CDR2, and LC-CDR3 within the light chain variable region amino acid sequence of SEQ ID NO: 151 (DDR1-9hu_Lc2). In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region having HC-CDR1, HC-CDR2, and HC-CDR3 within the heavy chain variable region amino acid sequence of SEQ ID NO: 151 (DDR1-9hu_Lc2). In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region having LC-CDR1, LC-CDR2, and LC-CDR3 in the light chain variable region amino acid sequence of SEQ ID NO: 150 (DDR1-9hu_Lv1) or SEQ ID NO: 151 (DDR1-9hu_Lc2), and a heavy chain variable region having HC-CDR1, HC-CDR2, and HC-CDR3 in the heavy chain variable region amino acid sequence of SEQ ID NO: 151 (DDR1-9hu_Lc2).

[0140] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region having the amino acid sequence of SEQ ID NO: 150 (DDR1-9hu_Lv1) or SEQ ID NO: 151 (DDR1-9hu_Lc2) and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 152 (DDR1-9hu_Hv). In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region having the amino acid sequence of SEQ ID NO: 150 (DDR1-9hu_Lv1) and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 152 (DDR1-9hu_Hv). In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region having the amino acid sequence of SEQ ID NO: 151 (DDR1-9hu_Lc2) and a heavy chain variable region having the amino acid sequence of SEQ ID NO: 152 (DDR1-9hu_Hv).

[0141] In some embodiments, the antibody or its antigen-binding fragment is a variant, and the variant's light chain variable region sequence and / or heavy chain variable region sequence 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, and the variant retains binding specificity and / or other functional properties to the DDR1 protein. In some embodiments, the variant's light chain variable region sequence and / or heavy chain variable region sequence has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conserved or non-conserved 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 LC-CDRs and / or HC-CDRs of the variant light chain variable region or variant heavy chain variable region compared to the parent LC-CDR or HC-CDR. In some embodiments, 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 light chain variable region and / or heavy chain variable region framework sequence compared to the parent light chain variable region sequence or heavy chain variable region sequence. In some embodiments, the antibody or its antigen-binding fragment has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conserved or non-conserved amino acid substitutions in the light chain variable region and / or heavy chain variable region framework sequence. The above-described variant forms apply to each of the light chain variable regions in Tables 3 and 5, and to each of the heavy chain variable regions in Tables 4 and 5.

[0142] In some embodiments, the antibody or its antigen-binding fragment contains a light chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the light chain variable region amino acid sequences selected from SEQ ID NOs: 108-128 in Table 3 and SEQ ID NOs: 150 and 151 in Table 5. In some embodiments, the antibody or its antigen-binding fragment contains a heavy chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the heavy chain variable region amino acid sequences selected from SEQ ID NOs: 129-149 in Table 4 and SEQ ID NO: 152 in Table 5. In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the light chain variable region amino acid sequences selected from SEQ ID NOs: 108-128 in Table 3 and SEQ ID NOs: 150 and 151 in Table 5, and a heavy chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the heavy chain variable region amino acid sequences selected from SEQ ID NOs: 129-149 in Table 4 and SEQ ID NO: 152 in Table 5.

[0143] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the light chain variable region amino acid sequence of SEQ ID NO: 112. In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the heavy chain variable region amino acid sequence of SEQ ID NO: 133. In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the light chain variable region amino acid sequence of SEQ ID NO: 112, and a heavy chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the heavy chain variable region amino acid sequence of SEQ ID NO: 133.

[0144] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the light chain variable region amino acid sequence of SEQ ID NO: 127. In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the heavy chain variable region amino acid sequence of SEQ ID NO: 148. In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the light chain variable region amino acid sequence of SEQ ID NO: 127, and a heavy chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the heavy chain variable region amino acid sequence of SEQ ID NO: 148.

[0145] In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the light chain variable region amino acid sequence of SEQ ID NO: 150 or 151. In some embodiments, the antibody or its antigen-binding fragment includes a heavy chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the heavy chain variable region amino acid sequence of SEQ ID NO: 152. In some embodiments, the antibody or its antigen-binding fragment includes a light chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the light chain variable region amino acid sequence of SEQ ID NO: 150 or 151, and a heavy chain variable region amino acid sequence having 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the heavy chain variable region amino acid sequence of SEQ ID NO: 152.

[0146] In some embodiments, the light chain variable region includes LC-CDR1, LC-CDR2, and LC-CDR3 as described herein (e.g., Table 1), or a specific light chain variable region (e.g., Tables 3 and 5), and the light chain variable region present in the antibody or its antigen-binding fragment is added to or ligated to all or part of the light chain constant region to form the light chain of the antibody or its antigen-binding fragment. In some embodiments, the light chain constant region is that of the species from which the antibody was isolated, e.g., the rabbit light chain constant region. In some embodiments, the light chain constant region added to or ligated to the light chain variable region is the kappa (κ) or lambda (λ) constant region as described herein. In some embodiments, the light chain constant region, if present, may be any one of the known λ subtypes, e.g., λ1, λ2, λ3, or λ4. In some embodiments, the light chain constant region is a lambda light chain constant region sequence. In some embodiments, the light chain constant region is a kappa light chain constant region sequence. In one preferred embodiment, the lambda or kappa constant region is a human lambda or human kappa constant region sequence.

[0147] In some embodiments, the heavy chain variable region includes HC-CDR1, HC-CDR2, and HC-CDR3 as described herein (e.g., Table 2), or each of specific heavy chain variable regions (e.g., Tables 4 and 5), and each of the heavy chain variable regions present in the antibody or its antigen-binding fragment is attached to or ligated to all or part of the heavy chain constant region to form the heavy chain of the antibody or its antigen-binding fragment. In some embodiments, the heavy chain constant domain is a rodent, primate, or other mammalian heavy chain constant region. In some embodiments, the heavy chain constant region ligated to or attached to the heavy chain variable region is a human heavy chain constant region. In some embodiments, the human heavy chain constant region includes at least one or all of the following: human CH1, human hinge, human CH2, and human CH3 domains. In some embodiments, the heavy chain constant region includes an Fc portion, where the Fc portion is a human IgG1, IgG2, IgG3, IgG4, or IgM isotype. In some embodiments, the human heavy chain constant region may have one or more mutations to alter the properties of the Fc constant region, e.g., stability, glycosylation, and Fc receptor binding, as further described below. In some embodiments, the anti-DDR1 antibody may be modified to reduce at least one constant region-mediated biological effector function compared to an unmodified antibody, e.g., reduced binding to one or more Fc receptors (FcγR), such as FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB. FcγR binding can be reduced by mutating the immunoglobulin constant region segment of the antibody in a specific region required for FcγR interaction (see, e.g., Canfield and Morrison, 1991, J. Exp. Med. 173:1483-1491, and Lund et al., 1991, J. Immunol. 147:2657-2662). A decrease in the antibody's FcγR binding ability can also reduce other effector functions that depend on FcγR interaction, such as opsonization, phagocytosis, and antigen-dependent cell-mediated cytotoxicity ("ADCC").

[0148] In some embodiments, the antibodies or antigen-binding fragments described herein include antibodies modified to acquire or improve at least one constant-region-mediated biological effector function compared to an unmodified antibody, for example, to enhance FcγR interactions (see, for example, U.S. Publication No. 2006 / 0134709). For example, the antibodies of this disclosure may have constant regions that bind to FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA, and / or FcγRIIIB with higher affinity than the corresponding wild-type constant regions.

[0149] Accordingly, the antibodies of this disclosure may have alterations in biological activity resulting in opsonization, phagocytosis, or an increase or decrease in ADCC. For example, a modification in an antibody that reduces ADCC activity is described in U.S. Patent No. 5,834,597. An exemplary ADCC-reducing variant corresponds to “Mutant 3” (also known as “M3”) in U.S. Patent No. 5,834,597, in which residues 234 and 237 (using EU numbering) are substituted with alanine. Variants of Mutant 3 (also known as “M3”) can be used in a number of antibody isotypes, e.g., IgG2. Additional substitutions that can modify FcγR binding and / or ADCC effector function include K322A substitutions or L234A and L235A double substitutions in the Fc region (see, e.g., Hezareh, et al. J. Virol., 2001, 75(24):12161-12168). In some embodiments, the antibodies of this disclosure have or lack low levels of fucose. Fucose-less antibodies, particularly at low doses, have correlated with enhanced ADCC activity (see, e.g., Shields et al., J. Biol. Chem., 2002, 277:26733-26740 and Shinkawa et al., J. Biol. Chem., 2003, 278:3466-73. Methods of preparing fucose-less antibodies include growth in rat myeloma YB2 / 0).

[0150] In some embodiments, the antibodies of this disclosure may include an entire modified (or variant) CH2 domain or Fc domain containing amino acid substitutions that increase binding to FcγRIIB and / or decrease binding to FcγRIIIA compared to binding to the corresponding wild-type CH2 or Fc domain. A variant CH2 or variant Fc domain is described in U.S. Patent Publication 2014 / 0377253, which is incorporated herein in its entirety. A variant CH2 or variant Fc domain typically contains one or more substitutions at positions 263, 266, 273, and 305, and the numbering of residues within the Fc domain is in the EU index similar to that of Kabat. In some embodiments, the anti-DDR1 antibody comprises one or more substitutions of the wild-type CH2 domain selected from V263L, V266L, V273C, V273E, V273F, V273L, V273M, V273S, V273Y, V305K, and V305W. In certain embodiments, one or more substitutions of the CH2 domain are selected from V263L, V273E, V273F, V273M, V273S, and V273Y for the CH2 domain of human IgG1. For example, one or more substitutions of the CH2 domain may be V273E. In another particular embodiment, the anti-DDR1 antibody of this disclosure comprises a variant CH2 domain containing the amino acid substitution V263L. Other examples of variant CH2 or variant Fc domains that can result in increased binding to FcγRIIB and / or decreased binding to FcγRIIIA compared to the binding of the corresponding wild-type CH2 or Fc region include, for example, S267E or S267E / L328F in human IgG1, as found in Vonderheide, et al. Clin. Cancer Res., 19(5), 1035-1043 (2013).

[0151] In some embodiments, anti-DDR1 antibodies include modifications that increase or decrease their binding affinity to the fetal Fc receptor, FcRn, by, for example, mutating immunoglobulin constant region segments in specific regions involved in FcRn interaction (see, for example, WO2005 / 123780). In certain embodiments, IgG-class anti-DDR1 antibodies are mutated such that at least one of the amino acid residues 250, 314, and 428 of the heavy chain constant region is replaced, either alone or in any combination thereof, for example, at positions 250 and 428, or at positions 250 and 314, or at positions 314 and 428, or at positions 250, 314, and 428, as well as in specific combinations of positions 250 and 428. Regarding position 250, the substituted amino acid residue may be any amino acid residue other than threonine, and includes, but is not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, valine, tryptophan, or tyrosine. Regarding position 314, the substituted amino acid residue may be any amino acid residue other than leucine, and includes, but is not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. At position 428, the substituted amino acid residue may be any amino acid residue other than methionine, and includes, but is not limited to, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine. An exemplary substitution known to modify Fc effector function is the Fc substitution M428L, which may occur in conjunction with the Fc substitution T250Q. Specific combinations of preferred amino acid substitutions are identified in Table 1 of U.S. Patent No. 7,217,797, which is incorporated herein by reference.Such mutations increase binding to FcRn, protecting the antibody from degradation and increasing its half-life.

[0152] In some embodiments, the antibody or its antigen-binding fragment is a single-chain antibody comprising LC-CDR and HC-CDR as disclosed herein. In some embodiments, the antibody or its antigen-binding fragment is a single-chain antibody comprising a light-chain variable region and a heavy-chain variable region as disclosed herein. In certain embodiments, the single-chain antibody comprises a clonal pair light-chain variable region and a heavy-chain variable region as disclosed herein.

[0153] In some embodiments, the LC-CDR and HC-CDR of the present disclosure, or antigen-binding fragments comprising a light chain variable region and a heavy chain variable region, are, as described herein, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized diabodies (dsdiabodies), single-chain antibody molecules (scFv), scFv dimers (bivalent diabodies), multispecific antibodies, single-domain antibodies (sdAb), camel antibodies or nanobodies, domain antibodies, or bivalent domain antibodies.

[0154] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody comprising the LC-CDR and HC-CDR disclosed herein, or a light chain variable region and a heavy chain variable region, where the Fc heavy chain constant region originates from a different species than the LC-CDR and HC-CDR, or the light chain variable region and heavy chain variable region disclosed herein. In some embodiments, the chimeric antibody comprises a human Fc region.

[0155] In some embodiments, the antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, and the framework regions of the light chain variable region including the LC-CDR and the heavy chain variable region including the HC-CDR of the Disclosure are replaced with human framework sequences. In some embodiments, the antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, and the framework regions of the light chain variable region selected from SEQ ID NOs: 108-128 and SEQ ID NOs: 150 and 151 are replaced with human light chain variable region framework sequences. In some embodiments, the antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, and the framework regions of the heavy chain variable region selected from SEQ ID NOs: 129-140 and SEQ ID NO: 152 are replaced with human heavy chain variable region framework sequences. In some embodiments, the antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, and the framework region of the light chain variable region selected from SEQ ID NOs: 108-128 and SEQ ID NOs: 150 and 151 is replaced with a human light chain variable region framework sequence, and the framework region of the heavy chain variable region selected from SEQ ID NOs: 129-140 and SEQ ID NO: 152 is replaced with a human heavy chain variable region framework sequence.

[0156] C. Exemplary epitopes and competing antigen-binding proteins In another embodiment, this disclosure provides epitopes to which anti-DDR1 antibodies bind. In some embodiments, the epitopes bound by the antibodies described herein are useful. In certain embodiments, the epitopes provided herein can be used to isolate antibodies or antigen-binding proteins that bind to DDR1. In certain embodiments, the epitopes provided herein can be used to generate antibodies or antigen-binding proteins that bind to DDR1. In certain embodiments, the epitopes or sequences containing the epitopes provided herein can be used as immunogens to generate antibodies or antigen-binding proteins that bind to DDR1. In certain embodiments, the epitopes or sequences containing the epitopes described herein can be used to interfere with the biological activity of DDR1.

[0157] In some embodiments, antibodies or antigen-binding fragments that bind to any of the epitopes are particularly useful. In some embodiments, the epitopes provided herein, when bound by an antibody, interfere with or inhibit the biological activity of DDR1. In some embodiments, the epitopes provided herein, when bound by an antibody, block the interaction between DDR1 and its binding partner.

[0158] In some embodiments, domains / regions containing residues that come into contact with or are embedded by the antibody can be identified by mutating specific residues within DDR1 and determining whether the antibody can bind to the mutated DDR1 protein. By producing a large number of individual mutations, it is possible to identify residues that play a direct role in binding or residues that are close enough to the antibody so that the mutation can affect the binding between the antibody and the antigen. From this knowledge of amino acids, it is possible to elucidate domains or regions of the antigen that contain residues that come into contact with the antigen-binding protein or residues that are covered by the antibody. Such domains may contain the binding epitopes of the antigen-binding protein.

[0159] In another aspect, the disclosure provides an antigen-binding protein that competes with one of the exemplary antibodies or antigen-binding fragments that bind to the epitopes described herein for specific binding to DDR1. Such an antigen-binding protein may also bind to the same epitope as one of the exemplary antibodies or antigen-binding fragments or overlapping epitopes. Antigen-binding proteins that compete with or bind to the same epitope as the exemplary antibodies are expected to exhibit similar functional properties. The exemplary antibodies include those described above, including antibodies having light and heavy chain variable regions (CDRs) shown in Tables 1 and 2, respectively, light and heavy chain variable regions shown in Tables 3 and 4, and light and heavy chain coding regions shown in Tables 8 and 9.

[0160] III. Polynucleotides, vectors, and host cells In another aspect, the disclosure provides a polynucleotide encoding an antibody that specifically binds to the DDR1 protein disclosed herein and its antigen-binding fragment, a vector comprising an expression vector containing the polynucleotide, and a host cell comprising, for example, a polynucleotide for the expression of the antibody or its antigen-binding fragment.

[0161] In particular, polynucleotides are isolated polynucleotides. Polynucleotides can be operably ligated to one or more heterologous regulatory sequences to create recombinant polynucleotides capable of controlling gene expression and expressing a target polypeptide. Expression constructs containing heterologous polynucleotides encoding the relevant polypeptide or protein can be introduced into suitable host cells to express the corresponding polypeptide.

[0162] As should be apparent to those skilled in the art, knowledge of protein sequences, for knowledge of all possible codons corresponding to various amino acids, provides a description of all polynucleotides that can encode the protein sequence of the subject. A very large number of nucleic acids encoding the aforementioned polypeptides can be made by selecting combinations based on possible codon selections, and all such variations are considered to be specifically disclosed for any and all of the polypeptides described herein.

[0163] In some embodiments, the polynucleotide encodes LC-CDR1, LC-CDR2, and / or LC-CDR3 of the light chain variable region disclosed herein, including the LC-CDRs listed in Table 1. In some embodiments, the polynucleotide encodes variants of LC-CDR1, LC-CDR2, and / or LC-CDR3 of the light chain variable region disclosed herein, including the LC-CDRs listed in Table 1, wherein one or more of the LC-CDRs of the variant have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof compared to the parent LC-CDR.

[0164] In some embodiments, the polynucleotide encodes HC-CDR1, HC-CDR2, and / or HC-CDR3 of the heavy chain variable region disclosed herein, including the HC-CDRs listed in Table 2. In some embodiments, the polynucleotide encodes variants of HC-CDR1, HC-CDR2, and / or HC-CDR3 of the heavy chain variable region disclosed herein, including the LC-CDRs listed in Table 2, wherein one or more of the HC-CDRs of the variant have one, two, or three amino acid substitutions, additions, deletions, or combinations thereof compared to the parent HC-CDR.

[0165] In some embodiments, the polynucleotides encoding LC-CDR1, LC-CDR2, and / or LC-CDR3 of the light chain variable region are selected from the polynucleotides shown in Table 6.

[0166] (Table 6) DNA sequences encoding the CDR of the light chain variable region of DDR1 antibodies TIFF0007883266000008.tif51136TIFF0007883266000009.tif204136

[0167] In some embodiments, the polynucleotides encoding HC-CDR1, HC-CDR2, and / or HC-CDR3 of the heavy chain variable region are selected from the polynucleotides shown in Table 7.

[0168] (Table 7) DNA sequences encoding the CDR of the heavy chain variable region of DDR1 antibodies TIFF0007883266000010.tif221150TIFF0007883266000011.tif89150

[0169] In some embodiments, the polynucleotide encodes at least one, two, or three LC-CDRs within the light chain variable region of the amino acid sequence of SEQ ID NOs: 108-128 or SEQ ID NOs: 150 or 151. In some embodiments, the polynucleotide encodes at least one, two, or three HC-CDRs within the heavy chain variable region of the amino acid sequence of SEQ ID NOs: 129-149 or SEQ ID NOs: 153.

[0170] In some embodiments, the polynucleotide encodes at least one, two, or three LC-CDRs in the light chain variable region of the amino acid sequence of SEQ ID NOs: 108-128 or SEQ ID NOs: 150 or 151, and at least one, two, or three HC-CDRs in the heavy chain variable region of the amino acid sequence of SEQ ID NOs: 129-149 or SEQ ID NOs: 153. In some embodiments, the selected LC-CDRs and HC-CDRs are those of a clone pair.

[0171] In some embodiments, the polynucleotide comprises at least one, two, or three polynucleotide sequences of the LC-CDR of each mAb shown in Table 6.

[0172] In some embodiments, the polynucleotide comprises at least 1, 2, or 3 of the polynucleotide sequences of the HC-CDRs of each mAb shown in Table 7.

[0173] In some embodiments, the polynucleotide comprises at least 1, 2, or 3 of the polynucleotide sequences of the LC-CDRs of each mAb shown in Table 6 and at least 1, 2, or 3 of the polynucleotide sequences of the HC-CDRs of each mAb shown in Table 7, and the selected LC-CDR and HC-CDR are those of the LC-CDR and HC-CDR of the clone pairs shown in Table 6 and Table 7, respectively.

[0174] In some embodiments, the polynucleotide encodes a light chain variable region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the light chain variable region amino acid sequence selected from SEQ ID NOs: 108 - 128 of Table 3 and SEQ ID NOs: 150 - 151 of Table 5.

[0175] In some embodiments, the polynucleotide encodes a heavy chain variable region having at least 80%, 85%, 9%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the heavy chain variable region amino acid sequence selected from SEQ ID NOs: 129 - 149 of Table 4 and SEQ ID NO: 153 of Table 5.

[0176] In some embodiments, the polynucleotide encodes a light chain variable region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a light chain variable region amino acid sequence selected from SEQ ID NOs: 108-128 of Table 3 or SEQ ID NOs: 150 and 151 of Table 5, and a heavy chain variable region having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98%, 99%, or more sequence identity to a heavy chain variable region amino acid sequence selected from SEQ ID NOs: 129-149 of Table 4 and SEQ ID NOs: 153 of Table 5.

[0177] In some embodiments, the polynucleotide encodes an antibody or antigen-binding fragment comprising a light chain variable region and a heavy chain variable region selected from: A light chain variable region (DDR1-1K) having the amino acid sequence of SEQ ID NO: 108, and a heavy chain variable region (DDR1-1H) having the amino acid sequence of SEQ ID NO: 129, A light chain variable region (DDR1-3K) having the amino acid sequence of SEQ ID NO: 109, and a heavy chain variable region (DDR1-3H) having the amino acid sequence of SEQ ID NO: 130, A light chain variable region (DDR1-5K) having the amino acid sequence of SEQ ID NO: 110, and a heavy chain variable region (DDR1-5H) having the amino acid sequence of SEQ ID NO: 131, A light chain variable region (DDR1-6K) having the amino acid sequence of SEQ ID NO: 111, and a heavy chain variable region (DDR1-6H) having the amino acid sequence of SEQ ID NO: 132, A light chain variable region (DDR1-9K) having the amino acid sequence of SEQ ID NO: 112, and a heavy chain variable region (DDR1-9H) having the amino acid sequence of SEQ ID NO: 133, A light chain variable region (DDR1-11K) having the amino acid sequence of SEQ ID NO: 113, and a heavy chain variable region (DDR1-11H) having the amino acid sequence of SEQ ID NO: 134, A light chain variable region (DDR1-12K) having the amino acid sequence of SEQ ID NO: 114, and a heavy chain variable region (DDR1-12H) having the amino acid sequence of SEQ ID NO: 135, A light chain variable region (DDR1-13K) having the amino acid sequence of SEQ ID NO: 115, and a heavy chain variable region (DDR1-13H) having the amino acid sequence of SEQ ID NO: 136, A light chain variable region (DDR1-14K) having the amino acid sequence of SEQ ID NO: 116, and a heavy chain variable region (DDR1-14H) having the amino acid sequence of SEQ ID NO: 137, A light chain variable region (DDR1-15K) having the amino acid sequence of SEQ ID NO: 117, and a heavy chain variable region (DDR1-15H) having the amino acid sequence of SEQ ID NO: 138, A light chain variable region (DDR1-17K) having the amino acid sequence of SEQ ID NO: 118, and a heavy chain variable region (DDR1-17H) having the amino acid sequence of SEQ ID NO: 139, A light chain variable region (DDR1-20K) having the amino acid sequence of SEQ ID NO: 119, and a heavy chain variable region (DDR1-20H) having the amino acid sequence of SEQ ID NO: 140, A light chain variable region (DDR1-21K) having the amino acid sequence of SEQ ID NO: 120, and a heavy chain variable region (DDR1-21H) having the amino acid sequence of SEQ ID NO: 141, A light chain variable region (DDR1-22K) having the amino acid sequence of SEQ ID NO: 121, and a heavy chain variable region (DDR1-22H) having the amino acid sequence of SEQ ID NO: 142, A light chain variable region (DDR1-23K) having the amino acid sequence of SEQ ID NO: 122, and a heavy chain variable region (DDR1-23H) having the amino acid sequence of SEQ ID NO: 143, A light chain variable region (DDR1-26K) having the amino acid sequence of SEQ ID NO: 123, and a heavy chain variable region (DDR1-26H) having the amino acid sequence of SEQ ID NO: 144, A light chain variable region (DDR1-28K) having the amino acid sequence of SEQ ID NO: 124, and a heavy chain variable region (DDR1-28H) having the amino acid sequence of SEQ ID NO: 145, A light chain variable region (DDR1-29K) having the amino acid sequence of SEQ ID NO: 125, and a heavy chain variable region (DDR1-29H) having the amino acid sequence of SEQ ID NO: 146, A light chain variable region (DDR1-32K) having the amino acid sequence of SEQ ID NO: 126, and a heavy chain variable region (DDR1-32H) having the amino acid sequence of SEQ ID NO: 147, A light chain variable region (DDR1-33K) having the amino acid sequence of SEQ ID NO: 127, and a heavy chain variable region (DDR1-33H) having the amino acid sequence of SEQ ID NO: 148, A light chain variable region (DDR1-34K) having the amino acid sequence of SEQ ID NO: 128, and a heavy chain variable region (DDR1-34H) having the amino acid sequence of SEQ ID NO: 149, A light chain variable region (DDR1-9hu_Lv) having the amino acid sequence of SEQ ID NO: 150, and a heavy chain variable region (DDR1-9hu_Hv) having the amino acid sequence of SEQ ID NO: 153, and A light chain variable region (DDR1-9hu_Lc2) having the amino acid sequence of SEQ ID NO: 151, and a heavy chain variable region (DDR1-9hu_Hv) having the amino acid sequence of SEQ ID NO: 153.

[0178] In some embodiments, the polynucleotide encoding the light chain variable region is selected from the polynucleotides shown in Table 8.

[0179] (Table 8) DNA sequences encoding the light chain variable region of anti-DDR1 antibodies TIFF0007883266000012.tif242154TIFF0007883266000013.tif229154TIFF0007883266000014.tif151154

[0180] In some embodiments, the polynucleotide encoding the heavy chain variable region is selected from the polynucleotides shown in Table 9.

[0181] (Table 9) DNA sequences encoding the heavy chain variable region of anti-DDR1 antibodies TIFF0007883266000015.tif232142TIFF0007883266000016.tif232148TIFF0007883266 000017.tif232146TIFF0007883266000018.tif232146TIFF0007883266000019.tif23292

[0182] In some embodiments, the polynucleotide encoding the humanized light chain variable region and / or humanized heavy chain variable region of antibody DDR1-9 (DDR1-9hu) is selected from the polynucleotides shown in Table 10.

[0183] (Table 10) Humanized DDR1-9hu antibody sequences - nucleic acids TIFF0007883266000020.tif101156

[0184] In some embodiments, the polynucleotide has sequence identity at the nucleotide level of at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with respect to a reference polynucleotide sequence selected from SEQ ID NOs: 2:58-278 and SEQ ID NOs: 301-302, and encodes a corresponding light chain variable region selected from SEQ ID NOs: 108-128 or SEQ ID NOs: 150-151.

[0185] In some embodiments, the polynucleotide has sequence identity at the nucleotide level of at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with respect to a reference polynucleotide sequence selected from SEQ ID NOs: 279-299 and SEQ ID NO: 300, and encodes a corresponding light chain variable region selected from SEQ ID NOs: 129-149 or SEQ ID NO: 152.

[0186] In some embodiments, the polynucleotide has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity at the nucleotide level with respect to the reference polynucleotide sequence of SEQ ID NO: 262 or 276, and encodes the corresponding light chain variable region of SEQ ID NO: 112 or SEQ ID NO: 126.

[0187] In some embodiments, the polynucleotide has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity at the nucleotide level with respect to the reference polynucleotide sequence of SEQ ID NO: 283 or 297, and encodes the corresponding light chain variable region of SEQ ID NO: 133 or 147.

[0188] In some embodiments, the polynucleotide has sequence identity at the nucleotide level of at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with respect to the reference polynucleotide sequence of SEQ ID NO: 301 or 302, and encodes the corresponding light chain variable region of SEQ ID NO: 150 or SEQ ID NO: 151.

[0189] In some embodiments, the polynucleotide has at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity at the nucleotide level with respect to the reference polynucleotide sequence of SEQ ID NO: 300, and encodes the corresponding light chain variable region of SEQ ID NO: 152.

[0190] In a further embodiment, the nucleic acids of the present disclosure include polynucleotides that hybridize to polynucleotides encoding the antibodies disclosed herein. In some embodiments, the polynucleotide hybridizes to a polynucleotide selected from SEQ ID NOs: 258-278 and SEQ ID NOs: 301-302 and encodes the light chain variable region of an antibody that specifically binds to the DDR1 protein. In some embodiments, the polynucleotide hybridizes to a polynucleotide selected from SEQ ID NOs: 279-299 and SEQ ID NO: 300 and encodes the heavy chain variable region of an antibody that specifically binds to the DDR1 protein.

[0191] Generally, the nucleic acid hybridizes under moderate or high stringency conditions to a nucleic acid that encodes the antibodies disclosed herein and also encodes antibodies that maintain the ability to specifically bind to the DDR1 protein. A first nucleic acid molecule is "hybridizable" to a second nucleic acid molecule if the single-stranded form of the first nucleic acid molecule can anneal to the second nucleic acid molecule under appropriate conditions of temperature and solution ionic strength (Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 3 rdSee ed., Cold Spring Harbor Press, Cold Spring Harbor, NY 2001). Temperature and ionic strength conditions determine the "stringency" of hybridization. Typical moderate-stringency hybridization conditions are 40% formamide with 5× or 6× SSC and 0.1% SDS at 42°C. High-stringency hybridization conditions are 50% formamide, 5× or 6× SSC (0.15M NaC1 and 0.015M Na-citrate), 42°C, or optionally, higher temperatures (e.g., 57°C, 59°C, 60°C, 62°C, 63°C, 65°C, or 68°C). Hybridization requires the two nucleic acids to contain complementary sequences, although base mismatches are also possible depending on the stringency of hybridization. The appropriate stringency for nucleic acid hybridization depends on the length and degree of complementarity of the nucleic acids, which are well-known variables in the art. The greater the degree of similarity or homology between two nucleotide sequences, the higher the stringency of the nucleic acids that can hybridize. For hybrids longer than 100 nucleotides, equations for calculating the melting temperature have been derived (see Sambrook et al. above). For hybridization with shorter nucleic acids, such as oligonucleotides, the location of the mismatch becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al. above).

[0192] In some embodiments, the polynucleotides disclosed herein can be manipulated in various ways to provide expression of encoded polypeptides, such as light-chain or heavy-chain variable regions, disclosed herein. In some embodiments, the polynucleotides are operably ligated to regulatory sequences for the expression of the polynucleotide and / or the corresponding polypeptide, including, among other things, a transcription promoter, a leader sequence, a transcription enhancer, a ribosome binding or entry site, a termination sequence, and a polyadenylation sequence. Manipulation of isolated polynucleotides prior to insertion into a vector may be desirable or necessary, depending on the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are known in the art. Guidance is provided in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Ed., Cold Spring Harbor Laboratory Press, New York (2001), and Current Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates (1998), updates to 2020.

[0193] In some embodiments, the polynucleotide may be part of an expression vector, where the vector and polynucleotide include one or more operablely linked control sequences to control the expression of the polynucleotide and / or the expression of the encoded polypeptide. The recombinant expression vector may be any vector (e.g., plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and result in the expression of a polynucleotide sequence. The choice of vector typically depends on the compatibility of the vector with the host cell into which the vector is introduced. The vector may be a linear or closed circular plasmid. Exemplary expression vectors include, among others, vectors based on the T7 or T7lac promoter (pACY:Novagen;pET), vectors based on the baculovirus promoter (e.g., pBAC), vectors based on the Ef1-α and HTLV promoters (e.g., pFUSE2;Invitrogen, CA,USA), vectors based on the CMV enhancer and human ferritin light chain gene promoter (e.g., pFUSE:Invitrogen, CA,USA), vectors based on the CMV promoter (e.g., pFLAG:Sigma, USA), and vectors based on the dihydrofolate reductase promoter (e.g., pEASE:Amgen, USA). Various vectors can be used for transient or stable expression of the polypeptide of interest.

[0194] In another embodiment, a polynucleotide encoding a polypeptide is operably ligated to one or more regulatory sequences for polypeptide expression in a host cell. Host cells used for polypeptide expression are well known in the art and include, but are not limited to, bacterial cells such as Escherichia coli and yeast cells, insect cells such as Drosophila S2 and Spodoptera Sf9 cells, animal cells such as Chinese hamster ovary (CHO), African green monkey kidney (COS), baby hamster kidney (BHK), mouse myeloma (e.g., NS0 and Sp2 / 0), and human embryonic kidney (HEK), as well as plant cells. Appropriate culture media and growth conditions for the aforementioned host cells are known in the art. In some embodiments, the host cell and expression vector are used to express the polypeptide of interest.

[0195] In some embodiments, host cells containing the expression vector and polynucleotide described herein are cultured in a suitable medium and under culture conditions suitable for the expression of the encoded polypeptide, for example, a polypeptide comprising an amino acid sequence selected from SEQ ID NOs: 108-128, SEQ ID NOs: 150 and 151, SEQ ID NOs: 129-149, and SEQ ID NO: 152. In some embodiments, an in vitro expression system can be used with the expression vector to express the polypeptide. Examples of in vitro expression systems include those based on Escherichia coli, rabbit reticulocytes, wheat germ, insect cells, and human cells. Whether expressed in host cells or in vitro, the expressed polypeptide can be isolated or purified as further described herein.

[0196] IV. Methods for preparing antibodies and modifying them In some embodiments, the monoclonal antibodies described herein can be prepared using standard methods, followed by screening, characterization, and functional evaluation. For example, the variable region can be sequenced and then subcloned into a human expression vector to generate a chimeric antibody gene, which can then be expressed and purified. These chimeric antibodies can be tested in antigen binding, signaling blockade, and xenograft experiments.

[0197] A. General methods Monoclonal antibodies that bind to DDR1 are understood to have several applications. These include the detection and diagnosis of cancer, as well as the manufacture of diagnostic kits for use in cancer therapy. In these contexts, such antibodies may be conjugated to diagnostic or therapeutic agents and used as capture agents or competitors in competitive assays, or used individually without attachment of additional agents. Antibodies may be mutated or modified, as will be further discussed below. Methods for preparing and characterizing antibodies are well known in the art (see, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; U.S. Patent No. 4,196,265).

[0198] Methods for producing monoclonal antibodies (MAbs) generally begin along the same line as methods for preparing polyclonal antibodies. The first step in both of these methods is immunization of a suitable host. As is well known in the art, a given composition for immunization can vary in its immunogenicity. Therefore, it is often necessary to enhance the host immune system, which can be achieved by conjugating a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole-limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins, such as ovalbumin, mouse serum albumin, or rabbit serum albumin, can also be used as carriers. Means for conjugating polypeptides to carrier proteins are known in the art and include glutaraldehyde, m-maleimidebencyl-N-hydroxysuccinimide ester, carbodiimide, and bis-biazoted benzidine. As is well known in the art, the immunogenicity of certain immunogen compositions can be enhanced by the use of nonspecific stimulants of the immune response known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a nonspecific stimulant of the immune response containing dead Mycobacterium tuberculosis), incomplete Freund's adjuvant, and aluminum hydroxide adjuvant.

[0199] The amount of immunogen composition used to produce polyclonal antibodies varies depending on the properties of the immunogen and the animals used for immunization. The immunogen can be administered via various routes (subcutaneous, intramuscular, intradermal, intravenous, and intraperitoneal). Polyclonal antibody production can be monitored by taking blood samples from the immunized animals at various points after immunization. A second booster injection may also be performed. The boosting and titration process is repeated until a suitable titer is obtained. Once the desired level of immunogenicity is achieved, the immunized animals can be bled, and the serum can be isolated and stored, and / or the animals can be used to produce MAb.

[0200] After immunization, somatic cells capable of producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generation protocol. These cells can be obtained from biopsied spleen or lymph nodes, or from circulating blood. Next, antibody-producing B lymphocytes from the immunized animals are fused with immortal myeloma cells, generally of the same species as the immunized animal or one of human or human / mouse chimeric cells. Myeloma cell lines suitable for use in the hybridoma-producing fusion procedure are preferably those that do not produce antibodies, have high fusion efficiency, and then have enzyme deficiencies that prevent them from growing in specific selective media that support only the growth of the desired fusion cells (hybridomas). As is known to those skilled in the art, any one of a large number of myeloma cells may be used (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells typically involve mixing somatic cells and myeloma cells in a 2:1 ratio in the presence of a substance (chemical or electrical) that promotes cell membrane fusion, although the ratio can vary from approximately 20:1 to approximately 1:1. Fusion methods using Sendai virus were described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), e.g., 37% (v / v) PEG, were described by Gefter et al. (1977). The use of electrically induced fusion methods is also appropriate (Goding, pp. 71-74, 1986). The fusion procedure typically involves 1 × 10⁻⁶ cells. -6 ~1 × 10 -8This produces viable hybrids at a low frequency. However, this does not pose a problem because viable fused hybrids differentiate from the parent injected cells (particularly injected myeloma cells, which would normally continue to divide indefinitely) by culturing them in selective media. Selective media generally contain agents that block the de novo synthesis of nucleotides in tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azazerin. Aminopterin and methotrexate block the de novo synthesis of both purines and pyrimidines, while azazerin blocks only purine synthesis. When aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as sources of nucleotides (HAT medium). When azazerin is used, the medium is supplemented with hypoxanthine. To eliminate EBV-transformed cell lines that have not fused to myeloma, ouabain is added if the B cell source is an Epstein-Barr virus (EBV)-transformed human B cell line.

[0201] In some embodiments, the preferred selection medium is HAT, or HAT containing ouabain. Only cells capable of manipulating the nucleotide salvage pathway can survive in HAT medium. Myeloma cells are deficient in key enzymes of the salvage pathway, such as hypoxanthine phosphoribosyltransferase (HPRT), and therefore cannot survive. B cells can manipulate this pathway, but they have a limited lifespan in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selection medium are hybrids formed from myeloma and B cells. When the source of B cells used for fusion is a lineage of EBV-transformed B cells, as here, ouabain is also used for drug selection of the hybrid, as EBV-transformed B cells are susceptible to drug death, but the myeloma partner used is selected to be resistant to EBV-transformed B cells.

[0202] Culture provides a population of hybridomas from which specific hybridomas are selected. Typically, hybridoma selection is performed by culturing cells by single-clonal dilution in microtiter plates, followed by testing the individual clonal supernatant for desired reactivity (after about 2-3 weeks). Assays must be highly sensitive, simple, and rapid, and include, for example, radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoconjugate assay, etc. The selected hybridomas can then be serially diluted or single-cell sorted by flow cytometry, cloned into individual antibody-producing cell lines, and then allowed to grow indefinitely to provide mAbs. Cell lines can be utilized for MAb production in two basic ways. Samples of hybridomas can be injected into animals (e.g., mice) (often intraperitoneally). Optionally, animals are primed with oils such as hydrocarbons, particularly pristane (tetramethylpentadecane), before injection. When using human hybridomas in this method, it is best to inject them into immunodeficient mice, such as SCID mice, to prevent tumor rejection. The injected animals develop tumors that secrete specific monoclonal antibodies produced by the fused cell hybrids. Then, animal fluids such as serum or ascites can be tapped to provide high concentrations of MAb. Individual cell lines can also be cultured in vitro, and MAb is naturally secreted into culture media where high concentrations are readily available. Alternatively, human hybridoma cell lines can be used in vitro to produce immunoglobulins in the cell supernatant. The cell lines can be adapted for growth in serum-free media to optimize the ability to recover high-purity human monoclonal immunoglobulins.

[0203] The MAb produced by either means may be further purified, if desired, by filtration, centrifugation, and various chromatographic methods such as FPLC or affinity chromatography. Monoclonal antibody fragments of this disclosure can be obtained from purified monoclonal antibodies by methods including digestion with enzymes such as pepsin or papain, and / or by cleavage of disulfide bonds by chemical reduction. Alternatively, monoclonal antibody fragments encompassed by this disclosure may be synthesized using an automated peptide synthesizer.

[0204] Furthermore, it is thought that monoclonals can be generated using molecular cloning approaches. Therefore, RNA can be isolated from hybridoma strains, antibody genes can be obtained by RT-PCR, and cloned into immunoglobulin expression vectors. Alternatively, a combined immunoglobulin phagemide library can be prepared from RNA isolated from cell lines, and phagemides expressing the appropriate antibody can be selected by panning with viral antigens. The advantages of this approach over conventional hybridoma techniques are approximately 10. 4 The fact that twice as many antibodies can be produced and screened in a single round, and that new specificities are generated by the combination of H and L chains, further increases the chances of finding the right antibody.

[0205] Other U.S. patents (each incorporated herein by reference) that teach the production of antibodies useful in this disclosure include U.S. Patent No. 5,565,332 describing the production of chimeric antibodies using a combination approach, U.S. Patent No. 4,816,567 describing recombinant immunoglobulin preparations, and U.S. Patent No. 4,867,973 describing antibody therapeutic conjugates.

[0206] B. Manipulation of antibody sequences In various embodiments, the sequence of the identified antibody may be manipulated for various reasons, such as improving expression, improving cross-reactivity, or reducing off-target binding. The following is a general description of related techniques for antibody manipulation.

[0207] Hybridomas may be cultured, then the cells may be lysed, and total RNA may be extracted. Random hexamers may be used with RT to generate cDNA copies of the RNA, and then PCR may be performed using a multiplex mixture of PCR primers expected to amplify all human variable gene sequences. The PCR product may be cloned into a pGEM-T Easy vector and then sequenced by automated DNA sequencing using standard vector primers. Binding and neutralization assays may be performed using antibodies collected from the hybridoma supernatant, purified by FPLC using a Protein G column. Recombinant full-length IgG antibodies may be produced by subcloning heavy and light chain Fv DNA from a cloning vector into an IgG plasmid vector, transfecting 293 Freestyle cells or CHO cells, and purifying and collecting the antibodies from the 293 or CHO cell supernatant.

[0208] The rapid availability of antibodies produced in the same host cells and cell culture processes as the final cGMP manufacturing process has the potential to shorten the duration of process development programs. Lonza developed a common method for the rapid production of small amounts (up to 50 g) of antibodies in CHO cells using pooled transfectants grown in CDACF medium. While slightly slower than true transient systems, the advantages include higher product concentrations and the use of the same host and process as the generating cell line. In an example of growth and productivity of a GS-CHO pool expressing a model antibody in a disposable bioreactor, i.e., a disposable bag bioreactor culture (5 L working volume) operated in fed-batch mode, a harvest antibody concentration of 2 g / L was achieved within 9 weeks from transfection.

[0209] The antibody molecule may include, for example, fragments produced by the proteolytic cleavage of an mAb (e.g., F(ab'), F(ab')2), or single-chain immunoglobulins that can be produced via recombinant means. Such antibody derivatives are monovalent. In one embodiment, such fragments can form a “chimeric” binding molecule with each other or with other antibody fragments or receptor ligands. Importantly, such a chimeric molecule may contain substituents that can bind to different epitopes of the same molecule.

[0210] 1. Antigen binding modification In the relevant embodiments, the antibody is a derivative of the disclosed antibody, for example, an antibody containing the same CDR sequence as that in the disclosed antibody (e.g., a chimeric or CDR-implanted antibody). Alternatively, modifications such as introducing conservative changes into the antibody molecule may be desired. When making such modifications, the hydrophobicity and hydrophilicity of amino acids may be considered. The importance of hydrophobicity and hydrophilicity of amino acids in conferring interactive biological function to proteins is generally understood in the art (Kyte and Doolittle, 1982). It is recognized that the relative hydrophobicity and hydrophilicity of amino acids contribute to the resulting secondary structure of the protein, which then defines the interaction between the protein and other molecules, such as enzymes, substrates, receptors, DNA, antibodies, antigens, etc.

[0211] It is also understood in the art that similar amino acid substitutions can be effectively made based on hydrophilicity. U.S. Patent No. 4,554,101, incorporated herein by reference, states that the maximum local mean hydrophilicity of a protein, governed by the hydrophilicity of its neighboring amino acids, correlates with the biological properties of the protein. As detailed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to amino acid residues: Basic amino acids: Arginine (+3.0), Lysine (+3.0), and Histidine (-0.5); Acidic amino acids: Aspartic acid (+3.0±1), Glutamic acid (+3.0±1), Asparagine (+0.2), and Glutamine (+0.2); Hydrophilic nonionic amino acids: Serine (+0.3), Asparagine (+ 0.2), glutamine (+0.2), and threonine (-0.4); sulfur-containing amino acids: cysteine ​​(-1.0) and methionine (-1.3); hydrophobic non-aromatic amino acids: valine (-1.5), leucine (-1.8), isoleucine (-1.8), proline (-0.5±1), alanine (-0.5), and glycine (0); hydrophobic aromatic amino acids: trypphan (-3.4), phenylalanine (-2.5), and tyrosine (-2.3).

[0212] It should be understood that amino acids can be substituted with other amino acids that have similar hydrophilicity and produce biologically or immunologically modified proteins. In such modifications, substitutions of amino acids with a hydrophilicity value of ±2 are preferred, those with a value of ±1 are particularly preferred, and those with a value of ±0.5 are even more preferred.

[0213] As outlined above, amino acid substitutions are generally based on the relative similarities of amino acid side-chain substituents, such as their hydrophobicity, hydrophilicity, charge, size, etc. Exemplary substitutions considering the various properties mentioned above are well known to those skilled in the art, and these include arginine and lysine, glutamic acid and aspartic acid, serine and threonine, glutamine and asparagine, as well as valine, leucine and isoleucine.

[0214] This disclosure also aims at isotype modification. Different functionalities can be achieved by modifying the Fc region to have different isotypes. For example, modification to IgG1 can increase antibody-dependent cytotoxicity, switching to class A can improve tissue distribution, and switching to class M can improve valency.

[0215] Modified antibodies can be prepared by any technique known to those skilled in the art, including expression using standard molecular biological techniques or chemical synthesis of polypeptides. Methods for recombinant expression are discussed elsewhere in this document.

[0216] 2.Fc region modification As described above, the antibodies disclosed herein may also be manipulated to modify one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or effector function (e.g., antigen-dependent cell-mediated cytotoxicity), typically by including modifications within the Fc region. Furthermore, the antibodies disclosed herein may be chemically modified (e.g., one or more chemical moieties may bind to the antibody) or modified to alter its glycosylation, in which case one or more functional properties of the antibody may also be modified. Each of these embodiments is described in further detail below. The numbering of residues within the Fc region is the numbering of the Kabat EU index. The antibodies disclosed herein also include antibodies having an Fc region that has been modified (or blocked) to provide an altered effector function. See, for example, U.S. Patent No. 5,624,821, WO2003 / 086310, WO2005 / 120571, and WO2006 / 0057702. Using such modifications, various immune system responses can be enhanced or suppressed, potentially leading to beneficial effects in diagnosis and therapy. Modifications to the Fc region include amino acid changes (substitutions, deletions, and insertions), glycosylation or deglycosylation, and the addition of multiple Fc regions. Changes to Fc can also alter the half-life of antibodies in therapeutic antibodies, enabling less frequent dosing and thus increasing convenience and reducing material usage. This mutation has been reported to eliminate heterogeneity in inter-heavy-chain disulfide crosslinks in the hinge region.

[0217] In some embodiments, the CH1 hinge region is modified to increase or decrease the number of cysteine ​​residues within the hinge region. Exemplary approaches are further described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues within the CH1 hinge region is modified, for example, to facilitate the assembly of the light and heavy chains, or to increase or decrease the stability of the antibody. In another embodiment, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, one or more of the following mutations may be introduced, as described in U.S. Patent No. 6,277,375: T252L, T254S, T256F. Alternatively, to increase the biological half-life, the antibody may be modified within the CH1 or CL region to include a salvage receptor-binding epitope obtained from two loops of the CH2 domain of the Fc region of IgG, as described in U.S. Patents No. 5,869,046 and No. 6,121,022. In further embodiments, the Fc region is modified by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be replaced with a different amino acid residue such that the antibody has a modified affinity for the effector ligand, while the antigen-binding ability of the parent antibody is preserved. The effector ligand whose affinity is modified may be, for example, the Fc receptor or the C1 component of complement. This approach is described in further detail in U.S. Patents 5,624,821 and 5,648,260.

[0218] In another example, one or more amino acid residues within positions 231 and 239 are modified to alter the antibody's ability to fix complement. This approach is further described in PCT Publication 94 / 29351. In yet another example, the Fc region is modified by modifying one or more amino acids at the following positions to increase or decrease the antibody's ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or increase or decrease the antibody's affinity for the Fcγ receptor: 238, 239, 243, 248, 249, 252, 254, 255, 256, 258, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 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 approach is further described in PCT Publication No. 00 / 42072. Furthermore, binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII, and FcRn have been mapped, and variants with improved binding have been described. Specific mutations at positions 256, 290, 298, 333, 334, and 339 have been shown to improve binding to FcγRIII. In addition, the following variant combinations have been shown to improve FcγRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A.

[0219] In one embodiment, the Fc region is modified to reduce the antibody's ability to mediate effector function and / or to increase anti-inflammatory properties by modifying residues 243 and 264. In one embodiment, the Fc region of the antibody is modified by changing the residues at positions 243 and 264 to alanine. In another embodiment, the Fc region is modified to reduce the antibody's ability to mediate effector function and / or to increase anti-inflammatory properties by modifying residues 243, 264, 267 and 328. In yet another embodiment, the antibody includes a specific glycosylation pattern. For example, a non-glycosylated antibody (i.e., the antibody lacks glycosylation) can be produced. The glycosylation pattern of the antibody can be modified, for example, to increase the antibody's affinity or avidity to an antigen. Such modifications can be achieved, for example, by modifying one or more glycosylation sites in the antibody sequence. For example, a variable region framework may result in the removal of one or more glycosylation sites, thereby leading to one or more amino acid substitutions that eliminate glycosylation at those sites. Such aglycosylation may increase the affinity or avidity of an antibody to an antigen. See, for example, U.S. Patents 5,714,350 and 6,350,861.

[0220] Antibodies may also be produced with glycosylation patterns containing low-fucosylated or non-fucosylated glycans; for example, low-fucosylated or non-fucosylated antibodies reduce the amount of fucosyl residues on the glycan. The antibody may also contain a glycan having an increased amount of bifid GlcNac structure. Such modification of the glycosylation pattern has been demonstrated to increase the ADCC capacity of the antibody. Such modification can be achieved, for example, by expressing the antibody in host cells genetically engineered to produce glycoproteins having a specific glycosylation pattern in the glycosylation pathway. These cells have been described in the art and can be used as host cells to express the recombinant antibody of the present invention, thereby producing an antibody with modified glycosylation. For example, the cell lines Ms704, Ms705, and Ms709 lack the fucosyltransferase gene, FUT8 (α(1,6)-fucosyltransferase), resulting in antibodies expressed in the Ms704, Ms705, and Ms709 cell lines lacking fucose on their carbohydrates. The Ms704, Ms705, and Ms709 FUT8- / - cell lines are constructed by targeted disruption of the FUT8 gene in CHO / DG44 cells using two recombinant vectors (see, for example, U.S. Patent Publication 2004 / 0110704). As another example, EP1176195 describes cell lines having a functionally disrupted FUT8 gene encoding fucosyltransferase, in which antibodies expressed in such cell lines exhibit hypofucosylation by reducing or eliminating α-1,6 binding-related enzymes. EP1176195 also describes cell lines with low or no enzymatic activity that add fucose to N-acetylglucosamine that binds to the Fc region of antibodies, such as the rat myeloma cell line YB2 / 0 (ATCC CRL 1662). PCT Publication 03 / 035835 describes the Lec13 cell line, a variant CHO cell line in which the ability to bind fucose to Asn(297)-linked carbohydrates is reduced, resulting in reduced fucosylation of antibodies expressed in its host cells.Antibodies with modified glycosylation profiles can also be produced in chicken eggs, as described in PCT Publication 06 / 089231. Alternatively, antibodies with modified glycosylation profiles can be produced in plant cells such as Lemna (U.S. Patent No. 7,632,983). Methods for antibody production in plant systems are disclosed in U.S. Patents No. 6,998,267 and No. 7,388,081. PCT Publication 99 / 54342 describes a cell line engineered to express a glycoprotein-modified glycosyltransferase (e.g., β(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such that the antibody expressed in the engineered cell line exhibits an increase in the bifidate GlcNac structure, resulting in increased ADCC activity of the antibody.

[0221] Alternatively, fucose residues in antibodies can be cleaved using fucosidase enzymes; for example, α-L-fucosidase removes fucosyl residues from antibodies. Antibodies disclosed herein further include those produced in lower eukaryotic host cells, particularly fungal host cells such as yeast and filamentous fungi, and genetically engineered to produce glycoproteins with mammalian or human-like glycosylation patterns. A particular advantage of these genetically modified host cells over currently used mammalian cell lines is their ability to control the glycosylation profile of intracellularly produced glycoproteins so that they can produce glycoprotein compositions in which specific N-glycan structures are dominant (see, for example, U.S. Patents 7,029,872 and 7,449,308). These genetically modified host cells are primarily used to produce antibodies with specific N-glycan structures.

[0222] In addition, fungi such as yeast or filamentous fungi lack the ability to produce fucosylated glycoproteins, so antibodies produced in such cells will lack fucose unless the cells are further modified to include an enzymatic pathway for producing fucosylated glycoproteins (see, for example, PCT International Publication 2008 / 112092). In certain embodiments, the antibodies disclosed herein are produced in lower eukaryotic host cells and further include antibodies containing fucosylated and non-fucosylated hybrids and complex N-glycans, including but not limited to bifid and polybranched species containing N-glycans such as GlcNAc(1-4)Man3GlcNAc2;Gal(1-4)GlcNAc(1-4)Man3GlcNAc2;NANA(1-4)Gal(1-4)GlcNAc(1-4)Man3GlcNAc2. In certain embodiments, the antibody compositions provided herein may comprise an antibody having at least one hybrid N-glycan selected from the group consisting of GlcNAcMan5GlcNAc2;GalGlcNAcMan5GlcNAc2; and NANAGalGlcNAcMan5GlcNAc2. In certain embodiments, the hybrid N-glycan is a dominant N-glycan species in the composition. In further embodiments, the hybrid N-glycan is a specific N-glycan species comprising about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the composition.

[0223] In certain embodiments, the antibody compositions provided herein include an antibody having at least one complex N-glycan selected from the group consisting of GlcNAcMan3GlcNAc2;GalGlcNAcMan3GlcNAc2;NANAGalGlcNAcMan3GlcNAc2;GlcNAc2Man3GlcNAc2;GalGlcNAc2Man3GlcNAc2;Gal2GlcNAc2Man3GlcNAc2;NANAGal2GlcNAc2Man3GlcNAc2; and NANA2Gal2GlcNAc2Man3GlcNAc2. In certain embodiments, the complex N-glycan is the dominant N-glycan species in the composition. In further embodiments, the complex N-glycan is a specific N-glycan species comprising about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the complex N-glycan in the composition. In certain embodiments, the N-glycan is fucosylated. Generally, fucose is located in an α1,3-linkage to GlcNAc at the reducing end of the N-glycan, an α1,6-linkage to GlcNAc at the reducing end of the N-glycan, an α1,2-linkage to Gal at the non-reducing end of the N-glycan, an α1,3-linkage to GlcNAc at the non-reducing end of the N-glycan, or an α1,4-linkage to GlcNAc at the non-reducing end of the N-glycan.

[0224] Therefore, in certain embodiments of the glycoprotein composition described above, the sugar form is either in the form of α1,3-linked or α1,6-linked fucose, or GlcNAc(Fuc)Man5GlcNAc2(Fuc), Man3GlcNAc2(Fuc), GlcNAcMan3GlcNAc2(Fuc), GlcNAc2Man3GlcNAc2(Fuc), GalGlcNAc2Man3GlcNAc2(Fuc), Gal2GlcNAc2Man3GlcNAc2(Fuc), NANAGal2GlcNAc2Man3GlcNAc2(Fuc), and NANA2Gal2GlcNAc2Man3GlcNAc2(Fuc), in order to generate a sugar form selected from the group consisting of Man5GlcNAc2(Fuc), GlcNAc(Fuc)Man5GlcNAc2, GlcNAc(Fuc)Man3GlcNAc2, GlcNAc2(Fuc1-2 To generate a sugar form selected from the group consisting of Man3GlcNAc2, GalGlcNAc2(Fuc1-2)Man3GlcNAc2, Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2, NANAGal2GlcNAc2(Fuc1-2)Man3GlcNAc2, and NANA2Gal2GlcNAc2(Fuc1-2)Man3GlcNAc2, it is located on α1,3-linked or α1,4-linked fucose, or to generate a sugar form selected from the group consisting of Gal(Fuc)GlcNAc2Man3GlcNAc2, Gal2(Fuc1-2)GlcNAc2Man3GlcNAc2, NANAGal2(Fuc1-2)GlcNAc2Man3GlcNAc2, and NANA2Gal2(Fuc1-2)GlcNAc2Man3GlcNAc2, it is located on α1,2-linked fucose.

[0225] In further embodiments, the antibody comprises, but is not limited to, a high-mannose N-glycan including Man8GlcNAc2, Man7GlcNAc2, Man6GlcNAc2, Man5GlcNAc2, Man4GlcNAc2, or an N-glycan consisting of a Man3GlcNAc2N-glycan structure. In the above further embodiments, the complex N-glycan further comprises fucosylated and non-fucosylated bifid and polybranched species. As used herein, the terms “N-glycan” and “sugar form” are interchangeable and refer, for example, to an N-linked oligosaccharide linked by an asparagine-N-acetylglucosamine linkage to an asparagine residue in a polypeptide. An N-linked glycoprotein contains an N-acetylglucosamine residue linked to the amide nitrogen of an asparagine residue in the protein.

[0226] C. Single chain antibody Single-chain variable fragments (scFvs) are fusions of the variable regions of the heavy and light chains of immunoglobulins, linked together with a short (usually serine, glycine) linker. This chimeric molecule retains the specificity of the original immunoglobulin despite the removal of the constant region and the introduction of the linker peptide. This modification typically preserves specificity without alteration. These molecules were historically created to facilitate phage display, where expressing the antigen-binding domain as a single peptide is highly convenient. Alternatively, scFvs can be created directly from subcloned heavy and light chains derived from hybridomas. Single-chain variable fragments lack the constant Fc region found in complete antibody molecules and therefore lack the common binding site (e.g., protein A / G) used to purify the antibody. These fragments can often be purified / immobilized using protein L, as protein L interacts with the variable region of the kappa light chain.

[0227] Flexible linkers generally consist of helical and spiral-promoting amino acid residues such as alanine, serine, and glycine. However, other residues can function similarly. Tang et al. (1996) used phage display as a means of rapidly selecting a tailored linker for single-chain antibodies (scFv) from a protein linker library. A random linker library was constructed in which the genes of the heavy and light chain variable domains were linked by segments encoding 18-amino acid polypeptides of variable compositions. The scFv repertoire (approximately 5 × 10⁶) 6 Several different members were displayed on filamentous phages and subjected to affinity selection using haptens. The selected population of variants showed a significant increase in binding activity but retained considerable sequence diversity. Subsequently, 1054 variants were screened to obtain catalytically active scFv efficiently produced in soluble form. Sequence analysis revealed that the only common feature of the selected tethers was V H The study revealed conserved proline in the linker of the two residues following the C-terminus, as well as large amounts of arginine and proline at other positions.

[0228] The recombinant antibodies of this disclosure may also include sequences or portions that enable receptor dimerization or multimerization. Such sequences include IgA-derived sequences that, together with the J chain, enable multimerization. Another multimerization domain is the Gal4 dimerization domain. In other embodiments, the chain may be modified with a drug such as biotin / avidin that enables the combination of two antibodies.

[0229] In another embodiment, single-chain antibodies can be produced by linking receptor light and heavy chains using non-peptide linkers or chemical units. Generally, the light and heavy chains are produced and purified in different cells and then linked together in an appropriate manner (i.e., the N-terminus of the heavy chain is linked to the C-terminus of the light chain via an appropriate chemical crosslink).

[0230] Crosslinking reagents are used to form molecular bridges that link the functional groups of two different molecules, for example, a stabilizer and a coagulant. However, it is also intended that heteromeric complexes consisting of dimers or polymers of the same analog or different analogs may be created. To link two different compounds stepwise, heterobifunctional crosslinking agents can be used to eliminate unwanted homopolymer formation.

[0231] An exemplary heterobifunctional crosslinking agent contains two reactive groups: one that reacts with a primary amine group (e.g., N-hydroxysuccinimide) and the other that reacts with a thiol group (e.g., pyridyl disulfide, maleimide, halogen, etc.). Via the primary amine reactive group, the crosslinking agent reacts with a lysine residue of one protein (e.g., a selected antibody or fragment), and via the thiol reactive group, the crosslinking agent already bound to the first protein may react with a cysteine ​​residue (free sulfhydryl group) of another protein (e.g., a selector).

[0232] It is preferable to use a crosslinking agent that has reasonable stability in the blood. Numerous types of disulfide bond-containing linkers are known and can be successfully used to conjugate targeted and therapeutic / prophylactic agents. Linkers containing sterically hindered disulfide bonds may provide greater stability in vivo and may be proven to prevent the release of targeted peptides before they reach the site of action. Therefore, these linkers are one type of linking agent.

[0233] Another crosslinking agent is SMPT, a bifunctional crosslinking agent containing a disulfide bond that is "sterically hindranced" by adjacent benzene rings and methyl groups. The steric hindrance of the disulfide bond is thought to protect the binding from attack by thiolate anions such as glutathione, which can be present in tissues and blood, thereby helping to prevent the detachment of the conjugate before the conjugated drug is delivered to its target site.

[0234] SMPT crosslinking agents, like many other known crosslinking agents, confer the ability to crosslink functional groups such as the SH group of cysteine ​​or primary amines (e.g., the epsilon-amino group of lysine). Another possible type of crosslinking agent is a heterobifunctional photoreactive phenyl azide containing a cleavable disulfide bond, such as sulfosuccinimidyl-2-(p-azidosalicylamide)ethyl-1,3'-dithiopropionate. The N-hydroxysuccinimidyl group reacts with primary amino groups, while the phenyl azide (in photodegradation) reacts non-selectively with any amino acid residue.

[0235] In addition to impaired crosslinking agents, unimpaired linkers may also be used according to this specification. Other useful crosslinking agents that are not thought to contain or generate protected disulfides include SATA, SPDP, and 2-iminothiolane (Wawrzynczak & Thorpe, 1987). The use of such crosslinking agents is well understood in the art. Another embodiment involves the use of flexible linkers.

[0236] U.S. Patent No. 4,680,338 describes a bifunctional linker useful for producing ligand conjugates with amine-containing polymers and / or proteins, particularly useful for forming antibody conjugates with chelators, drugs, enzymes, detectable labels, etc. U.S. Patents Nos. 5,141,648 and 5,563,250 disclose cleavable conjugates containing unstable bonds that can be cleaved under a variety of mild conditions. This linker is particularly useful in that the drug of interest can bind directly to the linker, and the active drug can be released upon cleavage. Specific applications include adding free amino or free sulfhydryl groups to proteins such as antibodies or drugs.

[0237] U.S. Patent No. 5,856,456 provides a peptide linker for use in linking polypeptide components to produce fusion proteins, such as single-chain antibodies. The linker is up to approximately 50 amino acids long and contains at least one charged amino acid (preferably arginine or lysine), followed by proline, and is characterized by higher stability and reduced aggregation. U.S. Patent No. 5,880,270 discloses an aminooxy-containing linker useful in various immunodiagnostic and separation techniques.

[0238] D. Purification In certain embodiments, the antibodies of this disclosure may be purified. As used herein, the term “purified” is intended to mean a composition that can be isolated from other components, and a protein is purified to any degree compared to its naturally achievable state. Thus, purified protein also means a protein that has been released from the environment in which it may naturally exist. Where the term “substantially purified” is used, this designation means a composition in which a protein or peptide forms a major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or more of the proteins in the composition.

[0239] Protein purification techniques are well known to those skilled in the art. These techniques, to some extent, involve the crude fractionation of the cellular environment into polypeptide and non-polypeptide fractions. After separating polypeptides from other proteins, the polypeptide of interest can be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suitable for the preparation of pure peptides are ion-exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, and isoelectric focusing. Other methods for protein purification include precipitation with ammonium sulfate, PEG, antibody, etc., or thermal denaturation, followed by centrifugation, gel filtration, reverse-phase, hydroxyl apatite, and affinity chromatography, as well as combinations of such techniques and other techniques.

[0240] When purifying the antibodies of this disclosure, it may be desirable to express the polypeptide in a prokaryotic or eukaryotic expression system and extract the protein using denaturing conditions. The polypeptide may be purified from other cellular components using an affinity column that binds to the tagged portion of the polypeptide. As is generally known in the art, the order in which the various purification steps are performed may be changed, or certain steps may be omitted, still resulting in a method suitable for preparing substantially purified proteins or peptides.

[0241] Generally, complete antibodies are fractionated using a substance that binds to the Fc portion of the antibody (i.e., protein A). Alternatively, the appropriate antibody can be simultaneously purified and selected using an antigen. Such methods often utilize a selection substance bound to a support such as a column, filter, or beads. The antibody is bound to the support, contaminants are removed (e.g., by washing), and the antibody is released by applying conditions (e.g., salt, heat).

[0242] Various methods for quantifying the purity of a protein or peptide will be known to those skilled in the art in light of this disclosure. These include, for example, determining the specific activity of an active fraction or assessing the amount of polypeptide in a fraction by SDS / PAGE analysis. Another method for assessing the purity of a fraction is to calculate the specific activity of the fraction and compare it to the specific activity of the initial extract to thus determine the degree of purity. The actual units used to express the amount of activity will, of course, depend on the specific assay technique chosen when performing the purification and whether the expressed protein or peptide exhibits detectable activity.

[0243] Polypeptide migration is known to sometimes be significantly altered under different SDS / PAGE conditions (Capaldi et al., 1977). Therefore, it should be understood that the apparent molecular weight of purified or partially purified expression products may change under different electrophoretic conditions.

[0244] V. Use and composition of anti-DDR1 antibodies A. Therapeutic methods and uses 1. Cancer treatment Hyperproliferative disorders can be associated with any disease in which cells begin to proliferate uncontrollably, but a typical example is cancer. One of the key elements of cancer is the disruption of the normal apoptotic cycle of cells, and therefore, drugs that inhibit cell growth are important therapeutic agents for treating these diseases. In this disclosure, the anti-DDR1 antibody or its antigen-binding fragment described herein can be used to reduce the number of cancer cells and therefore can be potentially used to treat various types of cancer cell lines. In some embodiments, the antibody or its antigen-binding fragment of this disclosure may be used to treat cancers, including solid tumors, in particular cancers that secrete the DDR1 protein or a part of it (such as the DDR1 extracellular domain), and / or cancers that have DDR1 present on the surface of cancer cells or cancer stem cells. In some embodiments, cancer or cancer cells that overexpress the DDR1 protein are selected for treatment with the antibody of this disclosure.

[0245] In some embodiments, the types of cancer and cancer cells that can be treated in accordance with this disclosure include, but are not limited to, cancers or cancer cells of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestinal tract, gingiva, head, kidney, liver, lung, nasopharynx, neck, ovaries, prostate, skin, stomach, pancreas, testes, tongue, cervix, or uterus. In addition, cancer may be, but are not limited to, the following histological types: malignant neoplasms, malignant tumors, carcinomas; undifferentiated carcinomas; giant cell carcinoma and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; piloma cell carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; malignant gastrinoma; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; cord-like adenoma; adenoid cystic carcinoma; adenocarcinoma of adenomatous polyp; familial Adenomatous polyposis; solid tumors; malignant carcinoid tumors; bronchiolalveolar adenocarcinoma; papillary adenocarcinoma; chromophobic carcinoma; eosinophilic carcinoma; affinity adenocarcinoma; basophilic carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary-follicular adenocarcinoma; non-encapsulated sclerosing carcinoma; adrenal cortical carcinoma; endometrial carcinoma; cutaneous adnexal carcinoma; apocrine gland carcinoma; sebaceous gland carcinoma; parotid gland carcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; invasive mammary gland Tubal carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; Paget's disease of the mammary gland; acinar cell carcinoma; adenosquamous cell carcinoma; adenosquamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant theca tumor; malignant granulosa cell tumor; malignant androblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extramammary paraganglioma; pheochromocytoma; gromangio sarcoma; malignant melanoma; achromatic melanoma; superficial spreading melanoma; malignant melanoma of a 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; mixed Müller's tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymal tumor; malignant Brenner tumor; malignant Philodes tumor; synovial sarcoma; malignant mesothelioma; dysplastic tumor; germ cell carcinoma; malignant teratoma; malignant ovarian goiter; choriocarcinoma; malignant mesonephroma; angiosarcoma; malignant hemangioendothelioma; Kaposi's sarcoma; malignant hemangioendothelioma; lymphangiosarcoma; osteosarcoma;Paraosteal osteosarcoma; chondrosarcoma; malignant chondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing's sarcoma; malignant odontogenic tumor; ameloblastoma; malignant ameloblastoma; ameloblastoma; malignant pineal tumor; chordoma; malignant glioma; ependymoma; astrocytoma; protoastrocytoma; fibrous astrocytoma; astroblastoma; glioma; oligodendroglioma; oligodendroglioma; primitive neuroectodermoma; cerebellar sarcoma; ganglion cell blastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignant meningioma; neurofibrosarcoma; malignant schwannoma; malignant Granuloma; malignant lymphoma; Hodgkin's disease; lateral granuloma; malignant small lymphocytic lymphoma; malignant large cell diffuse lymphoma; malignant follicular lymphoma; mycosis fungoides; other specific non-Hodgkin lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative bowel disease; leukemia; lymphocytic leukemia; plasma cell leukemia; erythrocyte leukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryocytic leukemia; myelosarcoma; and hairy cell leukemia. In certain embodiments, tumors may include osteosarcoma, angiosarcoma, rhabdomyosarcoma, leiomyosarcoma, Ewing's sarcoma, glioblastoma, neuroblastoma, or leukemia.

[0246] In some embodiments, antibodies or antigen-binding fragments are used to treat pancreatic cancer, lung cancer including small cell lung cancer and non-small cell lung cancer, 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, and bone cancer. In some embodiments, the cancer selected for treatment is breast cancer, including various breast cancer subtypes, as will be discussed further below.

[0247] In some embodiments, the target selected for treatment with an antibody or its antigen-binding fragment has cancer that secretes the DDR1 protein or a portion thereof, and / or has the DDR1 protein present on the cell surface of cancer cells. In some embodiments, a method for treating cancer includes the step of determining whether the cancer for treatment secretes the DDR1 protein and / or has the DDR1 protein expressed on the surface of cancer cells.

[0248] In some embodiments, the antibody or its antigen-binding fragment is used to treat hematological or blood cancers, including but not limited to malignant lymphoma, Hodgkin's disease, lateral granuloma, malignant small lymphocytic lymphoma, malignant large cell diffuse lymphoma, malignant follicular lymphoma, mycosis fungoides, other certain non-Hodgkin lymphomas, malignant histiocytosis, multiple myeloma, mast cell sarcoma, immunoproliferative bowel disease, leukemia, lymphocytic leukemia, plasma cell leukemia, erythrocyte leukemia, lymphosarcoma cell leukemia, myeloid leukemia, basophilic leukemia, eosinophilic leukemia, monocytic leukemia, mast cell leukemia, megakaryocytic leukemia, myelosarcoma, and leukemia.

[0249] In some embodiments, a subject selected for treatment with an antibody or its antigen-binding fragment has cancer that secretes the DDR1 protein or a portion thereof, and / or has the DDR1 protein present on the surface of cancer cells. In some embodiments, a method for treating cancer includes the step of determining whether the cancer for treatment secretes the DDR1 protein and / or has the DDR1 protein expressed on the surface of cancer cells. Thus, in some embodiments, a method for treating cancer in a subject includes determining the presence of secreted DDR1 protein in the subject or the presence of DDR1 protein on the surface of cancer cells, and if it is determined that the cancer cells have secreted DDR1 protein or have DDR1 present on the surface of cancer cells, the method includes treating the cancer in the subject by administering a therapeutically effective amount of the antibody or antigen-binding fragment disclosed herein.

[0250] 2. Treatment of breast cancer In some embodiments, the cancer for treatment is breast cancer, which typically originates from the breast, usually from the inner lining of the milk ducts or lobes. There are various types of breast cancer, differing in stage (extent), aggressiveness, and genetic makeup. With the best treatment, the 10-year disease-free survival rate varies from 98% to 10%. Treatment options include surgery, drugs (chemotherapy), and radiation. In the United States, there were 216,000 cases of invasive breast cancer in 2004, resulting in 40,000 deaths. Globally, breast cancer is the second most common cancer after lung cancer (10.4% of all cancers, regardless of gender) and the fifth leading cause of cancer death. In 2004, breast cancer caused 519,000 deaths worldwide (7% of all cancer deaths, nearly 1% of all deaths). Breast cancer is approximately 100 times more common in women than in men, but survival rates are similar for both sexes.

[0251] The first symptom, or subjective sign, of breast cancer is typically a lump that feels different from the surrounding breast tissue. According to the Merck Manual, more than 80% of breast cancer cases are discovered when a woman feels a lump. According to the American Association for Cancer Research, the first medical, or objective sign, of breast cancer detected by a physician is found by mammography. A lump found in the lymph nodes located in the armpit can also indicate breast cancer. Other indications for breast cancer may include changes in breast size or shape, skin retraction, nipple inversion, or spontaneous discharge from one nipple. Pain ("breast pain") is a less reliable tool for determining the presence or absence of breast cancer, but it can indicate other breast health problems.

[0252] When breast cancer cells invade the cutaneous lymphatic vessels and small lymphatic vessels of the breast skin, the appearance may resemble skin inflammation, and is therefore known as inflammatory breast cancer (IBC). Symptoms of inflammatory breast cancer include pain, swelling, warmth, and redness throughout the breast, as well as an orange, peeling texture, sometimes referred to as "peau d'orange." Another reported complex of breast cancer is Paget's disease of the breast. This syndrome presents as eczematous skin changes, such as redness and mild peeling of the skin around the nipple. As Paget's disease progresses, symptoms may include tingling, itching, increased sensitivity, burning, and pain. There may also be nipple discharge. Approximately half of women diagnosed with Paget's disease also have a lump in their breast.

[0253] Sometimes, breast cancer can manifest as a metastatic disease, that is, cancer that has spread beyond the original organ. Metastatic breast cancer will cause symptoms that depend on the location of the metastasis. Common sites of metastasis include the bones, liver, lungs, and brain. Unexplained weight loss, as well as symptoms of fever and chills, can sometimes be a sign of latent breast cancer. Bone or joint pain, as well as jaundice or neurological symptoms, can be symptoms of metastatic breast cancer. These symptoms are "nonspecific," meaning they could also be symptoms of many other diseases.

[0254] The main risk factors identified are sex, age, childbirth, hormones, high-fat diet, alcohol consumption, obesity, as well as environmental factors such as tobacco use, radiation, and shift work. While the etiology is unknown for 95% of breast cancer cases, approximately 5% of new breast cancers are attributed to hereditary syndromes. In particular, carriers of the breast cancer susceptibility genes BRCA1 and BRCA2 have a 30-40% increased risk of breast and ovarian cancer, depending on the part of the protein where the mutation occurs. Experts believe that 95% of hereditary breast cancers can be traced back to one of these two genes. Hereditary breast cancer can take the form of site-specific hereditary breast cancer, cancer affecting only the breast, or breast-ovarian and other cancer syndromes. Breast cancer can be inherited from both female and male relatives.

[0255] Breast cancer subtypes are typically classified immunohistochemically. The general definitions of subtypes are as follows: • Normal (ER+, PR+, HER2+, cytokeratin 5 / 6+, and HER1+) Luminal A (ER+ and / or PR+, HER2-) Luminal B (ER+ and / or PR+, HER2+) • Triple-negative (ER-, PR-, HER2-) • HER2+ / ER- (ER-, PR-, and HER2+) • Unclassified (ER-, PR-, HER2-, cytokeratin 5 / 6-, and HER1-)

[0256] In triple-negative breast cancer cells, cancer growth is not driven by estrogen or progesterone, or by growth signals derived from the HER2 protein. Similarly, such cancer cells do not respond to hormone therapies such as tamoxifen or aromatase inhibitors, or to therapies targeting the HER2 receptor, such as Herceptin®. It has been found that approximately 10–20% of breast cancers are triple-negative. It is important to identify these types of cancer and focus on therapies that can be used to treat triple-negative breast cancer so that the cost and toxic effects of treatments that are unlikely to be successful can be avoided. Like other forms of breast cancer, triple-negative breast cancer can be treated with surgery, radiation therapy, and / or chemotherapy. One particularly promising approach is "neoadjuvant" therapy, in which chemotherapy and / or radiation therapy are provided before surgery. Another drug therapy is the use of poly(ADP-ribose) polymerase, or PARP inhibitors.

[0257] While the screening techniques described above are useful in determining the likelihood of cancer, further testing is needed to confirm whether a lump detected by screening is cancerous, as opposed to benign alternatives such as simple cysts. In clinical practice, breast cancer is generally diagnosed using a “triple test” of clinical breast examination (a breast examination performed by a skilled physician), mammography, and fine-needle aspiration cytology. Both mammography and clinical breast examination, which are also used for screening, can indicate the approximate likelihood that a lump is cancerous and can also identify any other lesions. Fine-needle aspiration and cytology (FNAC), performed as an outpatient procedure using local anesthesia, involves an attempt to extract a small amount of fluid from the lump. Clear fluid indicates a very low probability that the lump is cancerous, while fluid mixed with blood may be transported for microscopic examination of cancerous cells. Using these three tools in combination allows for a highly accurate diagnosis of breast cancer. Other biopsy options include core biopsy, which removes a portion of the lump in the breast, and excisional biopsy, which removes the entire lump.

[0258] Breast cancer screening is an attempt to detect cancer in otherwise healthy individuals. The most common screening method for women is a combination of mammography and clinical breast examination. For women at higher risk than usual, such as those with a family history of serious cancer, additional tools may include genetic testing or magnetic resonance imaging of the breast.

[0259] Breast self-examination was a strongly advocated form of screening in the past, but it subsequently failed after several large-scale studies showed that it did not offer survival benefits to women and often caused considerable anxiety. This is likely because detectable cancers tended to be already relatively advanced, whereas other methods recommend identifying cancer at an earlier stage where curative treatment is more frequently possible.

[0260] X-ray mammography uses X-rays to examine the breasts for any featureless lumps or nodules. In some countries, regular mammography is recommended as a screening tool for women above a certain age.

[0261] Genetic testing for breast cancer typically involves testing for mutations in the BRCA gene. This is generally not recommended except for those at high risk of breast cancer.

[0262] When the location of the tumor is identified, the primary treatment for breast cancer is surgery, with possible use of adjuvant hormone therapy (using tamoxifen or aromatase inhibitors), chemotherapy, and / or radiation therapy. Currently, recommended postoperative treatment (adjuvant therapy) follows a pattern. Based on clinical criteria (age, type of cancer, size, metastasis), patients are broadly divided into high-risk and low-risk cases, and each risk category follows different treatment rules. Possible treatments include radiation therapy, chemotherapy, hormone therapy, and immunotherapy.

[0263] Targeted cancer therapy is a treatment that targets specific characteristics of cancer cells, such as proteins that allow cancer cells to grow rapidly or abnormally. Targeted therapies generally have a lower chance of damaging normal, healthy cells than chemotherapy. Some targeted therapies are antibodies that act like antibodies naturally produced by one's own immune system. These types of targeted therapies are sometimes called immunotargeted therapies.

[0264] Currently, there are three targeted therapies that doctors use to treat breast cancer. Herceptin® (trastuzumab) works against HER2-positive breast cancer by blocking the ability of cancer cells to receive chemical signals that instruct them to grow. Tykerb® (lapatinib) works against HER2-positive breast cancer by blocking certain proteins that can cause uncontrolled cell growth. Avastin® (bevacizumab) works by blocking the growth of new blood vessels on which cancer cells depend to grow and function.

[0265] Hormone (anti-estrogen) therapy is effective against hormone receptor-positive breast cancer in two ways: firstly, by reducing the amount of estrogen in the body, and secondly, by blocking the action of estrogen in the body. Most of the estrogen in a woman's body is produced by the ovaries. Estrogen promotes the growth of hormone receptor-positive breast cancer. Therefore, reducing the amount of estrogen or blocking its action helps shrink hormone receptor-positive breast cancer and reduce the risk of it recurring. Hormone therapy drugs are not effective against hormone receptor-negative breast cancer.

[0266] There are several types of hormone therapy drugs, including aromatase inhibitors, selective estrogen receptor modulators, and estrogen receptor downmodulators. In some cases, the ovaries and fallopian tubes may be surgically removed to treat hormone receptor-positive breast cancer or as a preventive measure for women at very high risk of developing breast cancer. The ovaries may also be temporarily closed using medication.

[0267] Treatment planning may also involve the use of PCR tests such as Oncotype DX, or microarray tests that predict breast cancer recurrence risk based on gene expression. In February 2007, the first breast cancer prediction test was officially approved by the U.S. Food and Drug Administration. This is a new genetic test that helps predict whether women with early-stage breast cancer will experience a recurrence after 5 or 10 years, and may influence how aggressively early tumors are treated.

[0268] Radiation therapy is also used to help destroy any cancer cells that may remain after surgery. When administered in the correct dose, radiation can reduce the risk of recurrence by 50-66%.

[0269] Accordingly, in some embodiments, the antibody or its antigen-binding fragment is used to treat breast cancer, including each of the breast cancer subtypes described above. In some embodiments, the antibody or its antigen-binding fragment of the Disclosure is used in combination with a second therapeutic agent, the second therapeutic agent comprising one or more agents described herein (e.g., radiotherapy, chemotherapy agents, hormones, and immunotherapy agents). In some embodiments, breast cancer or its subtypes that are determined to have DDR protein on the cell surface or to secrete DDR1 protein are treated with the antibody or antigen-binding fragment.

[0270] 3. Treatment of fibrous disorders In addition to expression in cancer, the DDR1 protein is also expressed in other organs, including the kidneys, lungs, gastrointestinal tract, skin, and brain, and is involved in fibrosis of the skin, lungs, and liver (Moll, et al. 2019, incorporated herein by reference). Therefore, in some embodiments, the anti-DDR1 antibody or its antigen-binding fragment of the present disclosure can be used alone or in combination with other therapies to treat fibrotic disorders. In some embodiments, the fibrotic disorder is organ fibrosis. In some embodiments, the fibrotic disorder is fibrosis of the skin, kidneys, liver, lungs, or heart. In some embodiments, the fibrotic disorder to be treated is, but is not limited to, hypertrophic cutaneous scarring, idiopathic pulmonary fibrosis, cirrhosis, and renal fibrosis.

[0271] In some embodiments, the fibrous disorder for treatment is pulmonary fibrosis. In some embodiments, the subject to be treated has interstitial lung disease. In some embodiments, the subject to be treated has idiopathic pulmonary fibrosis (IPF) or pulmonary scarring.

[0272] B. Formulation and Administration In another embodiment, the disclosure provides a pharmaceutical composition comprising an anti-DDR1 antibody and an antigen for generating it. Such a composition comprises a prophylactic or therapeutically effective amount of the antibody or a fragment thereof, and a pharmaceutically acceptable carrier. In certain embodiments, the term “pharmaceutically acceptable” means approved by a U.S. federal or state government regulatory authority or listed in the United States Pharmacopeia or other generally accepted pharmacopoeia for use in animals, particularly humans. The term “carrier” refers to a diluent, excipient, or vehicle administered with the therapeutic agent. Such a pharmaceutical carrier may be a sterile liquid, such as water and oil, and may be of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. When the pharmaceutical composition is administered intravenously, water is a specific carrier. Saline solutions, dextrose aqueous solutions, and glycerol aqueous solutions can also be used as liquid carriers, particularly in injectable solutions. Other suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, and ethanol.

[0273] If desired, the composition may also contain small amounts of wetting or emulsifying agents or pH buffers. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, etc. Oral formulations may contain standard pharmaceutical-grade carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, and magnesium carbonate. Examples of suitable pharmaceuticals are listed in "Remington's Pharmaceutical Sciences." Such compositions contain a prophylactically effective or therapeutically effective amount of antibody or a fragment thereof, preferably in a purified form, together with an appropriate amount of carrier, to provide a form for appropriate administration to the patient. The formulations must be adapted to a mode of administration, which may be delivered orally, intravenously, intra-arterially, orally, intranasally, by spray, bronchial inhalation, or by mechanical ventilation.

[0274] The antibodies of this disclosure described herein can be formulated for parenteral administration, for example, for injection via intradermal, intravenous, intramuscular, subcutaneous, intratumoral, or even intraperitoneal routes. Alternatively, the antibodies can be administered via direct local routes to the mucous membrane, for example, by nasal spray, inhalation, or nebulizer. Pharmaceutically acceptable salts include acidic salts and those formed with inorganic acids, such as hydrochloric acid or phosphoric acid, or organic acids, such as acetic acid, oxalic acid, tartaric acid, or mandelic acid. Salts formed with free carboxyl groups may be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, as well as organic bases such as isopropylamine, trimethylamine, 2-ethylaminoethanol, histidine, or procaine.

[0275] Generally, the components of the compositions of this disclosure are supplied separately or mixed together in unit dosage forms, and supplied as dry, lyophilized powders or water-free concentrates in sealed containers such as ampoules or sachets indicating the amount of activator. When the composition is administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical-grade water or saline. When the composition is administered by injection, ampoules of sterile water or saline for injection can be provided so that the components can be mixed before administration.

[0276] In some embodiments, the compositions of the present disclosure can be formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed from anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed from cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0277] In some embodiments, an antibody or its antigen-binding fragment is administered in an effective dose to treat a disease or disorder, particularly cancer such as breast cancer. The amount of anti-DDR1 antibody administered will depend on a variety of factors, including, but not limited to, the specific type of cancer being treated, the stage of the cancer being treated, the mode of administration, the frequency of administration, the desired therapeutic effect, and other parameters such as the patient's age, weight, and other characteristics.

[0278] The effective dosage for providing therapeutic benefits can be initially estimated from in vivo animal models or clinical trials. Suitable animal models for various diseases are known in the art, and determining effective doses for providing therapeutic benefits for specific modes and frequencies of administration is within the scope of the skills of those skilled in the art.

[0279] In some embodiments, the anti-DDR1 antibody or its antigen-binding fragment may be administered in a range of 0.0001 to 100 mg / kg body weight. Given the diversity of available antibody compositions and the different efficiencies of various administration routes, a wide range of dose variations is expected. These dose level variations can be adjusted using standard empirical routines for optimization, as is well understood in the art. In some embodiments, for the treatment of the indications described herein, the effective dose of the antibody of this disclosure may be in the range of about 0.001 to about 75 mg / kg body weight, 0.005 mg / kg to about 50 mg / kg body weight, about 0.01 mg / kg to about 30 mg / kg body weight, or about 0.01 to about 5 mg / kg body weight.

[0280] C. Cell therapy In another embodiment, the disclosure provides immune cells expressing a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen-binding fragment provided herein. In some embodiments, the CAR protein comprises, from the N-terminus to the C-terminus, a leader peptide, an anti-DDR1 heavy chain variable domain, a linker domain, an anti-DDR1 light chain variable domain, a human IgG1-CH2-CH3 domain, a spacer region, a CD28 transmembrane domain, and anti-DDR1 intracellular costimulatory signaling and CD3ζ intracellular T cell signaling domains.

[0281] Methods for immunotherapy are also provided, comprising the step of administering an effective amount of the immune cells of this disclosure. In one embodiment, a medical disease or disorder is treated by the transfer of an immune cell population that induces an immune response. In certain embodiments of this disclosure, cancer or infection is treated by the transfer of an immune cell population that induces an immune response. Methods for treating or delaying the progression of cancer in an individual are provided herein, comprising the step of administering an effective amount of antigen-specific cell therapy to the individual.

[0282] Immune cells may be T cells (e.g., regulatory T cells, CD4+ T cells, CD8+ T cells, or gamma delta T cells), NK cells, invariant NK cells, NKT cells, or macrophages. Methods for producing and manipulating immune cells, as well as methods for using and administering cells for adoptive cell therapy, in which case the cells may be autologous or allogeneic. Therefore, immune cells can be used in immunotherapy, for example, to target cancer cells.

[0283] Immune cells can be isolated from subjects, particularly human subjects. Immune cells can be obtained from healthy human subjects, healthy volunteers, or healthy donors. Immune cells can be obtained from subjects of interest, such as subjects suspected of having a specific disease or condition, subjects suspected of being predisposed to a specific disease or condition, or subjects receiving treatment for a specific disease or condition. Immune cells can be collected from any location present in the subject, including but not limited to blood, umbilical cord blood, spleen, thymus, lymph nodes, and bone marrow. Isolated immune cells can be used directly or stored for a period of time by means of freezing or other means.

[0284] Immune cells can be concentrated / purified from any tissue in which they reside, including blood (including blood collected by blood banks or umbilical cord blood banks), spleen, bone marrow, tissues removed and / or exposed during surgical procedures, and tissues obtained through biopsy procedures. The tissues / organs from which immune cells are concentrated, isolated, and / or purified can be isolated from both viable and non-viable subjects, the non-viable subject being an organ donor. In certain embodiments, immune cells are isolated from blood, such as peripheral blood or umbilical cord blood. In some embodiments, immune cells isolated from umbilical cord blood have enhanced immunomodulatory capacity, such as by CD4 or CD8-positive T cell suppression. In certain embodiments, immune cells are isolated from pooled blood, particularly pooled spinal blood, for enhanced immunomodulatory capacity. Pooled blood may come from two or more sources, such as 3, 4, 5, 6, 7, 8, 9, or 10 or more sources (e.g., donor subjects).

[0285] A population of immune cells may be obtained from a subject in need of treatment or from a subject suffering from a disease associated with reduced immune cell activity. Therefore, the cells are autologous to the subject in need of treatment. Alternatively, a population of immune cells may be obtained from a donor, preferably a histocompatibility-matched donor. The immune cell population can be collected from peripheral blood, umbilical cord blood, bone marrow, spleen, or any other organ / tissue where immune cells are present in the aforementioned subject or donor. Immune cells may be isolated from a pool of subjects and / or donors, for example, from pooled spinal blood.

[0286] When an immune cell population is obtained from a donor different from the target, the donor is preferably allogeneic, but the cells obtained are target-compatible in that they can be introduced into the target. Allogeneic donor cells may or may not be human leukocyte antigen (HLA) matched. To confer target compatibility, allogeneic cells can be treated to reduce their immunogenicity.

[0287] Immune cells can be genetically engineered to express antigen receptors such as engineered TCRs and / or chimeric antigen receptors (CARs). For example, host cells (e.g., autologous or allogeneic T cells) are modified to express T cell receptors (TCRs) that have antigen specificity for cancer antigens. In certain embodiments, NK cells are engineered to express TCRs. NK cells may be further engineered to express CARs. Multiple CARs and / or TCRs, such as those for different antigens, can be added to single cell types such as T cells or NK cells.

[0288] Suitable modification methods are known in the art. See, for example, Sambrook et al., above, and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and John Wiley & Sons, NY, 1994. For example, cells can be transduced to express T cell receptors (TCRs) with antigen specificity for cancer antigens using transduction techniques described in Heemskerk et al. (2008) and Johnson et al. (2009).

[0289] In some embodiments, the cell comprises one or more nucleic acids introduced via genetic engineering that encodes one or more antigen receptors, and genetically engineered products of such nucleic acids. In some embodiments, the nucleic acids are heterogeneous, i.e., not typically present in cells or samples obtained from cells, e.g., from another organism or cell, e.g., not typically found in the engineered cell and / or the organism from which such cells originate. In some embodiments, the nucleic acids are not naturally occurring, such as nucleic acids not found in nature (e.g., chimeras).

[0290] D. Combination therapy It may also be desirable to provide combination therapies using the antibodies of this disclosure in conjunction with additional anticancer therapies. These therapies are provided in combination doses effective in achieving a reduction in one or more disease parameters. This process may involve contacting cells / subjects with both drugs / therapies simultaneously, for example, by using a single composition or pharmacological formulation containing both drugs, or by simultaneously contacting cells / subjects with two different compositions or formulations (one composition containing an antibody and the other composition containing the other drug).

[0291] Alternatively, antibodies can precede or follow other treatments at intervals ranging from minutes to weeks. Generally, by ensuring that no significant time elapses between each delivery, the treatments can still exert a favorably combined effect on the cells / targets. In such cases, the aim is to bring the cells into contact with both modalities within approximately 12–24 hours of each other, within approximately 6–12 hours of each other, or with a delay of only about 12 hours. In some situations, it may be desirable to significantly extend the treatment period, with several tens of days (2, 3, 4, 5, 6, or 7) to several weeks (1, 2, 3, 4, 5, 6, 7, or 8) elapsed between each administration.

[0292] It may be desirable to administer more than one of the anti-DDR1 antibodies or other therapies. Various combinations can be used, and here are some examples, where the antibody is "A" and the other therapy is "B": A / B / AB / A / BB / B / AA / A / BB / A / AA / B / BB / B / B / AB / B / A / B A / A / B / BA / B / A / BA / B / B / AB / B / A / AB / A / B / AB / A / A / BB / B / B / A A / A / A / BB / A / A / AA / B / A / AA / A / B / AA / B / B / BB / A / B / BB / B / A / B

[0293] Other combinations are being explored. Using the methods and compositions of the present invention, target cells or sites can be brought into contact with an antibody and at least one other therapeutic agent to kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis, or otherwise reverse or reduce the malignant phenotype of tumor cells. These therapies are provided in combination amounts effective in inhibiting the death or proliferation of cancer cells. This process may involve bringing cells / sites / targets into contact with the drug / therapy simultaneously.

[0294] Specific agents intended for combination therapy with the antibodies disclosed herein include chemotherapy and hematopoietic stem cell transplantation. Chemotherapy may include cytarabine (ara-C) and anthracyclines (most often daunorubicin), high-dose cytarabine monotherapy, all-trans retinoic acid (ATRA) in addition to induction chemotherapy, usually in addition to anthracyclines, histamine dihydrochloride (Ceplene) and interleukin-2 (Proleukin) after completion of intensification therapy, gemtuzumab ozogamicin (Mylotarg) for relapsed AML patients aged 60 years or older who are not suitable for high-dose chemotherapy, clofarabine, and targeted therapies such as kinase inhibitors, farnesyltransferase inhibitors, decitabine, and MDR1 (multidrug resistance protein) inhibitors, or arsenic trioxide or relapsed acute promyelocytic leukemia (APL).

[0295] In certain embodiments, the agents for combination therapy include topoisomerase inhibitors, anthracycline topoisomerase inhibitors, anthracyclines, daunorubicin, nucleoside metabolism inhibitors, cytarabine, hypomethylating agents, low-dose cytarabine (LDAC), combinations of daunorubicin and cytarabine, daunorubicin and cytarabine liposomes for injection, Vyxeos®, azacitidine, Vidaza®, decitabine, all-trans retinoic acid (ATRA), arsenic, arsenic trioxide, histamine dihydrochloride, Ceplene®, interleukin-2, aldesleukin, Proleukin®, and getumuzumab. Ozogamicin, Mylotarg®, FLT-3 inhibitor, Midostaurin, Rydapt®, Clofarabine, Farnesyltransferase inhibitor, Decitabine, IDH1 inhibitor, Ivosidenib, Tibsovo®, IDH2 inhibitor, Enasidenib, Idhifa®, Smoothened (SMO) inhibitor, Glasdegib, Arginase inhibitor, IDO inhibitor, Epacadostat, BCL-2 inhibitor, Venetoclax, Venclexta®, Platinum complex derivative, Oxaliplatin, Kinase inhibitor, Tyrosine kinase inhibitor, PI3 kinase inhibitor, BTK inhibitor, Ibrutinib, IMBRUV The drug is one or more drugs selected from the group consisting of ICA®, acalabrutinib, CALQUENCE®, zanubrutinib, PD-1 antibody, PD-L1 antibody, CTLA-4 antibody, LAG3 antibody, ICOS antibody, TIGIT antibody, TIM3 antibody, CD40 antibody, 4-1BB antibody, CD47 antibody, SIRP1α antibody or fusion protein, E-selectin antagonists, antibodies that bind to tumor antigens, antibodies that bind to T cell surface markers, antibodies that bind to bone marrow cells or NK cell surface markers, alkylating agents, nitrosourea agents, antimetabolites, antitumor antibiotics, plant-derived alkaloids, hormone therapy drugs, hormone antagonists, aromatase inhibitors, and P-glycoprotein inhibitors. In some embodiments, the drugs used in combination therapy are drugs that have been previously used as therapies for specific indications, such as certain types of cancer.In some embodiments, the specific indication is breast cancer, and the drug used in combination with the antibody or its antigen conjugate is a drug that is acceptable for the treatment of breast cancer.

[0296] VI. Antibody Conjugates The antibodies of this disclosure may be conjugated to at least one drug to form an antibody conjugate. Conventional methods involve conjugating, covalently bonding, or complexing at least one desired molecule or part to an antibody molecule in order to increase its efficacy as a diagnostic or therapeutic agent. Such molecule or part may, but are not limited to, at least one effector or reporter molecule. Effector molecules include molecules having a desired activity, e.g., cytotoxic activity. Non-limiting examples of effector molecules conjugated to antibodies include toxins, antitumor agents, therapeutic enzymes, radionuclides, antivirals, chelators, cytokines, growth factors, and oligonucleotides or polynucleotides. In contrast, a reporter molecule is defined as any part that can be detected using an assay. Non-limiting examples of reporter molecules conjugated to antibodies include ligands such as enzymes, radiolabels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, photoaffinity molecules, colored particles, or biotin.

[0297] Antibody-drug conjugates have emerged as a groundbreaking approach to the development of cancer treatments. Antibody-drug conjugates (ADCs) contain a monoclonal antibody (MAb) covalently linked to a cell-killing drug. This approach combines the high specificity of the MAb to an antigen target with a potent cytotoxic drug, resulting in an "armed" MAb that delivers the payload (drug) to tumor cells enriched with the antigen. Targeted drug delivery also minimizes its exposure to normal tissue, leading to reduced toxicity and improved therapeutic index. This approach was validated by the FDA approval of two ADC drugs: ADCETRIS® (brentuximab vedotin) in 2011 and KADCYLA® (trastuzumab emtansine or T-DM1) in 2013. Currently, there are more than 30 ADC drug candidates in various stages of clinical trials for cancer treatment (Leal et al., 2014). As antibody engineering and linker-payload optimization become increasingly mature, the discovery and development of new ADCs increasingly depend on this approach and the identification and validation of new targets suitable for the generation of targeted MAbs. Two criteria for ADC targets are upregulation / high levels of expression in tumor cells and robust internalization.

[0298] Antibody conjugates are also preferably used as diagnostic agents. Antibody diagnostics generally fall into two classes: those for use in in vitro diagnostics such as various immunoassays, and those for use in in vivo diagnostic protocols commonly known as “antibody-directed imaging.” Many suitable imaging agents, as well as methods for their attachment to antibodies, are known in the art (see, for example, U.S. Patents 5,021,23, 4,938,948, and 4,472,509). Imaging sites used may include paramagnetic ions, radioisotopes, fluorescent dyes, NMR-detectable substances, and X-ray imaging agents.

[0299] In some embodiments, the portion that binds to the antibody or its antigen-binding fragment is a paramagnetic ion, which can be selected from, among others, chromium(III), manganese(II), iron(III), iron(II), cobalt(II), nickel(II), copper(II), neodymium(III), samarium(III), ytterbium(III), gadolinium(III), vanadium(II), terbium(III), dysprosium(III), holmium(III), and / or erbium(III), with gadolinium being particularly preferred. Ions useful in other contexts such as X-ray imaging include, but are not limited to, lanthanum(III), gold(III), lead(II), and especially bismuth(III).

[0300] For radioactive isotopes for therapeutic and / or diagnostic purposes, the isotope is astatine. 211 , 14 carbon, 51 chromium, 36 chlorine, 57 cobalt, 58 Cobalt, copper 67 , 152 EU, Gallium 67 , 3 Hydrogen, iodine 123 iodine 125 iodine 131 ,indium 111 , 59 iron, 32 Phosphorus, Rhenium 186 ,rhenium 188 , 75 selenium, 35 Sulfur, technetium 99m and / or yttrium 90 It can be selected from the following. 125 I is often preferred for use in certain embodiments, and technetium 99m and / or indium 111These are often preferred due to their suitability for low-energy and long-range detection. The radiolabeled monoclonal antibodies of this disclosure can be produced according to methods well known in the art. For example, monoclonal antibodies can be iodized by contact with sodium iodide and / or potassium iodide, as well as chemical oxidizing agents such as sodium hypochlorite, or enzymatic oxidizing agents such as lactoperoxidase. Monoclonal antibodies according to this disclosure can be produced by a ligand exchange process, for example, by reducing pertechnate with a stannous solution, chelating the reduced technetium on a Sephadex column, and applying the antibody to this column. 99m It can be labeled with . Alternatively, direct labeling techniques may be used, for example, by incubating the antibody with pertechnate, a reducing agent such as SNCl2, a buffer such as sodium potassium phthalate solution, and other components. Intermediate functional groups often used to bind radioisotopes, which exist as metal ions, to antibodies are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).

[0301] Fluorescent labels intended for use as conjugates include Alexa350, Alexa430, AMCA, BODIPY630 / 650, BODIPY650 / 665, BODIPY-FL, BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM, Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green 500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, Rhodamine Red, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and / or Texas Red.

[0302] Another type of antibody conjugate conjugate conjugate envisioned in this disclosure is primarily intended for in vitro use, in which the antibody is linked to a secondary binding ligand and / or enzyme (enzyme tag) that produces a colored product upon contact with a chromogenic substrate. Examples of preferred enzymes include urease, alkaline phosphatase, (horseradish) hydrogen peroxidase, or glucose oxidase. Preferred secondary binding ligands are biotin and avidin and streptavidin compounds. The use of such labels is well known to those skilled in the art and is described, for example, in U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241.

[0303] Another known method for site-specific binding of molecules to antibodies involves the reaction of the antibody with a hapten-based affinity label. Essentially, the hapten-based affinity label reacts with amino acids at the antigen-binding site, thereby disrupting this site and blocking the specific antigen reaction. However, this may not always be advantageous as it results in the loss of antigen binding by the antibody conjugate.

[0304] Molecules containing azide groups can also be used to form covalent bonds to proteins via reactive nitrene intermediates generated by low-intensity ultraviolet light (Potter and Haley, 1983). In particular, 2- and 8-azide analogs of purine nucleotides have been used as site-directed optical probes to identify nucleotide-binding proteins in crude cell extracts (Owens & Haley, 1987, Atherton et al., 1985). 2- and 8-azide nucleotides have also been used to map nucleotide-binding domains of purified proteins (Khatoon et al., 1989, King et al., 1989, Dholakia et al., 1989) and can be used as antibody conjugates.

[0305] Several methods for binding or conjugating antibodies to their conjugate moieties are known in the art. Some attachment methods involve the use of metal chelate conjugates with organic chelating agents such as diethylenetriaminepentaacetic anhydride (DTPA), ethylenetriaminetetraacetic acid, N-chloro-p-toluenesulfonamide, and / or tetrachloro-3α-6α-diphenylglycouryl-3 (U.S. Patent Nos. 4,472,509 and 4,938,948) attached to the antibody. Monoclonal antibodies may also be reacted with enzymes in the presence of coupling agents such as glutaraldehyde or periodates. Conjugates with fluorescein markers are prepared in the presence of these coupling agents or by reaction with isothiocyanates. U.S. Patent No. 4,938,948 describes a method for imaging breast tumors using a monoclonal antibody, and then conjugating the detectable imaging portion to the antibody using a linker such as methyl-p-hydroxybenzimidyl-3-(4-hydroxyphenyl)propionate.

[0306] In other embodiments, immunoglobulin derivatization is attempted by selectively introducing sulfhydryl groups into the Fc region of immunoglobulins using reaction conditions that do not alter the antibody binding site. Antibody conjugates produced according to this method have been disclosed to exhibit improved lifetime, specificity, and sensitivity (U.S. Patent No. 5,196,066, incorporated herein by reference). Site-specific binding of effector or reporter molecules, in which the reporter or effector molecule is conjugated to a carbohydrate residue within the Fc region, has also been disclosed in the literature (O'Shannessy et al., 1987). This approach has been reported to produce diagnostic and therapeutically promising antibodies currently undergoing clinical evaluation.

[0307] VII. Immunodetection Methods In further embodiments, the disclosure relates to immunodetection methods for binding, purifying, removing, quantifying, and otherwise detecting DDR1-associated cancers in general. While such methods can be applied in a conventional sense, another use is quality control and monitoring of vaccines and other viral stocks, where antibodies according to the disclosure can be used to assess the amount or integrity (i.e., long-term stability) of H1 antigen in the virus. Alternatively, the methods can be used to screen various antibodies for suitable / desired reactivity profiles.

[0308] Some immunoassay methods include, to name a few, enzyme-linked immunosorbent assays (ELISA), radioimmunoassays (RIA), immunoradioassays, fluorescence immunoassays, chemiluminescence assays, bioluminescence assays, and Western blotting. In particular, competitive assays for the detection and quantification of DDR1 are also provided. Steps of various useful immunoassay methods are described in scientific literature, for example, Doolittle and Ben-Zeev (1999), Gulbis and Galand (1993), De Jager et al. (1993), and Nakamura et al. (1987). Generally, an immunoconjugation method comprises obtaining a sample suspected of containing DDR1-related cancer and, optionally, contacting the sample with a first antibody according to this disclosure under conditions effective in enabling the formation of an immune complex.

[0309] These methods include methods for detecting or purifying DDR1 or DDR1-related cancer cells from a sample. The antibody is preferably bound to a solid support, such as in the form of a column matrix, and a sample suspected of containing DDR1-related cancer cells is applied to the immobilized antibody. Unwanted components are washed off the column while the DDR1-expressing cells remain immunocomplexed with the immobilized antibody, and then the organism or antigen is collected by removing it from the column.

[0310] The immunoconjugation method also includes methods for detecting and quantifying the amount of DDR1-related cancer cells or related components in a sample, as well as for detecting and quantifying any immune complexes formed during the conjugation process. This specification describes obtaining a sample suspected of containing DDR1-related cancer cells, contacting the sample with an antibody or its components that bind to DDR1, and subsequently detecting and quantifying the amount of immune complexes formed under specific conditions. With respect to antigen detection, the biological sample to be analyzed may be any sample suspected of containing DDR1-related cancer, such as tissue sections or specimens, homogenized tissue extracts, biological fluids including blood and serum, or secretions such as feces or urine.

[0311] Contacting a selected biological sample with antibodies for a sufficient period to allow the formation of immune complexes (primary immune complexes) under effective conditions generally involves simply adding the antibody composition to the sample and incubating the mixture for a sufficient period for the antibodies to form immune complexes, i.e., a sufficient period for them to bind to DDR1. After this time, the sample-antibody composition, such as tissue sections, ELISA plates, dot blots, or Western blots, is generally washed to remove any nonspecifically bound antibody species, allowing only antibodies specifically bound within the primary immune complex to be detected.

[0312] In general, the detection of immune complex formation is well known in the art and can be achieved by applying a number of approaches. These methods generally rely on the detection of labels or markers, such as radioactive, fluorescent, biological, and enzymatic tags. Patents relating to the use of such labels include U.S. Patents 3,817,837, 3,850,752, 3,939,350, 3,996,345, 4,277,437, 4,275,149, and 4,366,241. Of course, as is known in the art, further advantages can be found through the use of a second antibody and / or a secondary binding ligand such as a biotin / avidin ligand binding configuration.

[0313] The antibody used for detection may itself be bound to a detectable label, and this label can then be simply detected to determine the amount of primary immunocomplex in the composition. Alternatively, the first antibody that will bind within the primary immunocomplex may be detected by a second binding ligand having a binding affinity for the antibody. In these cases, the second binding ligand can be bound to a detectable label. The second binding ligand itself is often an antibody that can therefore be referred to as a “secondary” antibody. The primary immunocomplex is contacted with the labeled secondary binding ligand or antibody under effective conditions for a period sufficient to allow the formation of the secondary immunocomplex. The secondary immunocomplex is then generally washed to remove any nonspecifically bound labeled secondary antibody or ligand, and then any remaining label in the secondary immunocomplex is detected.

[0314] A further method involves a two-step approach to detecting primary immune complexes. As described above, a second binding ligand, such as an antibody with binding affinity to the antibody, is used to form a secondary immune complex. After washing, the secondary immune complex is again contacted with a third binding ligand or antibody having binding affinity to the second antibody, under effective conditions for a period sufficient to allow the formation of an immune complex (tertiary immune complex). The third ligand or antibody is linked to a detectable label, enabling the detection of the tertiary immune complex thus formed. This system may provide signal amplification if desired.

[0315] One immunoassay method uses two different antibodies. A first biotinylated antibody is used to detect a target antigen, and then a second antibody is used to detect biotin bound to complexed biotin. In this method, the sample to be tested is first incubated in a solution containing the antibody from the first step. If the target antigen is present, some of the antibodies bind to the antigen to form a biotinylated antibody / antigen complex. The antibody / antigen complex is then amplified by incubation in a series of solutions of streptavidin (or avidin), biotinylated DNA, and / or complementary biotinylated DNA, with each step adding further biotin sites to the antibody / antigen complex. The amplification step is repeated until a suitable level of amplification is achieved, at which point the sample is incubated in a solution containing the antibody from the second step against biotin. This second-step antibody is labeled with an enzyme that can be used, for example, to detect the presence of the antibody / antigen complex by histoenzymology using a chromogen substrate. Through appropriate amplification, macroscopically visible conjugates can be produced.

[0316] Another known method of immunodetection utilizes the immuno-PCR (polymerase chain reaction) methodology. The PCR method is similar to the Cantor method up to incubation with biotinylated DNA, but instead of using multiple rounds of streptavidin and biotinylated DNA incubation, the DNA / biotin / streptavidin / antibody complex is washed with a low-pH or high-salt buffer that releases the antibody. The resulting wash is then used to perform a PCR reaction with suitable primers that have appropriate controls. At least theoretically, the enormous amplification capacity and specificity of PCR can be utilized to detect single antigen molecules.

[0317] A.ELISA An immunoassay, in its simplest and most direct sense, is a binding assay. Certain preferred immunoassays include various types of enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in the art. Immunohistochemical detection using tissue sections is also particularly useful. However, it will be readily apparent that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analysis, and others can also be used.

[0318] In one exemplary ELISA, the antibody of this disclosure is immobilized on a selected surface exhibiting protein affinity, such as a well in a polystyrene microtiter plate. A test composition suspected to contain DDR1-associated cancer cells is then added to the well. After binding and washing to remove nonspecifically bound immune complexes, the bound antigen can be detected. Detection can be achieved by adding another anti-DDR1 antibody bound to a detectable label. This type of ELISA is a simple “sandwich ELISA”. Detection can also be achieved by adding a second anti-DDR1 antibody, followed by the addition of a third antibody having binding affinity to the second antibody, the third antibody being bound to a detectable label.

[0319] In another exemplary ELISA, a sample suspected of containing DDR1-associated cancer cells is immobilized on the surface of a well and then brought into contact with the anti-DDR1 antibody of this disclosure. After binding and washing to remove nonspecifically bound immune complexes, the bound anti-DDR1 antibody is detected. If the initial anti-DDR1 antibody is ligated to a detectable label, the immune complexes may be detected directly. Again, the immune complexes may be detected using a second antibody having a binding affinity to the first anti-DDR1 antibody, the second antibody being ligated to a detectable label.

[0320] Regardless of the format used, ELISAs share certain common characteristics, including coating, incubation, and binding, washing to remove nonspecifically bound species, and detection of bound immune complexes. These are described below.

[0321] When coating plates with either an antigen or an antibody, the plate wells are generally incubated with the antigen or antibody solution overnight or for a specified period of time. The plate wells are then washed to remove any incompletely adsorbed material. Next, any remaining available surface of the wells is "coated" with a nonspecific protein that is antigenically neutral with respect to the test antiserum. These include solutions of bovine serum albumin (BSA), casein, or milk powder. The coating blocks nonspecific adsorption sites on the immobilized surface, thus reducing background caused by the nonspecific binding of the antiserum to the surface.

[0322] In ELISA, it is more common to use secondary or tertiary detection methods rather than direct treatment. Therefore, proteins or antibodies are bound to wells, coated with a non-reactive material to reduce background, washed to remove unbound material, and then the immobilized surface is brought into contact with the biological sample to be tested under conditions effective in enabling immune complex (antigen / antibody) formation. Next, detection of the immune complexes requires a labeled secondary binding ligand or antibody, and a labeled tertiary antibody or a secondary binding ligand or antibody combined with a third binding ligand.

[0323] "Under conditions effective in enabling the formation of immune complexes (antigen / antibody)" means that the conditions preferably include diluting the antigen and / or antibody with a solution such as BSA, bovine gamma globulin (BGG), or phosphate-buffered saline (PBS) / Tween. These additives also tend to help reduce nonspecific background.

[0324] "Suitable" conditions also mean that the incubation is at a temperature or for a period of time sufficient to allow for effective bonding. The incubation step may typically be about 1 to 2 to 4 hours, preferably at a temperature of about 25°C to 27°C, or overnight at about 4°C.

[0325] Following all incubation steps in the ELISA, the contacted surfaces are washed to remove any uncomplexed material. Preferred washing procedures include washing with a solution such as PBS / Tween or borate buffer. Specific immunocomplexes are formed between the test sample and the first bound substance, and after subsequent washing, the development of trace amounts of immunocomplexes can be determined.

[0326] To provide a means of detection, the second or third antibody has a relevant label to enable detection. Preferably, this is an enzyme that produces color development when incubated with a suitable chromogenic substrate. Therefore, it would be desirable to contact or incubate the first and second immunocomplexes with urease, glucose oxidase, alkaline phosphatase, or hydrogen peroxidase conjugate antibodies for a certain period of time and under conditions favorable for the development of further immunocomplex formation (e.g., incubation for 2 hours at room temperature in a PBS-containing solution such as PBS-Tween).

[0327] After incubation with the labeled antibody and washing to remove unbound substances, the amount of label is quantified by incubation with a chromogenic substrate such as urea, bromocresol purple, 2,2'-azino-di(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS), or H2O2, for example, when peroxidase is used as the enzyme label. Quantification is then achieved by measuring the degree of color produced, for example, using a visible spectrum spectrophotometer.

[0328] B. Western blot Western blotting (or alternatively, protein immunoblotting) is an analytical technique used to detect specific proteins in a given tissue homogenate or extract sample. It uses gel electrophoresis to separate native or denatured proteins by polypeptide length (denatured conditions) or protein 3D structure (native / non-denatured conditions). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (detected) using antibodies specific to the target protein.

[0329] Samples can be taken from whole tissues or from cell cultures. Often, solid tissues are first mechanically broken down using a blender (for larger sample volumes), a homogenizer (for smaller volumes), or sonication. Cells may be released by one of the mechanical methods described above. However, it should be noted that bacteria, viruses, or environmental samples can be sources of proteins, and therefore Western blotting is not limited to cell studies. Various detergents, salts, and buffers can be used to promote cell lysis and solubilize proteins. Protease and phosphatase inhibitors are often added to prevent digestion of the sample by their own enzymes. Tissue preparation is often performed at low temperatures to avoid protein denaturation.

[0330] Proteins in a sample are separated using gel electrophoresis. Protein separation can be by isoelectric point (pI), molecular weight, charge, or a combination of these factors. The nature of the separation depends on the sample treatment and the properties of the gel. This is a very useful method for determining proteins. It is also possible to use a two-dimensional (2D) gel, spreading proteins from a single sample in two dimensions. Proteins are separated according to their isoelectric point (pH with a neutral net charge) in the first dimension and according to their molecular weight in the second dimension.

[0331] To create proteins accessible for antibody detection, they are transferred from the gel onto a membrane made of nitrocellulose or polyvinylidene fluoride (PVDF). The membrane is placed on top of the gel, and a stack of filter paper is placed on top of it. The entire stack is placed in a buffer and moved to the paper by capillary action, with the proteins being carried along with it. Another method for transferring proteins is called electroblotting, which uses an electric current to draw the proteins from the gel onto the PVDF or nitrocellulose membrane. The proteins move from the gel onto the membrane while maintaining their configuration within the gel. As a result of this blotting process, the proteins are exposed on a thin surface layer for detection (see below). Both types of membranes are chosen for their non-specific protein-binding properties (i.e., they bind all proteins equally well). Protein binding is based on hydrophobic interactions, as well as charged interactions between the membrane and the protein. Nitrocellulose membranes are cheaper than PVDF but are much more fragile and less resistant to repeated probing. The uniformity and overall effectiveness of protein transfer from the gel to the membrane can be confirmed by staining the membrane with Coomassie Brilliant Blue or Ponceau S dye. Once transferred, the protein is detected using a labeled primary antibody or an unlabeled primary antibody, and subsequently indirectly using labeled protein A or a secondary labeled antibody that binds to the Fc region of the primary antibody.

[0332] C. Immunohistochemistry In some embodiments, the antibodies of this disclosure may be used in conjunction with both fresh-frozen and / or formalin-fixed paraffin-embedded tissue blocks prepared for immunohistochemical (IHC) studies. Methods for preparing tissue blocks from these microparticle specimens have been successfully used in previous IHC studies of various prognostic factors and are well known to those skilled in the art (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990).

[0333] In short, frozen sections can be prepared by rehydrating 50 ng of frozen "pulverized" tissue in phosphate-buffered saline (PBS) at room temperature, pelletizing the particles by centrifugation, resuspending the particles in viscous embedding medium (OCT), inverting the capsule and / or pellet again by centrifugation, rapidly freezing in isopentane at -70°C, cutting the plastic capsule and / or removing the frozen tissue cylinder, fixing the tissue cylinder to a cryostat microtome chuck, and / or cutting 25 to 50 serial sections from the capsule. Alternatively, the entire frozen tissue sample may be used to cut serial sections.

[0334] Permanent sections may be prepared by a similar method, including rehydrating a 50 mg sample in a plastic microcentrifuge tube, pelletizing, resuspending in 10% formalin and fixing for 4 hours, washing / pelletizing, resuspending in warm 2.5% agar, pelletizing, cooling in ice water to harden the agar, removing the tissue / agar block from the tube, infiltrating and / or embedding the block in paraffin, and / or cutting up to 50 serial permanent sections. Again, the entire tissue sample may be replaced.

[0335] D. Immunoassay kits In further embodiments, this disclosure also envisions an immunodetection kit for use in conjunction with the immunodetection method described above. Since antibodies may be used to detect DDR1-associated cancer cells, antibodies may be included in the kit. Thus, the immunodetection kit comprises, in a suitable container means, a first antibody that binds to DDR1, and optionally an immunodetection reagent.

[0336] In certain embodiments, the antibody may be pre-bound to a solid support, such as a column matrix and / or a well of a microtiter plate. The immunodetection reagent in the kit may employ one of several forms, including a detectable label associated with or bound to a given antibody. Detectable labels associated with or bound to a secondary ligand are also intended. An exemplary secondary ligand is a secondary antibody having binding affinity to the first antibody.

[0337] Further preferred immunodetection reagents for use in this kit include a two-component reagent comprising a secondary antibody having binding affinity to a first antibody, and a third antibody having binding affinity to a second antibody, wherein the third antibody is linked to a detectable label. As described above, several exemplary labels are known in the art, and all such labels may be used in connection with this disclosure.

[0338] The kit may further contain appropriately divided compositions of DDR1, labeled or unlabeled, for use in creating standard curves for detection assays. The kit may contain antibody-labeled conjugates in either a fully conjugated form, an intermediate form, or separate parts that are conjugated by the user of the kit. The components of the kit may be packaged in either an aqueous medium or a lyophilized form.

[0339] The kit container means generally include at least one vial, test tube, flask, bottle, syringe or other container means that can contain, or preferably dispense, an antibody. The kits of this disclosure also typically include means for tightly sealing and containing the antibody, antigen, and any other reagent containers for commercial sale. Such containers may include injection-molded or blow-molded plastic containers that hold the desired vial.

[0340] E. Flow cytometry and FACS The antibodies disclosed herein can also be used in flow cytometry or FACS. Flow cytometry is a laser or impedance-based technique used in many detection assays, including cell counting, cell sorting, biomarker detection, and protein engineering. This technique allows for simultaneous multiparametric analysis of the physical and chemical properties of up to several thousand particles per second by suspending cells in a fluid flow and passing them through an electronic detector. Flow cytometry is routinely used in the diagnosis of disorders, particularly hematological cancers, but also has many other applications in basic research, clinical practice, and clinical trials.

[0341] Fluorescence-activated cell sorting (FACS) is a specialized type of cytometry. It provides a method for sorting heterogeneous mixtures of living cells into two or more containers, one cell at a time, based on the specific light scattering and fluorescence properties of each cell. Generally, this technique involves a cell suspension that is drawn into the center of a narrow flow of rapidly moving liquid. The flow is positioned so that there is a large separation between cells relative to its diameter. An oscillating mechanism causes the flow of cells to decompose into individual droplets. Just before the flow decomposes into droplets, the flow passes through a fluorescence measurement station where the fluorescence of each cell is measured. An electrocharging ring is positioned at the point where the flow decomposes into droplets. Charges are placed on the ring just before the fluorescence intensity is measured, and opposite charges are captured on the droplets as they collapse from the flow. The charged droplets then fall through an electrostatic deflection system that directs the droplets into containers based on their charge.

[0342] In certain embodiments, for use in flow cytometry or FACS, the antibody of this disclosure is labeled with a fluorophore and then conjugated to cells of interest to be analyzed by flow cytometry or sorted by a FACS instrument. [Examples]

[0343] VIII. Examples The following embodiments are included to demonstrate preferred embodiments of the present invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques that the inventors have found to work well in carrying out the present invention and can therefore be considered to constitute preferred modes for its implementation. However, those skilled in the art will understand that numerous modifications can be made in light of this disclosure to the specific embodiments disclosed, and that similar or comparable results can still be obtained without departing from the spirit and scope of the present invention.

[0344] Example 1 - Materials and Methods CRISPR KO. DDR1 sgRNA CRISPR / Cas9 All-in-One lentiviral vector set (ABM; catalog number K4331005) was used to knock out DDR1 in M-WNT, AT-3, and E0771 cells according to the manufacturer's instructions. Briefly, lentiviral packaging was performed by co-transfection of HEK293T cells with Lipofectamine 2000 (Life Technologies, catalog number 11668027) using the DDR1 KO vector and two helper vectors (psPAX2 and pMD2.G). After two days, the lentiviral supernatant was collected and used to infect target tumor cells. Single clones were harvested and expanded after antibiotic selection. Genomic DNA was extracted and sequenced from all selected KO clones to validate the desired mutations. The sgRNA sequences are as follows: sgRNA1, AAGCAGTGATGGAGATG (Sequence ID: 303); sgRNA2, TGTGTTCCCCAAAGAAG (Sequence ID: 304); sgRNA3, GACCATGCAGTTATCTG (Sequence ID: 305). Scrambled sgRNA sequences were used as a control.

[0345] Western blotting. Cells were lysed in Laemmli buffer for cell lysate preparation. Protein concentrations were assessed using the BCA protein assay kit (Pierce, 23225). Proteins were then run by SDS-PAGE and transferred to membranes according to an established protocol. Primary antibodies were anti-DDR1 (dilution: 1:1000; CST, 5583S) and anti-GAPDH (dilution: 1:5000; CST, 2118S).

[0346] To recover the protein in the prepared medium, the medium was collected, centrifuged at 6,000 rpm, and then passed through filter paper with a pore size of 0.45 μm to remove any cell debris. The medium was subjected to SDS-PAGE and subsequently immunoblotted with anti-DDR1 ECD antibody (dilution: 1:1000; R&D, AF2396).

[0347] qRT-PCR. RNA extraction and RT-qPCR were performed as previously described (Sun et al., 2018). Briefly, RNA was reverse transcribed using the ImProm-II reverse transcription system (Promega, A3800), and real-time PCR was set up using Luminaris Color HiGreen qPCR master mix (Thermo Fisher Scientific, K0364). The relevant primers were prepared using Primer Premier software. The primer sequences are as follows:

[0348] TIFF0007883266000021.tif32128

[0349] MTT. Tumor cells were seeded in 96-well plates and cultured for the specified period before analysis. On the day of harvest, MTT solution (3 mg / ml) was added to each well, and the plate was incubated for 1 hour. The medium was then removed, and the purple precipitate was dissolved with 100 μl of DMSO. The absorbance at 570 nm was measured for each well.

[0350] Tumor cell migration and invasion. For cell migration, tumor cells were suspended in serum-free culture medium and then seeded on top of a Transwell chamber. Medium containing 10% FBS was placed at the bottom of the chamber. After culturing the cells for 12 hours, they were analyzed.

[0351] For cell wetting, Matrigel matrix (Corning, 354483) was packed into the insert according to the manufacturer's instructions and incubated at 37°C for 30 minutes. Tumor cells were seeded into the upper chamber of the insert using 10% FBS-containing medium in the lower chamber. The cells were then incubated at 37°C for 20 hours. The cells on the upper side of the upper chamber were gently removed, and the cells on the lower side of the upper chamber were stained with crystal violet. Six random fields were counted under a standard microscope.

[0352] Mouse treatment and tumor studies. All animal experiments were approved by the Institutional Animal Care and Use Committee at the George Washington University. 8-week-old WT C57BL / 6 (Jackson Lab, 000664), Rag1 - / - (Jackson Lab, 002216) or nude (Jackson Lab, 002019) mice were used for tumor studies. E0771, AT-3, and M-Wnt cells were administered at a rate of 5 × 10⁶ per inoculation, respectively. 5 , 2×10 5 , and 2 × 10 5 The cells were injected in a volume of 100 μl into the mouse mammary gland fat pad. Tumor volume (0.5 × length × width) 2 The values ​​were measured using calipers on the instructed date. Upon tumor retrieval, the tumor was weighed, and the sample was used for immunophenotyping and IHC.

[0353] For the tumor transplant assay, first, tumor cells are processed using Rag1 - / - It was administered to mice. The tumor volume was approximately 200-300 mm². 3When the incubation period reached (usually 20 days after inoculation), 60 mg of tumor organoids were transplanted into WT C57BL / 6 mice. On day 12, tumor samples were collected for immunohistochemical staining.

[0354] For tumor re-challenge experiments, WT C57BL / 6 mice were first inoculated with 500,000 DDR1 KO E0771 or PBS alone into one side of the inguinal mammary gland fat pad. Thirty days later, the same mice were inoculated with 500,000 DDR1 WT E0771 tumor cells into both sides of the mammary gland fat pad. Tumor volume was measured as described above.

[0355] To treat with DDR1 antibody in vivo, the size is 100 mm 3 After the tumors grew larger, both homemade control IgG and anti-huDDR1 ECD antibodies were locally injected into the tumors at a dose of 10 mg / kg every other day until the end of the experiment.

[0356] Decellularization. E0771 cells were seeded at 2,000 cells per insert in inserts with a pore size of 5 μm (Costar, Corning Inc., 3422) and cultured for 2 days in DMEM + 10% FBS + 1% PS medium. The resulting ECM from DDR1 WT or KO cells was washed with PBS and decellularized by incubation at 37°C for 5 minutes in PBS containing 0.5% TritonX-100 and 20 mM NH4OH. The decellularized ECM was washed three times with PBS, followed by three rinses with distilled water, and immediately used in T cell migration experiments.

[0357] In Vitro CD8 + T cell isolation and migration assay. EasySep® mouse CD8 + Using the negative isolation kit (Stemcell, 19853) according to the manufacturer's manual, CD8 + T cells were isolated from the spleen cells of C57BL / 6 naive mice. CD8 +T cell migration assays were performed using a 6.5 mm polycarbonate membrane and a 5 μm pore size insert (Costar, Corning Inc., 3422). 500,000 purified CD8 cells were used. + T cells were added to the upper chamber and migrated for 2 hours at 37°C in the presence of recombinant CCL21 (100 ng / ml, R&D System, 4576C025CF) along with tumor conditioning medium in the bottom chamber. CD8 cells migrated to the bottom chamber. + T cells were quantified by flow cytometry. For huDDR1 antibody neutralization, the antibody was first co-incubated with conditioned medium at 37°C for 1 hour, and then the procedure described above was followed.

[0358] Second harmonic generation and immunofluorescence. Mouse mammary tumor tissue was embedded and stored in an optimal cutting temperature (OCT) compound at -80°C. Before cutting, the samples were kept at -20°C for at least 2 hours, and 20 μm thick sections were cut using a cryostat. The slides were thawed and incubated at 37°C for 30 minutes, then transferred to boiling antigen demasking solution (Vector Labs, H-3300) for 10 minutes. The samples were incubated with CD3e (BD, 553057) primary antibody and Alexa-488 (Life Technologies) secondary antibody. Each tumor section was added to fluorescent G medium (VWR) and mounted on a microscope coverslip (No. 1.5).

[0359] All samples were imaged using a Leica TCS SP8 multiphoton confocal microscope, with a 20x magnification, HC PL Apo, NA0.7 oil immersion objective lens used throughout the experiment.

[0360] Excitation wavelengths up to 840 nm (Erikson et al., 2007) were adjusted, and the SHG signal of collagen was detected using a 420 ± 5 nm narrow-band pass-emission filter. The SHG signal is generated when two incident photons interact with the non-centrosymmetric structure of collagen fibers, resulting in a photon with half the wavelength of the incident photons. 1024 × 1024 pixel images were acquired using LAS X software. Collagen measurements were performed using CT Fire software (freely available at loci.wisc.edu / software / ctfire). For collagen from tumor margin analysis, an area of ​​60 μm from the tumor boundary was taken.

[0361] Immunohistochemical staining (IHC). Mouse mammary tumor tissue was fixed overnight at 4°C in 10% buffered formalin (Fisher Scientific, 23-427098). The fixed tumor specimens were embedded in paraffin and cut into 4 μm sections for staining. The specimens were deparaffinized and rehydrated in PBS. The sections were boiled for 20 minutes in antigen demasking solution (Vector Labs, H-3300) and then blocked for 1 hour in 10% normal goat serum in PBS. Primary antibodies for CD8 (Biorbyt, orb10325) and CD4 (Sino Biological Inc., 50134-R001) were incubated overnight at 4°C. For detection of the primary antibodies, an ABC peroxidase detection system (Vector Labs, PK-6105) was used according to the manufacturer's instructions, with DAB (Vector Labs, SK-4105) as the substrate.

[0362] CD8 + T cell depletion and adoptive transfer. CD8 + Regarding T-cell depletion, C57BL / 6 mice were intraperitoneally administered 200 μg / mouse anti-mouse CD8 (clone 2.43, BioxCell, BE0061) or IgG2b isotype control (clone LTF-2, BioxCell, BE0090) two days prior to tumor inoculation, and then administered twice weekly.

[0363] CD8+ Regarding adoptive transfer of T cells, purified CD8 cells were used 17 days after tumor inoculation. + T cells (>90%), 5 × 10 6 Cell / mouse concentration, Rag1 with E0771 mammary tumor - / - I transferred it to the mouse.

[0364] Picrosilius red staining. Fixed breast tumor specimens were prepared and sectioned as previously described (Sun et al., 2018). Briefly, paraffin-embedded tumor tissue was cut into 4 μm slides and stained with a picrosilius red staining kit (Abcam, catalog number ab150681). The sections were deparaffinized, hydrated in distilled water, and coated with picrosilius red solution for 1 hour. The slides were then rinsed in acetic acid solution and dehydrated in absolute alcohol. The mounted slides were then examined under a standard microscope, and positive collagen fiber signals were quantified using ImageJ software.

[0365] ELISA. Type I collagen was diluted to a concentration of 50 μg / ml in PBS and added to a 96-well microtiter plate (50 μl / well). The plate was sealed and incubated overnight at room temperature, washed three times with wash buffer (R&D, WA126), and then blocked for 1 hour with 200 μl of reagent diluent (R&D, DY995). After three washes, 100 μl of conditional medium or recombinant ECD (used as standard, Sino Biological, 10730-H08H) was added to the plate at room temperature for 2 hours. Subsequently, it was washed three times, 100 μl of diluted anti-DDR1 N-terminal antibody (1:500, R&D, AF2396) was added, and incubated for 2 hours. After a 1-hour reaction with biotin-conjugated antibody, streptavidin-HRP was added to each well at a 1:2000 dilution (R&D, 893975) and incubated in the dark for 20 minutes. 100 μl of substrate solution (R&D, DY999) was added and incubated for another 20 minutes. After adding 50 μl of stop solution (R&D, DY994), the plate was analyzed at 450 nm using an ELISA reader.

[0366] Flow cytometry. Cells were stained for viability using Ghost Dye® Violet 450 (Tonbo Biosciences, 13-0863-T100) at a 1:1000 dilution in PBS at 4°C in the dark for 20 minutes, followed by washing with PBS. Samples were blocked with anti-CD16 / 32 at a 1:100 dilution (clone 2.4G2, Tonbo Biosciences, 70-0161-U100). The antibody was incubated at 4°C in the dark for 30 minutes. The following commercially available antibodies were used: CD45-BV645 (Invitrogen, 64-0451-82), CD3-eflour660 (eBiosciences, 50-0032-82), CD4-FITC (eBiosciences, 35-0042-U500), CD8-APC-Cy(trademark)7 (BD, 557654), CD44-BV786 (Biolegend, 103059), and CD62L-Pacific Blue (Biolegend, 104424). Data were acquired on a BD FACSCelesta flow cytometer and analyzed using FACSDiva or FlowJo software (BD).

[0367] Scanning electron microscopy. Both WT and DDR1 KO E0771 cells were examined in 0.1 × 10⁶ cells. 6 Cells were seeded at a density and cultured in DMEM (10% FBS) for 2 days to adhere evenly to the surface of a glass coverslip. Next, the cells were fixed with a 2.5% glutaraldehyde and 1% paraformaldehyde solution, followed by rinsing with OsO4 and water. The samples were then sequentially dehydrated with an alcohol gradient and dried at the critical point. After mounting on a SEM stub, the coverslips were coated with iridium and observed under a scanning electron microscope (Model FEI) with an ETD detector at a residence time of 10 ms and a magnification of 12,000.

[0368] Screening and generation of anti-DDR1 monoclonal antibodies (mAbs). Rabbits were immunized with human DDR1 extracellular domain (ECD) protein (Sino Biological, 10730-H08H), and anti-huDDR1 monoclonal antibodies were generated using the method described above (Gui et al., 2019b). Briefly, New Zealand white rabbits were administered 0.5 mg of recombinant human DDR1 ECD protein by intraperitoneal (ip) injection for priming and a series of 3-4 boosters after priming immunization at 3-week intervals. Memory B cells were isolated from PBMCs, and single B cells were cultured in 96-well cell culture plates for 10-14 days for antibody production. Cell culture supernatants were analyzed for DDR1 binding using ELISA, and positive hits were selected for antibody gene cloning and sequence analysis.

[0369] Positive cells were lysed from B cell culture wells, total RNA was isolated, and cDNA was synthesized using SuperScript reverse transcriptase II (Invitrogen) according to the manufacturer's suggestion. DNA sequences of the antibody variable regions from both the heavy and light chains were amplified by polymerase chain reaction (PCR) using a designed set of primers, and the variable regions of each antibody were cloned into vectors for sequencing. The cloned antibody variable sequences from both the heavy and light chains were fused with the constant regions of the IgG1 heavy chain and kappa light chain, respectively, to construct mammalian expression vectors for full-length recombinant antibody expression in human embryonic kidney (HEK) 293 (HEK293F) cells (Life Science Technologies). Monoclonal antibodies were purified from HEK293 cell culture medium to over 95% purity using a protein-A affinity resin, using a previously described method (O'Donnell et al., 2019). The purified antibodies were screened for neutralizing function in cell culture assays and antitumor activity in a mouse tumor model.

[0370] Statistics. Mean differences between two groups were compared using Student's t-tests. Mean differences between multiple groups were compared using one-way analysis of variance (ANOVA) and post-hoc multiple comparisons. Survival curves were analyzed by log-rank (Mantel-Cox) analysis. Pearson correlation analysis and all other statistics were performed using Graphpad Prism. P<0.05 was considered statistical significance. Data are presented as mean ± SEM.

[0371] Example 2 - Results The inventors specifically deleted Ddr1 in several mouse mammary tumor cells with basal-like / TNBC characteristics (E0771, AT-3, and M-Wnt; Figure 1a, Figures 5b-d). The knockout (KO) tumor cells showed no significant defects in cell proliferation, migration, or invasion in vitro (Figures 1b-d, Figures 5e-f). In addition, Ddr1-KO tumors in immunodeficient hosts proliferated at the same rate as wild-type (WT) control mice (Figure 1e and Figure 5g). In stark contrast, Ddr1-KO tumors in immunocompetent hosts (C57BL / 6) completely regressed within two weeks after inoculation in all three mammary tumor models tested (Figures 1f-h). This growth deletion of KO tumors in immunocompetent hosts resulted in an increased number of KO tumor cells being transplanted (0.5-20 × 10⁶ per inoculation). 6 In some individual cells (Figure 5h), the effect is not substantially relieved, and in immunodeficient hosts (Rag1 - / -) Initial growth and subsequent re - transplantation into immunocompetent naive mice also failed to rescue growth arrest (Fig. 1i - j). Furthermore, co - transplantation using equal numbers of parental WT and KO tumor cells resulted in robust tumor growth in immunocompetent hosts (Fig. 5i - k), indicating a dominant role of tumor DDR1. Injection of WT tumor cells into immunocompetent mice previously challenged with KO tumor cells in either the same or the contralateral mammary gland did not result in significant growth of the rechallenged parental tumor (Fig. 1k - m). This indicates that KO tumor cells can vaccinate the host against WT tumors. Taken together, these data strongly suggest that tumor DDR1 plays a characteristic role in tumor growth in immunocompetent hosts.

[0372] Immunohistochemistry (IHC) showed that CD4 + and CD8 + T cells were restricted to the peripheral regions of parental tumors after re - transplantation from immunodeficient to immunocompetent hosts (Fig. 2a). In contrast, these lymphocytes were abundant in both the margins and centers of Ddr1 - KO tumors (Fig. 2a). As a confirmation, flow cytometry showed that the total number of tumor - infiltrating CD8 + and CD4 + T cells (normalized by tumor weight) was substantially increased in the KO tumor group compared to parental controls (Fig. 2b - c). Interferon (IFN) - γ - producing CD8 + and CD4 + cells were also more abundant in KO tumors than in WT counterparts (Fig. 2d - e). Furthermore, effector and helper T cells were more potently activated in KO tumors compared to their parental controls (CD44 高 CD62L 低 )(Fig. 2f - g). However, when normalized by the corresponding total T - cell number, KO and parental controls were positive for Ki67 (Fig. 6a - b), IFNγ, or Gzmb (Fig. 2c - d) for CD4 + or CD8 +There was no difference in the percentage of T cells. This indicates that tumor DDR1 is likely to confer tumor exclusion of T cells without attenuating the proliferation or anti-tumor activity of T cells themselves.

[0373] To confirm the role of tumor DDR1 in antagonizing anti-tumor immunity, the inventors depleted CD8 + T cells in immunocompetent mice by antibody neutralization. Ddr1-KO tumors grew as robustly as isogenic WT controls in CD8 + cell-depleted hosts (Figure 2h–j, Figure 2e–f), as in the inventors' findings in immunodeficient hosts (Figure 1e). In reciprocal experiments, the inventors transplanted purified CD8 + T cells into immunodeficient mice and compared the growth of parental and KO tumors. Unlike sham-treated immunodeficient hosts, mice with transplanted CD8 + T cells produced significantly smaller KO tumors than parental tumors (Figure 2k–m, Figure 6g). Collectively, these results support the concept that tumor DDR1 plays an important role in suppressing T cell infiltration, although it is not necessarily required for the intrinsic growth of breast tumors.

[0374] T cell transport within tumors is a highly dynamic, multi-step process involving extravasation through blood vessels, tumor-induced chemomia, and passage through physical barriers based on the extracellular matrix (ECM) (Slaney et al., 2014; Sackstein et al., 2017; Ager et al., 2016). Histological analysis based on CD31 did not reveal significant immunovascular changes between WT and KO tumors (data not shown), nor did RNA-seq show differences in mRNA levels of T cell homing genes or chemokine coding genes that could explain enhanced immune infiltration in the Ddr1-KO group (Figure 6h-i). Given the affinity of DDR1 for collagen, we sought to test an alternative model in which tumor DDR1-collagen interaction would make tumors less susceptible to immune cell penetration. To this end, we first determined whether the immunomodulatory function of DDR1 depends on its kinase activity by introducing the following constructs into Ddr1-KO tumor cells (Figure 3a): (1) an empty vector (EV), (2) full-length mouse DDR1 (FL), (3) ΔKD, a cleaved variant of DDR1 lacking the intracellular kinase domain KD but retaining its transmembrane (TM) domain, and (4) ECD only. The loss of growth in KO tumors in immunocompetent hosts was rescued to a similar extent by ectopically expressed FL DDR1 and the two cleaved variants, ΔKD and ECD (Figure 3a). Further deletion analysis showed that the N-terminal discoidin homology domain (DS, Figure 3b), which is involved in collagen binding, completely rescued tumor growth in DDR1 KO cells (Figures 7a-b). Taken together, these data clearly demonstrate that tumor DDR1 suppresses antitumor immunity in a kinase-independent manner.

[0375] Based on the collagen-DS domain crystal structure (Letinger, 2014; Carafoli et al., 2012; Carafoli & Hohenester, 2013), we mutated several key collagen-binding amino acid residues in mouse DDR1 (W54, T58, D71, K113, E114, and S176, Figure 3b), corresponding to W53, T57, D70, K112, E113, and S175 in human DDR1. We also mutated R33, a residue located distal to the collagen-binding pocket and responsible for DDR1 transmembrane signaling. The collagen-binding affinity of WT and mutant ECD was validated in vitro by ELISA and co-IP (Figures 3c, 7c), and their ability to rescue KO tumor growth was evaluated in vivo (Figures 3d-k). As expected, R33A, located outside the collagen-binding region of ECD, retained the ability to bind to collagen and support tumor growth (10 / 10 tumors ...

Claims

1. A monoclonal antibody or its antigen-binding fragment, comprising a light chain variable region and a heavy chain variable region, that specifically binds to the discoidine domain receptor 1 (DDR1) protein, The LC-CDR1, LC-CDR2, and LC-CDR3 within the light chain variable region include the amino acid sequences of LC-CDR1, LC-CDR2, and LC-CDR3 of SEQ ID NO: 112, 150, or 151, and The HC-CDR1, HC-CDR2, and HC-CDR3 within the heavy chain variable region include the amino acid sequences of HC-CDR1, HC-CDR2, and HC-CDR3 of SEQ ID NO: 133 or 152. The monoclonal antibody or its antigen-binding fragment.

2. The LC-CDR1 contains the amino acid sequence of sequence number 11, The LC-CDR2 contains the amino acid sequence GVF, The LC-CDR3 contains the amino acid sequence of Sequence ID No. 12, The HC-CDR1 contains the amino acid sequence of Sequence ID: 57, The HC-CDR2 contains the amino acid sequence of SEQ ID NO: 58, and The HC-CDR3 contains the amino acid sequence of Sequence ID: 59, A monoclonal antibody or its antigen-binding fragment according to claim 1.

3. The LC-CDR1, LC-CDR2, and LC-CDR3 within the light chain variable region include the amino acid sequences of LC-CDR1, LC-CDR2, and LC-CDR3 as defined using the Kabat definition in SEQ ID NO: 150, and The HC-CDR1, HC-CDR2, and HC-CDR3 within the heavy chain variable region include the amino acid sequences of HC-CDR1, HC-CDR2, and HC-CDR3 as defined using the Kabat definition in Sequence ID No.

152. A monoclonal antibody or its antigen-binding fragment according to claim 1.

4. The LC-CDR1 contains the amino acid sequence of amino acids 24 to 34 of SEQ ID NO: 150, The LC-CDR2 contains the amino acid sequence of amino acids 50 to 56 of sequence number 150, The LC-CDR3 contains the amino acid sequence of amino acids 89 to 97 of SEQ ID NO: 150, The HC-CDR1 contains the amino acid sequence of amino acids 31-35 of SEQ ID NO: 152, The HC-CDR2 contains the amino acid sequence of amino acids 50 to 65 of SEQ ID NO: 152, and The monoclonal antibody or antigen-binding fragment according to claim 1, wherein the HC-CDR3 comprises the amino acid sequence of amino acids 98 to 110 of SEQ ID NO:

152.

5. The light chain variable region includes the amino acid sequence of sequence number 112, and The heavy chain variable region includes the amino acid sequence of Sequence ID: 133, A monoclonal antibody or its antigen-binding fragment according to claim 1.

6. The monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 5, wherein the monoclonal antibody is a rabbit antibody, a chimeric antibody, or a humanized antibody.

7. The monoclonal antibody or its antigen-binding fragment according to claim 6, wherein the monoclonal antibody is a rabbit antibody or a chimeric antibody.

8. The monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 4, wherein the monoclonal antibody is a humanized antibody.

9. The light chain variable region includes the amino acid sequence of sequence number: 150 or 151, and The heavy chain variable region includes the amino acid sequence of Sequence ID: 152, A monoclonal antibody or its antigen-binding fragment according to claim 1.

10. The light chain variable region includes the amino acid sequence of sequence number 150, and The heavy chain variable region includes the amino acid sequence of Sequence ID: 152, A monoclonal antibody or its antigen-binding fragment according to claim 1.

11. A monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 9, comprising a heavy chain of an IgG1, IgG2, IgG3, or IgG4 subtype.

12. IgG １ A monoclonal antibody or its antigen-binding fragment according to claim 10 or 11, comprising a subtype heavy chain.

13. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, comprising a kappa light chain.

14. The antigen-binding fragment is ScFv antibody, Fab fragment, F(ab') ２ A monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 13, which is a fragment or an Fv fragment.

15. A monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 and 9 to 10, wherein the humanized antibody comprises two heavy chains and two light chains.

16. The monoclonal antibody or antigen-binding fragment according to claim 15, wherein the two heavy chains are two IgG1 heavy chains.

17. The monoclonal antibody or antigen-binding fragment thereof according to claim 15 or 16, wherein the two light chains are two kappa light chains.

18. A monoclonal antibody or antigen-binding fragment according to any one of claims 15 to 17, wherein the two heavy chains are full-length heavy chains and the two light chains are full-length light chains.

19. A pharmaceutical composition comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18, and a pharmaceutically acceptable carrier.

20. An antibody-drug conjugate comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18, and an antitumor drug linked to the antibody or the antigen-binding fragment thereof.

21. A polynucleotide encoding a monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 18.

22. A host cell comprising the polynucleotide described in claim 21.

23. A pharmaceutical composition for treating or mitigating the effects of cancer in a subject where such treatment is necessary, comprising a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 1 to 18.

24. The pharmaceutical composition according to claim 23, which reduces or eradicates the tumor burden, reduces the number of tumor cells, or reduces the tumor size in the subject.

25. The pharmaceutical composition according to claim 23 or 24, wherein the cancer is a solid tumor or a tumor.

26. The pharmaceutical composition according to any one of claims 23 to 25, wherein the cancer is breast cancer.

27. ​​The pharmaceutical composition according to any one of claims 23 to 26, wherein the cancer cells of the cancer are identified as expressing secreted DDR1 protein and / or DDR1 protein on the surface of the cancer cells, or are determined to express it.

28. A monoclonal antibody or antigen-conjugated fragment according to any one of claims 1 to 18, for use in a method of treating or mitigating the effects of fibrosis in a subject, wherein the method comprises administering a therapeutically effective amount of the antibody or antigen-conjugated fragment to the subject.

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