Antibodies against collagen-containing triple helix repeat protein 1 (CTHRC1) and methods of use thereof

Abstract

Collagen-containing triple helix repeat protein 1 (CTHRC1) is identified from a subtractive hybridization cDNA library, so that a gene related to artery injury repair is found. The present invention relates to anti-CTHRC1 antibodies, compositions comprising said antibodies and methods of using such antibodies and compositions to prevent, diagnose and treat diseases or disorders such as, for example, cancer, bone diseases, fibrotic diseases, arthritis and osteoporosis.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 298,194, filed on January 10, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to antibodies and antibody domains that specifically bind to CTHCR1, compositions thereof, and methods of using the same. Background Art

[0004] Collagen triple helix repeat protein 1 (CTHRC1) was identified from a subtractive hybridization cDNA library to search for genes associated with arterial injury repair (Lindner, V. et al., Journal of Bone and Mineral Research, 2004). In addition, CTHRC1 was found to be involved in bone development. Notably, overexpression of CTHRC1 in vivo leads to structural disruption of chondrocytes in bone growth plates and incomplete formation of proteoglycan complexes on collagen fibrils, resulting in collagen structural disruption and severe deformity. Studies have further associated CTHRC1 with fibroblast activation and proper collagen organization in arterial and cardiac repair (LeClair, Renée J. et al., Circulation research, 2007; Ruiz-Villalba, Adrian et al., Circulation, 2020), as well as more general wound healing (J. Li et al., EBioMedicine, 2019). However, reports of CTHRC1 expression in normal adult homeostatic tissues are limited. For example, low levels of CTHRC1 expression are observed on smooth muscle cells, but injury is required to observe significant CTHRC1 expression (Leclair et al., Arterioscler. Thromb. Vasc. Biol., 2008).

[0005] CTHRC1 contains a short motif (12 Gly-XY repeats) commonly found in collagen-related proteins and is conserved across species (Mei et al., Mediators Inflamm., 2020). At the molecular level, a series of different reports have shown that CTHRC1 regulates many signaling pathways, including TGF-β (J. Li et al., EBioMedicine, 2019; Ni et al., CancerMed., 2018; Zhang et al., Oncogene, 2021), canonical Wnt / β-catenin (Hou et al., Oncotarget., 2015), non-canonical Wnt / PCP pathway (Yamamoto et al., Dev. Cell, 2008) and integrin / FAK (Y.-L. Chen et al., J. Ovarian Res., 2013; Guo et al., PLoS One, 2017). However, there is no consensus on its precise molecular mechanism of action. Thus, despite early reports indicating that CTHRC1 is critical for developmental bone growth and wound healing in adults and may function downstream of TGF-β / Wnt signaling, it remains a largely uncharacterized protein with significant gaps in the understanding of its precise function in humans.

[0006] In cancer, CTHRC1 overexpression has been reported in colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC), where CTHRC1 is associated with stage and poor survival (W. Liu et al., Oncology Letters, 2016; Ni et al., Cancer Med., 2018; Wang et al., Cancer Sci., 2012). Experiments using CRC, PDAC and ovarian cancer cell lines have shown that CTHRC1 promotes migration and invasive behavior, associated with metastasis (Guo et al., J. Ovarian Res., 2017; Ni et al., Cancer Med., 2018; Park et al., Carcinogenesis, 2013). Finally, in vivo data suggest that CTHRC1 is involved in angiogenesis, specifically, in CTHRC1 knockout mice, xenograft growth is impaired, and significant destruction of vascular organization is observed (Lee et al., Exp. & Mol. Med., 2016). In summary, several reports have implicated CTHRC1 in various pro-tumorigenic roles in cancer, but mechanistic details are scarce.

[0007] In addition to promoting cancer, CTHRC1 is also associated with fibrosis. Notably, recent work has associated CTHRC1 expression with pathological subgroups of fibroblasts in mouse models of pulmonary fibrosis, which can also be seen in the lungs of patients with idiopathic pulmonary fibrosis (IPF) (Tsukui et al., Nat. Commun., 2020). Evidence also suggests that CTHRC1 is upregulated in fibrotic liver disease (J. Li et al., EBioMedicine, 2019). Importantly, in a rodent model of chemically induced liver fibrosis, depletion of CTHRC1 by gene knockout inhibited the onset of fibrosis (J. Li et al., EBioMedicine, 2019). Therefore, taking these data together also suggests that CTHRC1 is involved in fibrosis and fibroblast biology, thus indicating that its pro-tumorigenic effects may also depend on fibroblasts in the tumor microenvironment. In addition to cancer, studies have also linked CTHRC1 to protective anti-inflammatory activity in rheumatoid arthritis (Jin et al., Bone, 2017), where the level of CTHRC1 in the blood can also distinguish between healthy people and patients with rheumatoid arthritis (Myngbay et al., Frontiers in Immunology, 2019). It is also suggested that CTHRC1 is a positive regulator of bone formation and is therefore protective in osteoporosis (Chen et al., Bone Research, 2019; Kimura et al., PloS One, 2008).

[0008] In summary, studies to date on CTHRC1 indicate that it plays a role in bone development, wound repair, cancer, fibrosis, arthritis and osteoporosis. However, despite these findings, the lack of any public CTHRC1 inhibitors has prevented further research on the causal role played by CTHRC1 in disease, let alone providing a rationale for developing therapeutic inhibitors against CTHRC1 and / or developing mAbs that target payloads to the tumor microenvironment based on CTHRC1 binding mAbs. Therefore, there is currently an unmet need for antibodies and antibody domains that specifically bind to CTHRC1. Summary of the invention

[0009] The present invention solves the above-mentioned shortcomings in the prior art by providing and characterizing a series of antibodies and antibody domains against CTHRC1 and methods of using them in the prevention, diagnosis and treatment of cancer, fibrosis and / or fibrotic diseases. It has been demonstrated that the anti-CTHRC1 antibodies of the present invention (i) selectively bind to CTHRC1, (ii) block cell adhesion to CTHRC1, (iii) are internalized in cells expressing CTHRC1 when binding to the cells, and / or iv) recruit CD8+T cells into the tumor microenvironment. Therefore, the specificity and functional effects of the subject anti-CTHRC1 antibodies and antibody domains are important in the context of cancer and fibrosis, particularly in disease states where CTHRC1 is upregulated.

[0010] In one aspect, the invention provides an anti-CTHRC1 antibody that binds to human CTHRC1. In embodiments, the anti-CTHRC1 antibody (i) selectively binds to CTHRC1, (ii) blocks cell adhesion to CTHRC1, and / or (iii) is internalized upon binding to a cell expressing CTHRC1.

[0011] In one embodiment, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 3, 5, 7 and 9. In one embodiment, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from Table 3.

[0012] In one embodiment, the anti-CTHRC1 antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 4, 6, 8 and 10. In one embodiment, the anti-CTHRC1 antibody comprises a light chain variable region comprising an amino acid sequence selected from Table 4.

[0013] In one embodiment, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 150-154; a CDR2 sequence selected from the group consisting of SEQ ID NOs: 180-184; and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 210-214.

[0014] In one embodiment, an anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO:150; a CDR2 sequence comprising SEQ ID NO:180; and a CDR3 sequence comprising SEQ ID NO:210.

[0015] In one embodiment, an anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO:151; a CDR2 sequence comprising SEQ ID NO:181; and a CDR3 sequence comprising SEQ ID NO:211.

[0016] In one embodiment, an anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO:152; a CDR2 sequence comprising SEQ ID NO:182; and a CDR3 sequence comprising SEQ ID NO:212.

[0017] In one embodiment, an anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO:153; a CDR2 sequence comprising SEQ ID NO:183; and a CDR3 sequence comprising SEQ ID NO:213.

[0018] In one embodiment, an anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO:154; a CDR2 sequence comprising SEQ ID NO:184; and a CDR3 sequence comprising SEQ ID NO:214.

[0019] In one embodiment, the anti-CTHRC1 antibody comprises a light chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 240-244; a CDR2 sequence selected from the group consisting of SEQ ID NOs: 270-274; and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 300-304.

[0020] In one embodiment, an anti-CTHRC1 antibody comprises a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO:240; a CDR2 sequence comprising SEQ ID NO:270; and a CDR3 sequence comprising SEQ ID NO:300.

[0021] In one embodiment, an anti-CTHRC1 antibody comprises a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO:241; a CDR2 sequence comprising SEQ ID NO:271; and a CDR3 sequence comprising SEQ ID NO:301.

[0022] In one embodiment, an anti-CTHRC1 antibody comprises a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO:242; a CDR2 sequence comprising SEQ ID NO:272; and a CDR3 sequence comprising SEQ ID NO:302.

[0023] In one embodiment, an anti-CTHRC1 antibody comprises a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO:243; a CDR2 sequence comprising SEQ ID NO:273; and a CDR3 sequence comprising SEQ ID NO:303.

[0024] In one embodiment, an anti-CTHRC1 antibody comprises a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO:244; a CDR2 sequence comprising SEQ ID NO:274; and a CDR3 sequence comprising SEQ ID NO:304.

[0025] In one embodiment, an anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 150; a CDR2 sequence comprising SEQ ID NO: 180; and a CDR3 sequence comprising SEQ ID NO: 210; and further comprises a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 240; a CDR2 sequence comprising SEQ ID NO: 270; and a CDR3 sequence comprising SEQ ID NO: 300.

[0026] In one embodiment, an anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 151; a CDR2 sequence comprising SEQ ID NO: 181; and a CDR3 sequence comprising SEQ ID NO: 211; and further comprises a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 241; a CDR2 sequence comprising SEQ ID NO: 271; and a CDR3 sequence comprising SEQ ID NO: 301.

[0027] In one embodiment, an anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 152; a CDR2 sequence comprising SEQ ID NO: 182; and a CDR3 sequence comprising SEQ ID NO: 212; and further comprises a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 242; a CDR2 sequence comprising SEQ ID NO: 272; and a CDR3 sequence comprising SEQ ID NO: 302.

[0028] In one embodiment, an anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 153; a CDR2 sequence comprising SEQ ID NO: 183; and a CDR3 sequence comprising SEQ ID NO: 213; and further comprises a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 243; a CDR2 sequence comprising SEQ ID NO: 273; and a CDR3 sequence comprising SEQ ID NO: 303.

[0029] In one embodiment, an anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 154; a CDR2 sequence comprising SEQ ID NO: 184; and a CDR3 sequence comprising SEQ ID NO: 214; and further comprises a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 244; a CDR2 sequence comprising SEQ ID NO: 274; and a CDR3 sequence comprising SEQ ID NO: 304.

[0030] In one embodiment, the binding affinity (K) of the anti-CTHRC1 antibody to CTHRC1 is D ) is less than 10 nM, preferably less than 5 nM, more preferably less than 1 nM.

[0031] In one embodiment, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 2. In one embodiment, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 3 and a light chain variable region comprising SEQ ID NO: 4. In one embodiment, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 5 and a light chain variable region comprising SEQ ID NO: 6. In one embodiment, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8. In one embodiment, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 9 and a light chain variable region comprising SEQ ID NO: 10.

[0032] In one embodiment, the present invention provides an anti-CTHRC1 antibody that competes for binding to the CTHRC1 epitope with an antibody comprising a heavy chain variable region comprising SEQ ID NO: 1, 3, 5, 7 or 9 and a light chain variable region comprising SEQ ID NO: 2, 4, 6, 8 or 10.

[0033] The anti-CTHRC1 antibodies of the present invention include, for example, monoclonal antibodies, antibody fragments (including Fab, Fab', F(ab')2 and Fv fragments), single-chain antibodies, diabodies, single domain antibodies, chimeric antibodies, humanized antibodies, human antibodies and antibodies that competitively inhibit the binding (binding to the CTHRC1 epitope) of an antibody comprising a heavy chain variable region comprising SEQ ID NO: 1, 3, 5, 7 or 9 and a light chain variable region comprising SEQ ID NO: 2, 4, 6, 8 or 10.

[0034] In some embodiments, the anti-CTHRC1 antibodies of the invention further comprise a human subgroup III heavy chain framework consensus sequence. In one embodiment of these antibodies, these antibodies further comprise a human κI light chain framework consensus sequence.

[0035] In one embodiment, the anti-CTHRC1 antibody inhibits or neutralizes one or more human CTHRC1 functions.

[0036] In one embodiment, the anti-CTHRC1 antibody is a chimeric antibody, a humanized antibody or a human antibody. In one embodiment, the anti-CTHRC1 antibody is a monoclonal antibody. In one embodiment, the anti-CTHRC1 antibody is an antibody fragment. In one embodiment, the anti-CTHRC1 antibody is a single-chain variable fragment. In one embodiment, the anti-CTHRC1 antibody is an antibody-drug conjugate (ADC). In one embodiment, the anti-CTHRC1 antibody is a radioconjugate.

[0037] In some embodiments, the anti-CTHRC1 antibody or fragment thereof elicits little immunogenic response to the anti-CTHRC1 antibody or fragment thereof in a subject, such as a human subject. In some embodiments, the present invention provides a humanized antibody that elicits and / or is expected to elicit minimal or no human anti-mouse antibody response (HAMA). In one example, the antibody of the present invention elicits an anti-mouse antibody response at or below a clinically acceptable level.

[0038] In some aspects, the present invention provides nucleic acids comprising DNA encoding any anti-CTHRC1 antibody or portion thereof, or CAR or portion thereof described herein. In some embodiments, the nucleic acid comprises any one or more of SEQ ID NO:100-109. In embodiments, the present invention provides vectors comprising nucleic acids encoding any anti-CTHRC1 antibody or portion thereof, or CAR or portion thereof described herein. In some embodiments, the vector comprises any one or more of SEQ ID NO:100-109. In embodiments, the present invention provides host cells comprising any such vectors. For example, the host cell can be a CHO cell, an Escherichia coli (E.coli) cell, or a yeast cell. Further provided is a method for producing any polypeptide described herein, and comprising culturing the host cell under conditions suitable for expressing the desired polypeptide, and recovering the desired polypeptide from the cell culture.

[0039] In one aspect, the present invention provides a method for preparing an antibody of the present invention. In an embodiment, the present invention provides a method for preparing a CTHRC1 antibody (which includes its full length and fragments as defined herein), the method comprising expressing a recombinant vector of the present invention encoding the antibody (or its fragment) in a suitable host cell, and recovering the antibody.

[0040] In one aspect, the present invention provides a CAR-modified immune cell, such as a CAR-T cell or a CAR-NK cell, or a CAR-macrophage, comprising a chimeric antigen receptor capable of binding to a CTHRC1 epitope.

[0041] In one aspect, the present invention provides a CAR-modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage, comprising a chimeric antigen receptor, wherein the chimeric antigen receptor comprises the heavy chain variable region of an anti-CTHRC1 antibody as disclosed herein and the light chain variable region of an anti-CTHRC1 antibody as disclosed herein.

[0042] In one aspect, the present invention provides a CAR-modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage, comprising an anti-CTHRC1 antibody. In one embodiment, the anti-CTHRC1 antibody is an antibody fragment. In one embodiment, the anti-CTHRC1 antibody is a scFv.

[0043] In one aspect, the present invention provides a method for inhibiting the growth of a cell directly or in the form of a complex, the cell displays a CTHRC1 epitope (e.g., a CTHRC1 tumor epitope), the method comprising contacting the cell with an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell (e.g., CAR-T or CAR-NK cell, or CAR-macrophage). In one embodiment, the anti-CTHRC1 antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioactive conjugate. In an embodiment, contacting the cell comprises administering a therapeutically effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell to the patient.

[0044] In one aspect, the present invention provides a method for inhibiting the metastasis of a tumor directly or in the form of a complex, the tumor displays a CTHRC1 epitope (e.g., a CTHRC1 tumor epitope), the method comprising contacting the cells of the tumor with an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell (e.g., CAR-T or CAR-NK cell, or CAR-macrophage). In one embodiment, the anti-CTHRC1 antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioactive conjugate. In an embodiment, contacting the cell comprises administering a therapeutically effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell to the patient.

[0045] In one aspect, the present invention provides a method of inducing cell death, the cell displays a CTHRC1 epitope (e.g., a CTHRC1 tumor epitope), the method comprising contacting the cell with an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell (e.g., CAR-T or CAR-NK cell, or CAR-macrophage). In one embodiment, the anti-CTHRC1 antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioactive conjugate. In an embodiment, contacting the cell comprises administering to the patient a therapeutically effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell.

[0046] In one aspect, the present invention provides a method of reducing the size of a tumor, the tumor comprising a cell displaying a CTHRC1 epitope (e.g., a CTHRC1 tumor epitope), the method comprising contacting the cell with an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell (e.g., CAR-T or CAR-NK cell, or CAR-macrophage). In one embodiment, the anti-CTHRC1 antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioactive conjugate. In an embodiment, contacting the cell comprises administering a therapeutically effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell to the patient.

[0047] In one aspect, the present invention provides a method of inhibiting the vascularization of a tumor, the tumor comprising cells displaying a CTHRC1 tumor epitope, the method comprising contacting the cell with an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell (such as a CAR-T or CAR-NK cell, or a CAR-macrophage). In one embodiment, the anti-CTHRC1 antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioactive conjugate. In an embodiment, contacting the cell comprises administering a therapeutically effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell to the patient.

[0048] In one aspect, the present invention provides a method for showing cell inhibitory activity on tumor cells or cancer-associated fibroblasts displaying CTHRC1, the method comprising contacting cells / cancer-associated fibroblasts with anti-CTHRC1 antibodies of the present invention or CAR-modified immune cells (such as CAR-T or CAR-NK cells, or CAR-macrophages). In one embodiment, the anti-CTHRC1 antibody is used in the form of ADC. In one embodiment, the ADC comprises a radioactive conjugate. In an embodiment, contacting cells includes administering a therapeutically effective amount of anti-CTHRC1 antibodies of the present invention or CAR-modified immune cells to a patient.

[0049] In one aspect, the present invention provides a method for preventing and inhibiting immune cells in a tumor microenvironment, the method comprising contacting at least one cell of the tumor microenvironment with an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell (such as a CAR-T or CAR-NK cell, or a CAR-macrophage). In one embodiment, the anti-CTHRC1 antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioactive conjugate. In an embodiment, contacting a cell comprises administering to a patient a therapeutically effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell.

[0050] In one aspect, the present invention provides a method for enhancing the infiltration of anti-tumor immune cells in a tumor microenvironment in vivo, the method comprising contacting at least one cell of the tumor microenvironment with an anti-CTHRC1 antibody of the present invention, preferably in combination with cellular immunotherapy, such as allogeneic or autologous T or NK cell therapy. In an embodiment, contacting the cell comprises administering a therapeutically effective amount of an anti-CTHRC1 antibody to a patient, together with administering cellular immunotherapy, such as CAR-T or CAR-NK cell therapy.

[0051] In an embodiment of the present invention, the cell displaying the CTHRC1 tumor epitope is a cancer cell.

[0052] In one aspect, the present invention provides a method for treating or preventing a cell proliferative disorder associated with increased expression and / or display of CTHRC1, the method comprising administering to a subject an effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage. In one embodiment, the anti-CTHRC1 antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioactive conjugate. In one embodiment, the cell proliferative disorder is cancer.

[0053] In one aspect, the present invention provides a method for inhibiting tumor metastasis in a subject with cancer, the method comprising administering to the subject an effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage. In one embodiment, the anti-CTHRC1 antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioactive conjugate.

[0054] In one aspect, the present invention provides a method for reducing the tumor size of a subject with cancer, the method comprising administering to the subject an effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage. In one embodiment, the anti-CTHRC1 antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioactive conjugate.

[0055] In one embodiment, the subject is a human subject. In one embodiment, the cancer is selected from the group consisting of adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colon adenocarcinoma, B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, myeloid leukemia, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, pancreatic adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, melanoma, gastric adenocarcinoma, testicular germ cell cancer, thymoma, uterine corpus cancer, and uterine carcinosarcoma.

[0056] In one aspect, the present invention provides a method of inhibiting and / or reducing fibrosis in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage. In an embodiment, the anti-CTHRC1 antibody is administered in combination with radiotherapy.

[0057] In one embodiment, the present invention provides a method for treating a subject with a fibrotic disease, the method comprising administering to the subject an effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage. In one embodiment, the anti-CTHRC1 antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioactive conjugate. In an embodiment, the subject is a human subject.

[0058] In an embodiment, the fibrotic disease may be selected from the group consisting of idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, pulmonary hypertension, renal fibrosis, keratosis, non-alcoholic fatty liver disease (NASH), scleroderma, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis and systemic lupus erythematosus.

[0059] In one aspect, the present invention provides a pharmaceutical composition comprising an anti-CTHRC1 antibody and a pharmaceutically acceptable carrier. In one aspect, the present invention provides a pharmaceutical composition comprising a CAR-modified immune cell (such as a CAR-T or CAR-NK cell or a CAR-macrophage) of the present invention and a pharmaceutically acceptable carrier. In one embodiment, the anti-CTHRC1 antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioactive conjugate.

[0060] In one aspect, the present invention provides a method for preparing an anti-CTHRC1 antibody. In one aspect, the present invention provides a method for preparing a CAR-modified immune cell disclosed herein. In one embodiment, the present invention provides a method for preparing an ADC comprising an anti-CTHRC1 antibody.

[0061] In one aspect, the invention provides a method for determining the presence of CTHRC1 (e.g., a CTHRC1 epitope, e.g., a CTHRC1 tumor epitope) in a subject or a biological sample from a subject. In one embodiment, the method comprises contacting the sample with an anti-CTHRC1 antibody and determining the binding of the anti-CTHRC1 antibody to the sample, wherein the binding of the anti-CTHRC1 antibody to the sample indicates the presence of a CTHRC1 epitope in the sample.

[0062] In one aspect, the invention provides a method for diagnosing a cell proliferative disorder (e.g., cancer) associated with (i) an increase in cells expressing CTHRC1 (e.g., breast cancer cells, ovarian cancer cells, melanoma cells, liver cells, kidney cells, pancreatic cells, or glioblastoma cells), or (ii) an increase in CTHRC1 expression in a tumor. In embodiments, the method comprises detecting the presence of a CTHRC1 epitope (e.g., a CTHRC1 tumor epitope) in a subject or a biological sample from a subject.

[0063] In one aspect, the invention provides a method for determining the prognosis of a subject diagnosed with cancer, the method comprising detecting the presence of a CTHRC1 epitope (e.g., a CTHRC1 tumor epitope) in a subject or a biological sample from a subject. In one embodiment, the method involves detecting the presence of a CTHRC1 epitope in a subject or a biological sample from a subject after the subject has received a therapeutic agent for treating cancer.

[0064] In one aspect, the present invention provides the use of a CTHRC1 antibody or CAR-modified immune cell of the present invention, preferably a CAR-T or CAR-NK cell or a CAR macrophage, for the preparation of a pharmaceutical agent for the therapeutic and / or prophylactic treatment of a disease or disorder (such as cancer, a tumor and / or a cell proliferative disorder, fibrosis and / or a fibrotic disease).

[0065] In one aspect, the invention provides the use of a nucleic acid of the invention for the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease or disorder, such as cancer, a tumor and / or a cell proliferative disorder, fibrosis and / or a fibrotic disease.

[0066] In one aspect, the present invention provides use of an expression vector of the present invention for the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease or disorder (such as cancer, a tumor and / or a cell proliferative disorder, fibrosis and / or a fibrotic disease).

[0067] In one aspect, the invention provides use of a host cell of the invention for the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease or disorder, such as cancer, a tumor and / or a cell proliferative disorder, fibrosis and / or a fibrotic disease.

[0068] In one aspect, the invention provides the use of an article of manufacture of the invention for the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease or disorder, such as cancer, a tumor and / or a cell proliferative disorder, fibrosis and / or a fibrotic disease.

[0069] In one aspect, the invention provides use of a kit of the invention for the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease or disorder, such as cancer, a tumor and / or a cell proliferative disorder, fibrosis and / or a fibrotic disease.

[0070] Also provided herein are kits and methods of using the same. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 is a heat map illustrating enzyme-linked immunosorbent assay (ELISA) data showing the identification of 12 clones that selectively bind to human and / or rat CTHRC1.

[0072] Figure 2 is a bar graph illustrating the evaluation of cell adhesion to CTHRC1, periostin and the ECM protein fibronectin in a panel of fibroblast cell lines (BJ, skin; CCD-8Lu, lung; CCD18Co, colon) and cancer cell lines (SKOV3, ovarian; Mia PaCa2, pancreatic; HCT116, colorectal).

[0073] Figure 3 is a bar graph illustrating cell adhesion of ovarian cancer cells to CTHRC1 and the ECM proteins vitronectin and fibronectin following treatment with various integrin blocking antibodies.

[0074] Figure 4 is a bar graph illustrating selective blockade of cell adhesion by CTHRC1S-M5 (AB987) and CTHRC1S-M23 (AB988) in ovarian cancer cells.

[0075] Figure 5A-Figure 5B CTHRC1 mRNA is the top-ranked marker for cancer-associated fibroblasts (CAFs) in cancer-rich, immune-cold tumor samples. Aggregated tumor samples were profiled by cancer scRNA studies to generate large scRNA maps. Samples were then divided into T cell-rich, cancer-poor samples (immune-hot) and cancer-rich, T cell-poor samples (immune-cold) ( Figure 5A ). The genes expressed by CAFs in the two groups were compared to determine which genes were most associated with cancer-enriched samples (Wilcoxon rank). These genes were then screened to be those specifically expressed by CAFs in all samples (top 500 ranked; Wilcoxon rank). CTHRC1 was the 11th highest ranked gene in the analysis ( Figure 5B ).

[0076] Figure 6It is shown that CTHRC1 is highly upregulated in cancer compared to normal adjacent tissues, as determined by a large number of RNA measurements obtained through analysis of The Cancer Genome Atlas. In a range of indications profiled, the highest levels of CTHRC1 expression were observed in solid cancers, with particular significance in breast, lung, ovarian, pancreatic, sarcoma, melanoma, and uterine carcinosarcoma (P<0.001 in all cases tested). Overall, this suggests that CTHRC1 expression is highest in more desmoplastic, matrix-rich cancers, consistent with the concept that it is primarily a CAF secretory target.

[0077] Figure 7 A series of survival graphs are depicted showing CTHRC1 survival curves for various solid tumors, where CTHRC1 is associated with poorer survival. The values ​​are derived from The Cancer Genome Atlas. The online tool GEPIA was used to calculate and plot the survival curves.

[0078] Figure 8 Illustration, CTHRC1 levels increase with stage in liver cancer (left) and colorectal cancer (right) as well as other indications (not shown). In both cases, p<0.05; Student's T-test between stage I and stage IV. Stages II and III show intermediate CTHRC1 expression levels.

[0079] Figure 9A-9C The known stromal target FAP ( Fig. 9A ) and LRRC15( Fig. 9B ) and CTHRC1( Fig. 9C ). The histogram highlights that there is a significant therapeutic window for targeting CTHRC1 in pancreatic cancer based on the bulk RNA measurements. This window is similar to or even larger than the windows for the known / explored stromal targets FAP and LRRC15.

[0080] Fig.10is a dataset that illustrates that very high levels of CTHRC1 expression are observed in CAFs of many solid cancers, and cancer epithelial expression is seen in breast, pancreatic, lung, ovarian, and skin cancers. In contrast, minimal CTHRC1 expression is observed in normal tissues. The data are based on previously generated large integrated single-cell RNA-seq maps to enable detection of gene expression at the single-cell level in a variety of cancer and normal tissue samples (Swechha, 2021). The data highlight the potential large therapeutic window for blocking CTHRC1 and the value of using mAbs against CTHRC1 to target payloads (e.g., ADCs) to the tumor microenvironment.

[0081] Fig.11 Is to use Fig.10 Discussing the same profile, a dataset was performed against LRRC15, a known non-toxic stromal target, where an antibody ADC has been engineered and proven to be safe in the clinic. Low levels of LRRC15 were observed in normal tissues compared to CTHRC1. In the cancerous single cell RNA (scRNA) dataset, it was observed that, unlike CTHRC1, LRRC15 expression was more selectively localized to specific CAFs in specific breast cancers, and low levels of expression were also observed on sarcoma cancer cells.

[0082] Fig.12 is a graph illustrating quantitative ELISA of CTHRC1 in monocultures and cocultures. Supernatants from a different set of monocultures (fibroblasts or cancer cells) and cocultures (fibroblasts and cancer cells) were profiled for CTHRC1 levels. CTHRC1 expression levels were low in fibroblast monocultures (BJ, CCD18-Co) and upregulated in cocultures, suggesting that an interaction between fibroblasts and cancer cells drives CTHRC1 expression.

[0083] Fig.13 A series of images of tissues from three mouse models probed with a CTHRC1-specific mAb are shown. Extensive staining was observed within the tumor region, suggesting that the CTHRC1 protein localizes to the cancer region in vivo.

[0084] Fig.14 A series of images from tissues of three human cancers are shown, showing changes in the CTHRC1 expression pattern at the protein level, indicating different expression dynamics. In head and neck cancer and melanoma cancer samples, CTHRC1 is localized to the interface between cancer and matrix tissue. In pancreatic cancer, CTHRC1 is observed to be widely expressed in areas with dense CAF / matrix tissue.

[0085] Figure 15A-15BThis indicates that CTHRC1 expression was observed in human cancer cell lines ( Fig.15A ), and CTHRC1 mAbs M14 and M23 bound to the surface of a range of human cancer cell lines expressing CTHRC1 ( Fig. 15B ).like Fig.13 As shown, binding of the same mAbs to EMT6 cancer cells expressing CTHRC1 in vivo was observed. As illustrated, the higher affinity mAb M14 showed a higher level of cell surface binding than the lower affinity mAb M23.

[0086] Figures 16A-16D is a graph showing that CTHRC1 mAb is rapidly internalized by cancer cells in the presence or absence of 50 nM exogenous CTHRC1. Internalization is much faster in SKOV3 ovarian cells expressing high levels of CTHRC1 and is comparable to that in KP4 pancreatic cells ( Figure 16C-16D ), exogenous CTHRC1 also had a smaller effect on the internalization rate ( Figure 16A-16B ). Consistent with a higher level of surface binding, CTHRC1S-M14 ( Fig.16A and Fig. 16C ) than CTHRC1S-M23( Fig. 16B and Fig.16D ) is internalized faster.

[0087] Fig.17 Shown is a diagram illustrating that CTHRC1mAb is internalized by mouse EMT6 and 4T1 cancer cells without the addition of exogenous CTHRC1. It was observed that internalization occurred faster in EMT6 cancer cells than in 4T1 cells. This is consistent with the cancer cells in this model having more mesenchymal appearance. Similar to human cell lines, a greater internalization rate was observed for CTHRC1S-M14 (left) compared to CTHRC1S-M23 (right).

[0088] Fig.18 Shown is a graph illustrating that CTHRC1 mAb conjugated to MMAE (Vedotin) resulted in selective killing of SKOV3 cells.

[0089] Figure 19A-Figure 19C Illustration of the efficacy of CTHRC1 tested in the syngeneic mouse mammary tumor model EMT6. Three different anti-CTHRC1 antibodies are shown, specifically M5 ( Fig.19A )、M14( Fig.19B ) and M14( Fig.19C ) results.

[0090] Figure 20A-20B Demonstrating the efficacy of anti-CTHRC1 (clone M5) in the PD-1 resistant Pan02 pancreatic cancer model.

[0091] Fig.21 The effect of preconditioning cells with anti-CTHRC1 antibody on CD8 T cell infiltration is illustrated. DETAILED DESCRIPTION

[0092] General techniques

[0093] Unless otherwise indicated, the practice of the present invention will adopt the conventional techniques of molecular biology (including recombinant technology), microbiology, cell biology, biochemistry and immunology, which are within the technical scope of the art. Such techniques are fully explained in the literature, such as "Molecular Cloning: A Laboratory Manual", Second Edition (Sambrook et al., 1989); "Oligonucleotide Synthesis" (MJ Gait compiled, 1984); "Animal Cell Culture" (RI Freshney compiled, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Current Protocols in Molecular Biology" (FM Ausubel et al. compiled, 1987, and regularly updated); "PCR: The Polymerase Chain Reaction", (Mullis et al. compiled, 1994); "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988); "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

[0094] Those skilled in the art will recognize that there are many methods and materials similar or equivalent to those described herein that can be used for the practice of the present invention. In fact, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined as follows.

[0095] II. Definitions

[0096] For purposes of interpreting this specification, the following definitions shall apply and, where appropriate, terms used in the singular shall include the plural and vice versa. If any of the stated definitions conflict with any document incorporated herein by reference, the definitions stated below shall prevail.

[0097] Unless otherwise indicated, as used herein, the term "collagen triple helix repeat-containing protein 1 (CTHRC1)" refers to any native CTHRC1 from any vertebrate source, including mammals, such as primates (e.g., humans, primates) and rodents (e.g., mice and rats). The term encompasses several isoforms (see, e.g., SEQ ID NOs: 97-99). Human CTHRC1 is encoded by a nucleotide sequence corresponding to GenBank Accession No. NG031985.

[0098] The term "collagen triple helix repeat-containing protein 1" encompasses "full-length", unprocessed CTHRC1 as well as any form of CTHRC1 produced by processing in cells. The term encompasses naturally occurring variants of CTHRC1, such as splice variants, allelic variants, and isoforms. The CTHRC1 polypeptides described herein can be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. "Native sequence CTHRC1 polypeptides" include polypeptides having the same amino acid sequence as the corresponding CTHRC1 polypeptides derived from nature. Such native sequence CTHRC1 polypeptides can be isolated from nature, or can be manufactured by recombinant or synthetic means. The term "native sequence CTHRC1 polypeptide" specifically encompasses naturally occurring truncated forms or secreted forms (e.g., extracellular domain sequences) of specific CTHRC1 polypeptides, naturally occurring variant forms of the polypeptide (e.g., alternative splicing forms), and naturally occurring allelic variants. In certain embodiments of the present invention, the native sequence CTHRC1 polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acid sequence shown in the attached disclosure.

[0099] As used herein, "modification" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, wherein the change is caused by a sequence change involving the amino acid residue / position. For example, a typical modification includes a residue (or at the position) being replaced with another amino acid (e.g., conservative or non-conservative replacement), inserting one or more (usually less than 5 or 3) amino acids adjacent to the residue / position, and deleting the residue / position. "Amino acid substitution" or its variants refers to replacing an existing amino acid residue in a predetermined (starting) amino acid sequence with a different amino acid residue. Typically, compared to a polypeptide comprising an initial (or "wild type") amino acid sequence, modification results in a change in at least one physical and biochemical activity of the variant polypeptide. For example, in the case of an antibody, the physical and biochemical activity of the change can be binding affinity, binding capacity, and / or binding effect to a target molecule.

[0100] The term "antibody" is used in the broadest sense and specifically covers, for example, a single anti-CTHRC1 monoclonal antibody (including agonists, antagonists, neutralizing antibodies, full-length or complete monoclonal antibodies), anti-CTHRC1 antibody compositions with multi-epitope specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies formed by at least two complete antibodies (e.g., bispecific antibodies, as long as they exhibit the desired biological activity), single-chain anti-CTHRC1 antibodies, and fragments of anti-CTHRC1 antibodies (see below) (including Fab, Fab', F(ab')2 and Fv fragments), diabodies, single domain antibodies (sdAb), as long as they exhibit the desired biological or immunological activity. Anti-CTHRC1 antibodies, and in particular fragments, also include portions of anti-CTHRC1 antibodies that can be used as targeting arms (and combinations of portions of anti-CTHRC1 antibodies, such as scFv), which are directed to, for example, CTHRC1 tumor epitopes in chimeric antigen receptors of CAR-T cells, CAR-NK cells, or CAR-macrophages. Such fragments are not necessarily proteolytic fragments, but rather portions of the polypeptide sequence that can confer affinity to the target. The term "immunoglobulin" (Ig) is used interchangeably herein with antibody. The antibody can be, for example, a human antibody, a humanized antibody, and / or an affinity matured antibody.

[0101] The terms "anti-CTHRC1 antibody," "CTHRC1 antibody," and "antibody that binds to CTHRC1" are used interchangeably. The anti-CTHRC1 antibody preferably binds with sufficient affinity to allow the antibody to be used as a diagnostic and / or therapeutic agent, whether isolated or as part of a fusion protein, cell, or cell composition.

[0102] In one embodiment, the CTHRC1 antibody is used herein to specifically refer to an anti-CTHRC1 monoclonal antibody, which (i) comprises a heavy chain variable domain of any one of SEQ ID NOs: 1, 3, 5, 7 and 9, and / or a light chain variable domain of any one of SEQ ID NOs: 2, 4, 6, 8 and 10; or (ii) comprises one, two, three, four, five or six of the CDRs shown in Table 3 or Table 4.

[0103] An "isolated antibody" is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that may interfere with the therapeutic use of the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0104] The basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the 4-chain unit is generally about 150,000 daltons. Each L chain is connected to the H chain by a covalent disulfide bond, and the two H chains are connected to each other by one or more disulfide bonds according to the H chain isotype. Each H chain and L chain also have regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) of each of the α and γ chains, and four CH domains of μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at the other end. VL is aligned with VH, and CL is aligned with the first constant domain (CH1) of the heavy chain. It is believed that specific amino acid residues form the interface between the light chain and the heavy chain variable domain. VH and VL are paired together to form a single antigen binding site. For the structure and properties of different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6.

[0105] L chains from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domains. Immunoglobulins are classified into different classes or isotypes based on the amino acid sequence of the constant domain of their heavy chains (CH). There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0106] The "variable region" or "variable domain" of an antibody refers to the amino terminal domain of the heavy chain or light chain of an antibody. The variable domain of the heavy chain may be referred to as "VH" or "V H The variable domain of the light chain may be referred to as "VL" or "V L ”. These domains are usually the most variable parts of antibodies and contain the antigen binding site.

[0107] The term "variable" refers to the fact that certain segments of the variable domain are very different in sequence between antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody to its specific antigen. However, variability is not evenly distributed within the 110 amino acids of the variable domain. Instead, the V region consists of relatively invariant fragments known as framework regions (FRs), which have 15-30 amino acids and are separated by shorter regions of extreme variability known as "hypervariable regions" each of which is 9-12 amino acids long. The variable domains of natural heavy and light chains each contain four FRs, mainly in a β-folded configuration, connected by three hypervariable regions, which form a loop connecting the β-folded structure and forming a part of the β-folded structure in some cases. The hypervariable regions in each chain are tightly bound together by the FRs and contribute to the formation of the antigen binding site of the antibody together with the hypervariable regions from the other chain (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0108] A "complete" antibody is an antibody that comprises an antigen binding site as well as CL and at least heavy chain constant domains CH1, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the complete antibody has one or more effector functions.

[0109] "Antibody fragments" include a portion of a complete antibody, preferably an antigen binding region or one or more variable regions of a complete antibody. Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng.8(10):1057-62(1995)); single-chain antibody molecules; and multispecific antibodies formed by antibody fragments. In one embodiment, the antibody fragment includes the antigen binding site of the complete antibody, thereby retaining the ability to bind to the antigen. Anti-CTHRC1 antibody fragments also include portions of anti-CTHRC1 antibodies that can be used as targeting arms (and combinations of portions of anti-CTHRC1 antibodies, such as scFv), which are directed to CTHRC1 tumor epitopes in, for example, chimeric antigen receptors of CAR-T cells or CAR-NK cells, or CAR-macrophages. Such fragments are not necessarily proteolytic fragments, but portions of polypeptide sequences that can confer target affinity.

[0110] Papain digestion of antibodies produces two identical antigen-binding fragments, known as "Fab" fragments, and a residual "Fc" fragment (the name reflects the ability to crystallize easily). The Fab fragment consists of an intact L chain as well as the variable region domains of the H chain (VH) and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of antibodies produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with divalent antigen-binding activity and is still able to cross-link antigen. The Fab' fragment differs from the Fab fragment in having several additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab'-SH is herein the name for Fab' in which the cysteine ​​residues of the constant domains carry free thiol groups. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0111] The Fc fragment comprises the carboxyl terminal portions of two H chains held together by disulfide bonds. The effector functions of an antibody are determined by sequences in the Fc region, which is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[0112] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a heavy chain variable region domain and a light chain variable region domain in tight non-covalent association. In a single-chain Fv (scFv) substance, a heavy chain variable domain and a light chain variable domain can be covalently linked by a flexible peptide linker, so that the light chain and the heavy chain can associate in a "dimer" structure similar to that in a double-chain Fv substance. Six hypervariable loops (3 loops each from the H chain and the L chain) are produced by the folding of these two domains, which contribute amino acid residues to antigen binding and give antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three CDRs specific for an antigen) has the ability to recognize and bind to an antigen, although the affinity is lower than that of the entire binding site.

[0113] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form a desired structure for antigen binding. For a review of sFv, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, see below. In one embodiment, an anti-CTHRC1 antibody-derived scFv is used as a targeting arm for CAR-T cells, CAR-NK cells, or CAR-macrophages disclosed herein.

[0114] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., except for possible naturally occurring mutations that may be present in small amounts, the individual antibodies constituting the population are identical. Monoclonal antibodies are highly specific and are directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations including different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. The advantage of monoclonal antibodies in addition to their specificity is that they can be synthesized without being contaminated by other antibodies. The modifier "monoclonal" should not be interpreted as requiring antibodies to be produced by any ad hoc method. For example, monoclonal antibodies that can be used in the present invention can be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be prepared using recombinant DNA methods in bacteria, eukaryotic animals or plant cells (e.g., U.S. Patent No. 4,816,567). "Monoclonal antibodies" can also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature, 352:624-8 (1991) and Marks et al., J. Mol. Biol., 222:581-97 (1991).

[0115] The term "hypervariable region", "HVR" or "HV" when used herein refers to a region of an antibody variable domain that is hypervariable in sequence and / or forms a structurally defined loop. Typically, an antibody comprises six hypervariable regions; three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Many descriptions of hypervariable regions are in use and are covered herein. The Kabat complementarity determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers to the position of the structural loops (Chothia and Lesk J. Mol. Biol. 196: 901-917 (1987)). When numbering using the Kabat numbering convention, the ends of the Chothia CDR-H1 loop vary between H32 and H34, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable region represents a compromise between the Kabat CDR and Chothia structural loops and is used by the AbM antibody modeling software of Oxford Molecular. The "contact" hypervariable region is based on an analysis of available complex crystal structures. The residues from each of these hypervariable regions are as follows.

[0116]

[0117] The hypervariable region may comprise the following "extended hypervariable region": 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 (L3) in VL; and 26-35B (H1), 50-65, 47-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al., supra.

[0118] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0119] The term "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refer to the numbering system used for heavy chain variable domains or light chain variable domains in Kabat et al., supra for antibody compilations. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to shortening of the FR or CDR of the variable domain or insertion into the FR or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). For a given antibody, the Kabat numbering of the residues can be determined by sequence alignment with the "standard" Kabat numbering in homologous regions of the antibody sequence.

[0120] When referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is generally used (e.g., Kabat et al., supra). When referring to residues in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise indicated herein, reference to residue numbering in the variable domain of an antibody means residue numbering by the Kabat numbering system.

[0121] A "blocking" antibody or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of an antigen to which it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. In one embodiment, an anti-CTHRC1 antibody is provided that is an antagonist antibody.

[0122] An antibody that "binds" an antigen or epitope of interest is one that binds to the antigen or epitope with sufficient affinity to be measurably different from nonspecific interactions. Specific binding can be measured, for example, by measuring the binding of a molecule compared to the binding of a control molecule, which is typically a molecule of similar structure that does not have binding activity.

[0123] Antibodies that inhibit tumor cell growth are antibodies that cause measurable cancer cell growth inhibition. In one embodiment, anti-CTHRC1 antibodies are capable of inhibiting the growth of cancer cells displaying CTHRC1 tumor epitopes. As mentioned herein, CTHRC1 tumor epitopes include CTHRC1 epitopes that can be bound by anti-CTHRC1 antibodies or fragments thereof as disclosed herein, or CTHRC1 epitopes that can be at least partially bound by antibodies or other molecules that compete with anti-CTHRC1 antibodies as disclosed herein for binding to the epitope. Compared to appropriate controls, preferred growth inhibitory anti-CTHRC1 antibodies inhibit the growth of tumor cells expressing CTHRC1 by greater than 20%, preferably about 20% to about 50%, and even more preferably greater than 50% (e.g., about 50% to about 100%), which controls are typically tumor cells that have not been treated with the antibody being tested.

[0124] Anti-CTHRC1 antibodies can (i) inhibit tumor metastasis in vivo; (ii) inhibit tumor growth in vivo; (iii) reduce tumor size in vivo; (iv) inhibit tumor vascularization in vivo; (v) exhibit cytotoxic activity against tumor cells and cancer-associated fibroblasts expressing CTHRC1 in vivo; (vi) exhibit cytostatic activity against tumor cells or cancer-associated fibroblasts expressing CTHRC1 in vivo; or (vii) prevent the inhibition of immune cells in the tumor microenvironment in vivo.

[0125] The term "antagonist" is used in the broadest sense and includes any molecule that partially or completely blocks, inhibits or neutralizes the biological activity of the antigen. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of natural CTHRC1 polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. The method for identifying CTHRC1 polypeptide antagonists may include contacting the CTHRC1 polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities typically associated with the CTHRC1 polypeptide.

[0126] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), skin cancer, melanoma, lung cancer (including small cell lung cancer), non-small cell lung cancer ("NSCLC"), lung adenocarcinoma and lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, stomach or gastric cancer (including gastrointestinal cancer), pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), glioblastoma, cervical cancer, ovarian cancer (e.g., high-grade serous ovarian cancer), liver cancer (e.g., hepatocellular carcinoma (HCC)), bladder cancer (e.g., urothelial bladder cancer), testicular (germ cell tumor) cancer, hepatoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney or renal cancer (e.g., renal cell carcinoma, Wilms' tumor or Wilms' tumor), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. Other examples of cancer include, but are not limited to, adrenocortical carcinoma, bile duct carcinoma, colon adenocarcinoma, B-cell lymphoma, esophageal cancer, glioblastoma multiforme, renal clear cell carcinoma, renal papillary cell carcinoma, myeloid leukemia, lung adenocarcinoma, lung squamous cell carcinoma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, gastric adenocarcinoma, thymoma, uterine corpus carcinoma, and uterine carcinosarcoma.

[0127] The term "metastatic cancer" refers to a cancerous state in which cancer cells of a tissue of origin spread from the original site through blood or lymphatic vessels to one or more sites elsewhere in the body, forming one or more secondary tumors in one or more organs other than the tissue of origin. A prominent example is metastatic breast cancer.

[0128] As used herein, "CTHRC1-associated cancer" is a cancer associated with overexpression of the CTHRC1 gene or gene product and / or associated with display of CTHRC1 tumor epitopes. Suitable control cells can be, for example, cells from an individual without cancer or non-cancerous cells from a subject with cancer.

[0129] The methods of the invention include methods for treating a subject having cancer, particularly cancers associated with expression of the CTHRC1 tumor epitope. The methods of the invention also include methods for modulating certain cell behaviors, particularly cancer cell behaviors, particularly cancer cells displaying the CTHRC1 tumor epitope.

[0130] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0131] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all precancerous and cancerous cells and tissues.

[0132] As used herein, the term "fibrotic disease" refers broadly to many different diseases characterized by the development of fibrosis in organs, examples include, but are not limited to, idiopathic pulmonary fibrosis (IPF) and scleroderma. The term "fibrosis" refers to the development of fibrous connective tissue as a representative response to injury or damage. Fibrosis can occur as part of normal healing or in response to excessive tissue deposition that occurs as part of a pathological process.

[0133] As used herein, the terms "predictive" and "prognostic" are also interchangeable. In one sense, methods for predicting or prognosticating allow a person practicing the predictive / prognostic methods of the invention to select patients who are considered (usually before treatment, but not necessarily) to be more likely to respond to treatment with an anti-cancer agent, preferably an anti-CTHRC1 antibody of the invention or a CAR engineered cell.

[0134] III. Compositions and Methods of the Invention

[0135] A. Anti-CTHRC1 Antibodies

[0136] In one embodiment, the invention provides anti-CTHRC1 antibodies useful as therapeutic agents herein.Exemplary antibodies include polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, and human antibodies.

[0137] 1. Polyclonal antibodies

[0138] Polyclonal antibodies can be produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (particularly when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized. For example, the antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using bifunctional or derivatizing agents such as maleimidobenzoyl sulfosuccinimide ester (conjugated through cysteine ​​residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R'N=C=NR, wherein R and R' are different alkyl groups.

[0139] Animals are immunized against the antigen, immunogenic conjugate or derivative by combining, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the antibody titer of the serum is determined. The animals are boosted until the titer reaches a stable level. The conjugates can also be prepared as protein fusions in recombinant cell culture. In addition, aggregating agents such as alum are suitably used to enhance the immune response.

[0140] 2. Monoclonal antibodies

[0141] Monoclonal antibodies (mAbs) against the antigen of interest can be prepared by using any technique known in the art. These techniques include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), human B cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4: 72), and EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Selective lymphocyte antibody method (SLAM) (Babcook, JS et al., A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc Natl Acad Sci SA, 1996. 93 (15): 7843-8 pages.) and (McLean G et al., 2005, J Immunol. 174 (8): 4768-78). Such antibodies may belong to any immunoglobulin class, including IgG, IgM, IgE, IgA and IgD and any subclass thereof. Hybridomas for producing mAbs of the present invention may be cultured in vitro or in vivo.

[0142] Monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (US Pat. No. 4,816,567).

[0143] In the hybridoma method, mice or other suitable host animals such as hamsters are immunized as described above to obtain lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pages 59-103 (Academic Press, 1986)).

[0144] The hybridoma cells thus prepared are seeded and grown in an appropriate culture medium that may contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as the fusion partner). For example, if the parental myeloma cells lack the enzyme, hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), then the selective culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of cells lacking HGPRT.

[0145] Preferred fusion partner myeloma cells are those that fuse efficiently, support the stable high-level production of antibodies by the cells that produce the antibodies selected, and are sensitive to the selective medium selected for the unfused parental cells. Preferred myeloma cell lines are murine myeloma cell lines, such as those derived from MOPC-21 and MPC-11 mouse tumors, which are available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA; and SP-2 and derivatives, such as X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Va., USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984); and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0146] Culture medium in which hybridoma cells are grown is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0147] The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis as described in Munson et al., Anal. Biochem. 107:220 (1980).

[0148] Once hybridoma cells producing antibodies of the desired specificity, affinity and / or activity are identified, clones can be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals, for example by injecting the cells intraperitoneally into mice.

[0149] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as affinity chromatography (e.g., using protein A or protein G-agarose) or ion exchange chromatography, hydroxyapatite chromatography, gel electrophoresis, dialysis, and the like.

[0150] DNA encoding monoclonal antibodies is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells are used as a preferred source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into a host cell, such as an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce antibody protein, to obtain the synthesis of the monoclonal antibody in a recombinant host cell. Review articles on recombinant expression of DNA encoding antibodies in bacteria include Skerra et al., Curr. Opinion in Immunol. 5: 256-62 (1993) and Plückthun, Immunol. Rev. 130: 151-88 (1992).

[0151] In another embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries produced using the techniques described in McCafferty et al., Nature, 348: 552-54 (1990). Clackson et al., Nature, 352: 624-28 (1991) and Marks et al., J. Mol. Biol., 222: 581-97 (1991) describe the use of phage libraries to separate mouse antibodies and human antibodies, respectively. Subsequent publications describe the production of high-affinity (nM range) human antibodies as a strategy for constructing very large phage libraries by chain shuffling (Marks et al., Bio / Technology, 10: 779-83 (1992)) and combined infection and in vivo recombination (Waterhouse et al., Nuc. Acids. Res. 21: 2265-6 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0152] The DNA encoding the antibody can be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting the human heavy and light chain constant domains (CH and CO sequences for the homologous mouse sequences (U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequences can replace the constant domains of the antibody, or they can replace the variable domains of one antigen-binding site of the antibody to produce a chimeric bivalent antibody comprising one antigen-binding site having specificity for an antigen and another antigen-binding site having specificity for a different antigen.

[0153] 3. Chimeric, humanized and human antibodies

[0154] In some embodiments, the anti-CTHRC1 antibody is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-5 (1984). In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate such as a monkey) and a human constant region. In another example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from the class or subclass of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0155] In some embodiments, chimeric antibodies are humanized antibodies. Generally, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Generally, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or part thereof) are derived from non-human antibodies, and FR (or part thereof) are derived from human antibody sequences. Humanized antibodies are optionally also comprised of at least a portion of human constant regions. In some embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies from which CDR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0156] The anti-CTHRC1 antibodies of the present invention may comprise humanized antibodies or human antibodies. The humanized form of non-human (e.g., mouse or rabbit) antibodies is a chimeric immunoglobulin, an immunoglobulin chain or a fragment thereof (e.g., Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of an antibody) containing a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins (receptor antibodies), wherein residues from the complementary determining regions (CDRs) of the receptor are replaced by residues from non-human species (donor antibodies) such as mice, rats or rabbits with the desired specificity, affinity and ability. In some cases, the Fv framework residues of human immunoglobulins are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are neither found in the receptor antibody nor in the CDR or framework sequences introduced. In general, humanized antibodies will include substantially all of at least one, usually two variable domains, wherein all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the FR regions are FR regions of the consensus sequence of human immunoglobulins. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin (Jones et al., Nature, 321:522-5 (1986); Riechmann et al., Nature, 332:323-9 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-6 (1992)).

[0157] The humanized antibodies of the present invention may include one or more human and / or human consensus non-hypervariable region (e.g., framework) sequences in their heavy chain and / or light chain variable domains. In some embodiments, there are one or more additional modifications in human and / or human consensus non-hypervariable region sequences. In one embodiment, the heavy chain variable domain of an antibody of the present invention includes a consensus framework sequence for humans, which in one embodiment is a consensus framework sequence for subgroup III. In one embodiment, an antibody of the present invention includes a consensus framework sequence for a variant subgroup III modified at at least one amino acid position.

[0158] As known in the art, and as described in more detail herein, the amino acid position / boundary of the hypervariable region of the antibody can vary, depending on the context and various definitions known in the art (as described below). Some positions in the variable domain can be regarded as hybrid hypervariable positions, because these positions can be considered to be in the hypervariable region under a set of standards, and are considered to be outside the hypervariable region under a different set of standards. One or more of these positions can also be found in the hypervariable region of extension (as further defined below). The invention provides antibodies comprising modifications in these hybrid hypervariable positions. In one embodiment, these hypervariable positions include one or more positions 26-30, 33-35B, 47-49, 57-65, 93, 94 and 101-102 in the heavy chain variable domain. In one embodiment, these hybrid hypervariable positions include one or more of positions 24-29, 35-36, 46-49, 56 and 97 in the light chain variable domain. In one embodiment, the antibody of the present invention is included in the human variant human subgroup consensus framework sequence modified at one or more hybrid hypervariable positions.

[0159] The antibodies of the invention may comprise any suitable human or human consensus light chain framework sequence, so long as the antibody exhibits the desired biological properties (e.g., desired binding affinity). In one embodiment, the antibodies of the invention comprise at least a portion (or all) of the framework sequence of a human κ light chain. In one embodiment, the antibodies of the invention comprise at least a portion (or all) of the human κ subgroup I framework consensus sequence.

[0160] Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced therein from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from the "import" variable domain. Humanization can be carried out substantially according to the method of Winter and colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by replacing the corresponding sequence of a human antibody with a rodent CDR or CDR sequence. Therefore, this type of "humanized" antibody is a chimeric antibody (U.S. Patent No. 4,816,567), in which substantially less than a complete human variable domain has been replaced by a corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0161] When the antibody is intended for human therapeutic use, the choice of human variable domains (both light and heavy chains) to be used in making the humanized antibodies is very important to reduce antigenicity and HAMA response (human anti-mouse antibody). Reduction or elimination of the HAMA response is an important aspect of the clinical development of suitable therapeutic agents (see, e.g., Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al., Transplantation (1986), 41:572; Shawler et al., J. Immunol. (1985), 135:1530; Sears et al., J. Biol. Response Mod. (1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352; Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res. (1990), 50:1495). As described herein, the present invention provides humanized antibodies such that the HAMA response is reduced or eliminated. Variants of these antibodies can be further obtained using conventional methods known in the art, some of which are further described below. According to the so-called "best fit" method, the sequence of the variable domain of a rodent antibody is screened for the entire library of known human variable domain sequences. The human V domain sequence closest to the rodent is confirmed, and the human framework region (FR) therein is accepted for humanized antibodies (Sims et al., J. Immunol. 151: 2296 (1993); Chothia et al., J. Mol. Biol., 196: 901 (1987)). Another method uses a specific framework region derived from the consensus sequence of all human antibodies of a specific light chain or heavy chain subgroup. The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89: 4285 (1992); Presta et al., J. Immunol. 151: 2623 (1993)).

[0162] For example, the amino acid sequence from an antibody as described herein can be used as a diversified starting (parent) sequence for a framework and / or hypervariable sequence. The selected framework sequence to which the starting hypervariable sequence is connected is referred to herein as an acceptor human framework. Although the acceptor human framework can be derived from or is derived from human immunoglobulin (its VL and / or VH region), it is preferred that the acceptor human framework is derived from or is derived from a human consensus framework sequence, because such frameworks have been shown to have minimal immunogenicity or no immunogenicity in human patients.

[0163] In cases where the acceptor is derived from a human immunoglobulin, a human framework sequence can optionally be selected based on its homology to the donor framework sequence by aligning the donor framework sequence with various human framework sequences in a collection of human framework sequences and selecting the most homologous framework sequence as the acceptor.

[0164] In one embodiment, the human consensus framework herein is from or derived from a VH subgroup III and / or VL kappa subgroup I consensus framework sequence.

[0165] Although the receptor can be identical in sequence to the selected human framework sequence, whether it is from a human immunoglobulin or a human consensus framework, the invention contemplates that the receptor sequence may contain pre-existing amino acid substitutions relative to the human immunoglobulin sequence or the human consensus framework sequence. These pre-existing substitutions are preferably minimal; typically only four, three, two or one amino acid differences relative to the human immunoglobulin sequence or the consensus framework sequence.

[0166] The hypervariable region residues of the non-human antibody are incorporated into the VL and / or VH acceptor human framework. For example, residues corresponding to Kabat CDR residues, Chothia hypervariable loop residues, Abm residues and / or contact residues can be incorporated. Optionally, the following extended hypervariable region residues are incorporated: 24-34 (L1), 50-56 (L2) and 89-97 (L3), 26-35B (H1), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3).

[0167] Although "incorporation" of hypervariable region residues is discussed herein, it will be appreciated that this can be accomplished in a variety of ways, for example, nucleic acid encoding the desired amino acid sequence can be produced by mutating a nucleic acid encoding a mouse variable domain sequence such that the framework residues are changed to acceptor human framework residues, or by mutating a nucleic acid encoding a human variable domain sequence such that the hypervariable domain residues are changed to non-human residues, or by synthesizing a nucleic acid encoding the desired sequence, etc.

[0168] As described herein, hypervariable region grafted variants can be generated by Kunkel mutagenesis of nucleic acids encoding human receptor sequences, using separate oligonucleotides for each hypervariable region. Kunkel et al., Methods Enzymol. 154: 367-382 (1987). Appropriate changes can be introduced into the framework and / or hypervariable regions to correct and reconstruct appropriate hypervariable region-antigen interactions using conventional techniques.

[0169] Phage(mid) display (also referred to herein as phage display in some cases) can be used as a convenient and rapid method for producing and screening many different potential variant antibodies in a library generated by sequence randomization. However, methods for preparing and screening altered antibodies are available to the skilled person.

[0170] Phage (mid) display technology provides a powerful tool for the production and selection of novel proteins that bind to ligands such as antigens. The use of phage (mid) display technology allows the generation of large libraries of protein variants that can quickly sort those sequences that bind to target molecules with high affinity. Usually, the nucleic acid encoding the variant polypeptide is fused with the nucleic acid sequence encoding the viral coat protein such as gene III protein or gene VIII protein. Monovalent phagemid display systems have been developed in which the nucleic acid sequence encoding the protein or polypeptide is fused with the nucleic acid sequence encoding a portion of the gene III protein. (Bass, S., Proteins, 8: 309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3: 205 (1991)). In the monovalent phagemid display system, the gene fusion is expressed at a low level, and the wild-type gene III protein is also expressed, so that the infectivity of the particles is retained. Methods for generating peptide libraries and screening these libraries have been disclosed in numerous patents (eg, U.S. Pat. No. 5,723,286, U.S. Pat. No. 5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,427,908, and U.S. Pat. No. 5,498,530).

[0171] Libraries of antibodies or antigen-binding polypeptides have been prepared in a variety of ways, including by inserting random DNA sequences to change a single gene or by cloning a family of related genes. Methods for displaying antibodies or antigen-binding fragments using phage (mid) display have been described in U.S. Patents Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727. Libraries of antibodies or antigen-binding proteins expressing desired properties are then screened.

[0172] Methods for substituting selected amino acids into template nucleic acids are well established in the art, some of which are described herein. For example, hypervariable region residues can be substituted using the Kunkel method (e.g., Kunkel et al., Methods Enzymol. 154:367-382 (1987)).

[0173] The sequence of the oligonucleotide includes one or more of the codon sets designed for the hypervariable region residues to be changed. A codon set is a set of different nucleotide triplet sequences for encoding the desired variant amino acids. A codon set can be represented by a symbol to refer to an equimolar mixture of a specific nucleotide or nucleotides, as shown below according to the IUB code.

[0174] IUB Code

[0175] G Guanine

[0176] A Adenine

[0177] Tthymine

[0178] C-Cytosine

[0179] R(A or G)

[0180] Y(C or T)

[0181] M(A or C)

[0182] K(G or T)

[0183] S(C or G)

[0184] W(A or T)

[0185] H(A or C or T)

[0186] B(C or G or T)

[0187] V(A or C or G)

[0188] D(A or G or T)H

[0189] N(A or C or G or T)

[0190] For example, in the codon set DVK, D can be the nucleotide A or G or T; V can be A or G or C; and K can be G or T. This codon set can present 18 different codons and can encode the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

[0191] Standard methods can be used to synthesize oligonucleotides or primer sets. One group of oligonucleotides can be synthesized, for example, by solid phase synthesis, containing all possible combinations of nucleotide triplets representing the codon groups provided and by encoding the sequence of the required amino acid group. The synthesis of oligonucleotides with selected nucleotide "degeneracy" at certain positions is well known in the art. Such nucleotide groups with certain codon groups can be synthesized using commercial nucleic acid synthesizers (available from, for example, Applied Biosystems, Foster City, Calif.), or can be obtained from commercial sources (for example, deriving from Life Technologies, Rockville, Md.). Therefore, a group of oligonucleotides with a specific codon group synthesized will generally include a plurality of oligonucleotides with different sequences, and its difference is established by the codon groups in the entire sequence. The oligonucleotides used according to the present invention have sequences that allow hybridization with the variable domain nucleic acid templates, and can also include restriction enzyme sites for cloning purposes.

[0192] In one method, the nucleic acid sequence encoding the variant amino acid can be produced by oligonucleotide-mediated mutagenesis. This technology is well known in the art, as described by Zoller et al., Nucleic Acids Res. 10: 6487-6504 (1987). In short, the nucleic acid sequence encoding the variant amino acid is produced by hybridizing an oligonucleotide set encoding the desired codon set with a DNA template, wherein the template is a single-stranded form of a plasmid containing a variable region nucleic acid template sequence. After hybridization, a DNA polymerase is used to synthesize a complete second complementary strand of the template, which will therefore be incorporated into the oligonucleotide primer and will contain the codon set provided by the oligonucleotide set.

[0193] Typically, oligonucleotides of at least 25 nucleotides in length are used. The best oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide encoding the mutation. This ensures that the oligonucleotide will correctly hybridize with the single-stranded DNA template molecule. It is easy to synthesize oligonucleotides using technology known in the art, such as Crea et al., Proc. Nat'l. Acad. Sci. USA, 75:5765 (1978) describes the technology.

[0194] DNA templates are generated from those vectors derived from bacteriophage M13 vectors (the commercially available M13 mp 18 and M13 mp 19 vectors are suitable) or those vectors containing a single-stranded phage replication origin as described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus, the DNA to be mutated can be inserted into one of these vectors to generate a single-stranded template. The generation of single-stranded templates is described in Sambrook et al., supra, Sections 4.21-4.41.

[0195] In order to change the native DNA sequence, the oligonucleotide is hybridized with the single-stranded template under suitable hybridization conditions.Then add a DNA polymerase, typically the Klenow fragment of T7 DNA polymerase or DNA polymerase I, to use the oligonucleotide as a synthetic primer to synthesize the complementary strand of the template.So form a heteroduplex molecule, so that a chain of DNA encodes the mutant form of gene 1, and the natural unaltered sequence of another chain (original template) encoding gene 1.Then this heteroduplex molecule is transformed into a suitable host cell, typically a prokaryotic cell, such as Escherichia coli JM101.After cell growth, they are spread on an agarose plate, and the oligonucleotide primers labeled with 32-phosphate radioactivity are used to screen, to confirm the bacterial colonies containing mutant DNA.

[0196] The method just described above can be modified so that homoduplex molecules are produced, wherein both chains of the plasmid contain mutations. The modifications are as follows: a single-stranded oligonucleotide is annealed to a single-stranded template as described above. A mixture of three deoxyribonucleotides, i.e., deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTT), is combined with a modified thiodeoxyribocytosine (available from Amersham) called dCTP-(aS). This mixture is added to the template-oligonucleotide complex. After DNA polymerase is added to this mixture, a chain of DNA identical to the template is produced except for the mutated bases. In addition, this new DNA chain will contain dCTP-(aS) instead of dCTP, which is used to protect it from restriction endonuclease digestion. After the template chain of the double-stranded heteroduplex is nicked with an appropriate restriction enzyme, the template chain can be digested with ExoIII nuclease or another appropriate nuclease to pass through the region containing the site to be mutagenized. The reaction is then stopped, leaving only partially single-stranded molecules. A complete double-stranded DNA homoduplex is then formed using a DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell.

[0197] As previously noted, the sequence of the oligonucleotide set has sufficient length to hybridize with the template nucleic acid, and may also, but not necessarily, contain restriction sites. The DNA template can be produced by those vectors derived from the phage M13 vector or by a vector containing a single-stranded phage replication origin as described in Viera et al., Meth. Enzymol., 153:3 (1987). Therefore, the DNA to be mutated must be inserted into one of these vectors to produce a single-stranded template. The generation of a single-stranded template is described in Sambrook et al., above, Sections 4.21-4.41.

[0198] According to another approach, antigen binding can be restored during humanization of an antibody by selecting a repaired hypervariable region (see, e.g., U.S. Application Serial No. 11 / 061,841, filed February 18, 2005). The method comprises incorporating a non-human hypervariable region onto an acceptor framework and further introducing one or more amino acid substitutions in one or more hypervariable regions without modifying the acceptor framework sequence. Alternatively, the introduction of one or more amino acid substitutions may be accompanied by modifications in the acceptor framework sequence.

[0199] According to another method, library can be produced by providing upstream and downstream oligonucleotide groups, each group has a plurality of oligonucleotides with different sequences, and different sequences are established by the codon groups provided in the sequence of oligonucleotide.Upstream and downstream oligonucleotide groups and variable domain template nucleic acid sequences can be used for polymerase chain reaction to produce " library " of PCR product.PCR product can be referred to as " nucleic acid box ", because they can adopt the molecular biology technique of establishment to merge with other relevant or unrelated nucleotide sequences (for example, viral coat protein and dimerization domain).

[0200] The sequence of the PCR primer includes one or more of the codon sets designed for solvent accessible and highly diverse positions in the hypervariable region. As described above, a codon set is a set of different nucleotide triplet sequences for encoding the desired variant amino acids.

[0201] Antibody selectors that meet the desired criteria selected by appropriate screening / selection steps can be isolated and cloned using standard recombinant techniques.

[0202] It is further important that the antibody is humanized, retaining high binding affinity to antigen and other favorable biological properties. In order to achieve this goal, according to a preferred method, humanized antibodies are prepared by analyzing the process of parental sequences and various conceptual humanized products using three-dimensional models of parental sequences and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs that illustrate and display the possible three-dimensional conformational structures of selected candidate immunoglobulin sequences can be obtained. Inspection of these displays allows analysis of the possible effects of residues in the functioning of candidate immunoglobulin sequences, i.e., analysis of the residues that affect the ability of candidate immunoglobulins to bind their antigens. In this way, FR residues can be selected from receptors and imported sequences and combined to obtain desired antibody characteristics, such as realizing an increased affinity for the target antigen. In general, hypervariable region residues directly and most substantially participate in affecting antigen binding.

[0203] Various forms of humanized anti-CTHRC1 antibodies are contemplated. For example, the humanized antibody can be an antibody fragment, such as Fab. Alternatively, the humanized antibody can be a complete antibody, such as a complete IgG1 antibody.

[0204] As an alternative to humanization, human antibodies can be produced. For example, it is now possible to produce transgenic animals (e.g., mice) that can produce a complete human antibody repertoire in the absence of endogenous immunoglobulin production after immunization. For example, homozygous deletions of antibody heavy chain joining region (JH) genes in chimeric and germline mutant mice have been described to result in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-8 (1993); Bruggemann et al., Year in Immuno. 7:33 (1993); U.S. Pat. Nos. 5,545,806; 5,569,825; 5,591,669; 5,545,807; and WO 97 / 17852).

[0205] Alternatively, phage display technology (McCafferty et al., Nature 348:552-53 (1990)) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene libraries from unimmunized donors. According to this technology, antibody V domain genes are cloned in-frame into major or minor coat protein genes of filamentous phages (such as M13 or fd) and displayed as functional antibody fragments on the surface of phage particles. Because filamentous particles contain single-stranded DNA copies of phage genomes, selection based on the functional properties of antibodies also leads to selection of genes encoding antibodies that exhibit these properties. Therefore, phages simulate some characteristics of B cells. Phage display can be performed in a variety of forms, and reviews are found in, for example, Johnson, Kevin S and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). V gene segments from several sources can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a variety of anti-oxazolone antibodies from a small random combinatorial library of V genes from the spleen of immunized mice. V gene libraries from unimmunized human donors can be constructed and antibodies to a variety of antigens (including self-antigens) can be isolated essentially according to the techniques described by Marks et al., J. Mol. Biol. 222:581-97 (1991) or Griffith et al., EMBO J. 12:725-34 (1993) (see also U.S. Pat. Nos. 5,565,332 and 5,573,905).

[0206] As discussed above, human antibodies can also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275).

[0207] In another embodiment, the antibodies disclosed herein are human monoclonal antibodies. Such human monoclonal antibodies against CTHRC1 can be generated using transgenic or transchromosomal mice that carry parts of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include those referred to herein as HuMAb Mouse, TM and KM Mouse TM mice, and are collectively referred to herein as "human Ig mice".

[0208] HuMAb Mouse TM(Medarex, Inc.) contains a human immunoglobulin gene minilocus encoding unrearranged human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, as well as targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg et al., (1994) Nature 368(6474):856-9). Thus, the mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgGκ monoclonal antibodies (Lonberg, N. et al. (1994), supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13:65-93; and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci. 764:536-46). HuMAb Mouse is further described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-4; Choi et al. (1993) Nature Genetics 4:117-23; Chen, J. et al. (1993) EMBO J. 12:21-830; Tuaillon et al., (1994) J. Immunol. 152:2912-20; Taylor, L. et al. (1994) International Immunology 6:579-91; and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-51. TMThe preparation and use of mice, and the genomic modifications carried by such mice, the contents of all of which are hereby expressly incorporated by reference in their entirety. See also, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; U.S. Pat. No. 5,545,807; PCT Publications WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884, and WO 99 / 45962; and PCT Publication WO 01 / 14424.

[0209] In another embodiment, the human antibodies of the present disclosure can be produced using mice carrying human immunoglobulin sequences on transgenes and transchromosomes, such as mice carrying human heavy chain transgenes and human light chain transchromosomes. This mouse is referred to herein as a "KM Mouse." TM ” and is described in detail in PCT publication WO 02 / 43478.

[0210] In addition, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to generate anti-CTHRC1 antibodies of the present disclosure. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used; such mice are described in, for example, U.S. Patent Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963.

[0211] In addition, alternative transchromosomal animal systems expressing human immunoglobulin genes are available in the art and can be used to produce anti-CTHRC1 antibodies of the present disclosure. For example, mice carrying both human heavy chain transchromosomes and human light chain transchromosomes, referred to as "TC mice", can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-7. As another example, cows carrying human heavy and light chain transchromosomes have been described in the art (e.g., Kuroiwa et al. (2002) Nature Biotechnology 20:889-94 and PCT Application No. WO 2002 / 092812) and can be used to produce anti-CTHRC1 antibodies of the present disclosure. Other examples of transgenic animals that can be used to produce anti-CTHCR1 antibodies include OmniRat TM and OmniMouseTM (See, e.g., Osborn M. et al. (2013) Journal of Immunology 190:1481-90; Ma B. et al. (2013) Journal of Immunological Methods 400-401:78-86; Geurts A. et al. (2009) Science 325:433, U.S. Pat. No. 8,907,157; ​​European Patent No. 2152880B1; European Patent No. 2336329B1). Yet another example includes the use of Technology (see, e.g., U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, ). In short, The technology involves producing a transgenic mouse with a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, so that the mouse produces antigen-binding proteins, such as antibodies, comprising human variable regions and mouse constant regions in response to antigenic stimulation. DNA encoding the variable regions of the heavy and light chains of the antibodies is separated and operably linked to DNA encoding human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0212] 4. Antibody fragments

[0213] In some cases, there are advantages to using antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and can improve entry into solid tumors.

[0214] A variety of techniques for producing antibody fragments have been developed. Traditionally, these fragments are obtained via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-7 (1992); and Brennan et al., Science, 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli, thus allowing large amounts of these fragments to be easily produced. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab′-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab′)2 fragments (Carter et al., Bio / Technology 10: 163-7 (1992)). According to another method, F(ab′)2 fragments can be isolated directly from recombinant host cell cultures. Fab and F(ab')2 fragments with increased in vivo half-life are described in U.S. Patent No. 5,869,046, which contain salvage receptor binding epitope residues. Other techniques for producing antibody fragments will be apparent to the skilled person. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv) (see WO93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458). Fv and sFv are the only species with complete binding sites lacking constant regions; therefore, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins can be constructed to produce fusions of effector proteins at the amino or carboxyl termini of sFv (see Antibody Engineering, Borrebaeck, ed., supra. Antibody fragments can also be "linear antibodies", for example, as described in U.S. Patent No. 5,641,870.

[0215] In one embodiment, scFv derived from anti-CTHRC1 antibodies is used for CAR-modified immune cells, such as CAR-T or CAR-NK cells, or CAR-macrophages. Anti-CTHRC1 antibody fragments include portions of anti-CTHRC1 antibodies that can be used as targeting arms (and combinations of portions of anti-CTHRC1 antibodies, such as scFv), which are directed to CTHRC1 tumor epitopes in chimeric antigen receptors of CAR-T or CAR-NK cells or CAR-macrophages. Such fragments are not necessarily proteolytic fragments, but portions of polypeptide sequences that can confer target affinity.

[0216] 5. Bispecific Antibodies

[0217] Bispecific antibodies are antibodies that have binding specificity to at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of CTHRC1 protein as described herein. Other such antibodies can combine the CTHRC1 binding site with the binding site of another protein. Alternatively, the anti-CTHRC1 arm can be combined with an arm that binds to a trigger molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD3) or an Fc receptor (FcγR) of IgG, such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), so that the cell defense mechanism is concentrated and positioned to the cells expressing CTHRC1. Bispecific antibodies can also be used to position cytotoxic agents to cells expressing CTHRC1. These antibodies have a CTHRC1 binding arm and an arm that binds a cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloids, ricin A chain, methotrexate or a radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (eg, F(ab')2 bispecific antibodies).

[0218] WO 96 / 16673 describes bispecific anti-ErbB2 / anti-FcγRIII antibodies, and US Pat. No. 5,837,234 discloses bispecific anti-ErbB2 / anti-FcγRI antibodies. Bispecific anti-ErbB2 / Fcα antibodies are shown in WO 98 / 02463. US Pat. No. 5,821,337 teaches bispecific anti-ErbB2 / anti-CD3 antibodies.

[0219] Methods for preparing bispecific antibodies are known in the art. The traditional production method of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities (Millstein et al., Nature 305:537-9 (1983)). Due to the random assortment of immunoglobulin heavy chains and light chains, these hybridomas (quadruple tumors) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule usually completed by the affinity chromatography step is quite cumbersome, and the product yield is low. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J.10:3655-3659 (1991).

[0220] 6. Antibody variants and modifications

[0221] a) Substitution, insertion and deletion variants

[0222] In addition to the anti-CTHRC1 antibodies described herein, it is contemplated that anti-CTHRC1 antibody variants may be prepared. Anti-CTHRC1 antibody variants may be prepared by introducing appropriate nucleotide changes into the encoding DNA, and / or by synthesizing the desired antibody or polypeptide. It will be appreciated by those skilled in the art that amino acid changes may alter the post-translational processing of the anti-CTHRC1 antibody, such as changing the number or position of glycosylation sites, or changing membrane anchoring characteristics.

[0223] The variation of the anti-CTHRC1 antibodies described herein can be carried out, for example, using any of the techniques and guidelines for conservative and non-conservative mutations described in, for example, U.S. Pat. No. 5,364,934. The variation can be a substitution, deletion or insertion of one or more codons encoding an antibody or polypeptide, which results in a change in the amino acid sequence compared to a native sequence antibody or polypeptide. Optionally, the variation is by replacing at least one amino acid with any other amino acid in one or more domains of the anti-CTHRC1 antibody. By comparing the sequence of the anti-CTHRC1 antibody with the sequence of a homologous known protein molecule and minimizing the number of amino acid sequence changes made in a highly homologous region, a guide for determining which amino acid residues can be inserted, substituted or deleted without adversely affecting the desired activity can be established. Amino acid substitution can be the result of replacing one amino acid with another amino acid having a similar structure and / or chemical property, such as replacing leucine with serine, i.e., conservative amino acid substitution. Insertion or deletion can be optionally within the range of about 1 to 5 amino acids. The variation allowed can be determined by systematically inserting, deleting or replacing amino acids in the sequence and testing the activity exhibited by the obtained variants by the full-length or mature native sequence.

[0224] Anti-CTHRC1 antibody fragments are provided herein. For example, when compared to a full-length native antibody or protein, such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues. Certain fragments lack amino acid residues that are not essential for the desired biological activity of the anti-CTHRC1 antibody.

[0225] Anti-CTHRC1 antibody fragments can be prepared by any of a variety of conventional techniques. The desired peptide fragments can be chemically synthesized. Alternative methods involve producing antibodies or polypeptide fragments by enzymatic digestion, for example by treating proteins with enzymes known to cut proteins at sites defined by specific amino acid residues, or by digesting DNA with suitable restriction enzymes and separating the desired fragments. Another suitable technique involves separating and amplifying DNA fragments encoding the desired antibody or polypeptide fragments by polymerase chain reaction (PCR). Oligonucleotides that limit the desired ends of the DNA fragment are used at the 5' and 3' primers in PCR. Preferably, the anti-CTHRC1 antibody fragment shares at least one biological activity and / or immunological activity with the natural anti-CTHRC1 antibodies disclosed herein.

[0226] In specific embodiments, conservative substitutions of interest are shown under the heading of preferred substitutions in Table 1. If such substitutions result in a change in biological activity, more substantial changes, i.e., the exemplary substitutions named in Table 1, or as further described below with reference to amino acid classes, are introduced and the products screened.

[0227] Table 1

[0228]

[0229]

[0230] Substantial modification of the function or immunological properties of anti-CTHRC1 antibodies is achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, such as a folded or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into several groups based on common side chain properties:

[0231] (1) Hydrophobic: norleucine, met, ala, val, leu, ile;

[0232] (2) neutral hydrophilic: cys, ser, thr;

[0233] (3) Acidic: asp, glu;

[0234] (4) Basic: Asn, Gln, His, Lys, Arg;

[0235] (5) Residues that affect chain orientation: gly, pro; and

[0236] (6) Aromatic: trp, tyr, phe.

[0237] Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class. Such substituted residues may also be introduced at conservative substitution sites or, more preferably, at the remaining (non-conservative) sites.

[0238] The variation can be produced by methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction-selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London Ser A, 317:415 (1986)) or other known techniques can be performed on cloned DNA to generate anti-CTHRC1 antibody variant DNA.

[0239] Scanning amino acid analysis can also be used to confirm one or more amino acids along a continuous sequence. Preferred scanning amino acids include relatively small neutral amino acids. Such amino acids include alanine, glycine, serine and cysteine. Alanine is usually the preferred scanning amino acid in this group because it eliminates side chains other than the β-carbon and is unlikely to change the main chain conformation of the variant (Cunningham and Wells, Science, 244: 1081-5 (1989)). Alanine is also usually preferred because it is the most common amino acid. In addition, it is often found in buried and exposed positions (Creighton, The Proteins, (WH Freeman & Co., NY); Chothia, J. Mol. Biol., 150: 1 (1976)). If alanine substitution does not produce a sufficient amount of variants, isomeric amino acids can be used.

[0240] Any cysteine ​​residue that is not involved in maintaining the correct conformation of the anti-CTHRC1 antibody may also be substituted, usually with serine, to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, cysteine ​​bonds may be added to the anti-CTHRC1 antibody to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

[0241] Particularly preferred types of substitution variants involve replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody or a human antibody). Typically, the resulting variants selected for further development have improved biological properties relative to the parent antibody from which they are produced. A convenient method for producing such substitution variants involves affinity maturation using phage display. In short, several hypervariable region sites (e.g., 6-7 sites) are mutated to produce all possible amino substitutions at each site. The antibody variants thus produced are displayed as fusions of the gene III product of M13 packaged within each particle from filamentous phage particles in a monovalent manner. As disclosed herein, the biological activity (e.g., binding affinity) of the phage-displayed variants is then screened. In order to confirm the candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to confirm the hypervariable region residues that contribute significantly to antigen binding. Alternatively or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to confirm the contact points between the antibody and the CTHRC1 polypeptide. Such contact residues and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants is subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0242] Nucleic acid molecules encoding amino acid sequence variants of anti-CTHRC1 antibodies are prepared by various methods known in the art. These methods include, but are not limited to, separation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of variants or non-variant forms of anti-CTHRC1 antibodies prepared earlier.

[0243] b) Modification

[0244] Covalent modification of anti-CTHRC1 antibodies is included within the scope of the present invention. One type of covalent modification includes reacting targeted amino acid residues of the anti-CTHRC1 antibody with an organic derivatizing agent that is capable of reacting with selected side chains or N-terminal or C-terminal residues of the anti-CTHRC1 antibody. Derivatization with a bifunctional agent is useful, for example, for crosslinking anti-CTHRC1 antibodies to water-insoluble support matrices or surfaces for use in methods for purifying anti-CTHRC1 antibodies, and vice versa. Commonly used cross-linking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters (e.g., the ester with 4-azidosalicylic acid), homobifunctional imino esters (including disuccinimidyl esters such as 3,3'-dithiobis(succinimidyl propionate)), difunctional maleimides (such as bis-N-maleimido-1,8-octane), and agents such as methyl-3-[(p-azidophenyl)dithio]propionimidate.

[0245] Other modifications include deamidation of glutamine and asparagine residues to the corresponding glutamyl and asparagine residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0246] Another type of covalent modification of anti-CTHRC1 antibodies included within the scope of the present invention includes altering the native glycosylation pattern of the antibody or polypeptide. For the purposes of this article, "altering the native glycosylation pattern" means deleting one or more carbohydrate moieties present in the native sequence anti-CTHRC1 antibody (by removing potential glycosylation sites or by chemical and / or enzymatic means to delete glycosylation) and / or adding one or more glycosylation sites not present in the native sequence anti-CTHRC1 antibody. In addition, the phrase includes qualitative changes in the glycosylation of the native protein, involving changes in the nature and proportion of the various carbohydrate moieties present.

[0247] Glycosylation of antibodies and other polypeptides is usually N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid except proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0248] Adding glycosylation sites to the anti-CTHRC1 antibody is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above tripeptide sequences (for N-linked glycosylation sites). The sequence of the original anti-CTHRC1 antibody may also be altered by adding one or more serine or threonine residues to the sequence or replacing the sequence of the original anti-CTHRC1 antibody with one or more serine or threonine residues (for O-linked glycosylation sites). The anti-CTHRC1 antibody amino acid sequence may optionally be altered by changes at the DNA level, particularly by mutating the DNA encoding the anti-CTHRC1 antibody at a preselected base, so that codons are generated that will translate into the desired amino acids.

[0249] Another method of increasing the number of carbohydrate moieties on the anti-CTHRC1 antibody is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, for example, in WO 87 / 05330 published September 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0250] Removal of carbohydrate moieties present on anti-CTHRC1 antibodies can be accomplished chemically or enzymatically or by mutational substitution of codons encoding amino acid residues that serve as glycosylation targets. Chemical deglycosylation techniques are known in the art and are described, for example, by Hakimuddin et al., Arch. Biochem. Biophys., 259: 52 (1987) and by Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by using a variety of endo- and exo-glycosidases, as described by Thotakura et al., Meth. Enzymol., 138: 350 (1987).

[0251] c) Fc region variants

[0252] It may be desirable to modify the antibodies of the invention with respect to effector function, for example to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively or additionally, cysteine ​​residues may be introduced in the Fc region to allow interchain disulfide bond formation in this region. The homodimeric antibody thus produced may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (see Caron et al., J. Exp Med. 176: 1191-5 (1992); Shopes, BJ Immunol. 148: 2918-22 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53: 2560-5 (1993). Alternatively, the antibody may be engineered to have dual Fc regions, which may have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-30 (1989). In order to increase the serum half-life of an antibody, a salvage receptor binding epitope can be incorporated into an antibody (particularly an antibody fragment), for example as described in U.S. Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of an IgG molecule.

[0253] d) Cysteine ​​engineered antibody variants

[0254] In certain embodiments, it may be desirable to produce cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In specific embodiments, the substituted residues are present at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thus localized to accessible sites of the antibody and can be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates, as further described herein. Cysteine ​​engineered antibodies can be produced as described, e.g., in U.S. Patent No. 7,521,541.

[0255] e) Immunoconjugates

[0256] The subject matter disclosed herein also provides immunoconjugates, which include antibodies disclosed herein conjugated to one or more cytotoxic agents (such as chemotherapeutic agents or drugs), growth inhibitory agents, proteins, peptides, toxins (e.g., protein toxins, enzymatic toxins of bacterial, fungal, plant or animal origin or fragments thereof), or radioactive isotopes. For example, the antibodies of the disclosed subject matter can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other binding molecules (such as another antibody, antibody fragment, peptide or binding mimetic).

[0257] In certain embodiments, the immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody of the disclosure is conjugated to one or more drugs, including but not limited to maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent No. 0 425 235). Bl); auristatins, such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483 and 5,780,588 and 7,498,298); dolastatin; calicheamicin or its derivatives (see U.S. Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001 and 5,877,296; Hinman et al., Cancer Res. 53:3336-3342 (1993); and Lode et al., Cancer Res. 58:2925-2928 (1998)); anthracyclines, such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13:477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj. Chem. 16:717-721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Pat. No. 6,630,579); methotrexate; vindesine; taxanes, such as docetaxel, paclitaxel, larotaxel, tesetaxel, and octataxel; trichothecenes; and CC1065. In certain embodiments, the immunoconjugate includes an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, a non-binding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modicin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curculin, crotonin, saponin inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and tricyclic deoxyenol toxins.

[0258] In certain embodiments, the immunoconjugate includes an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes can be used to prepare radioconjugates. Non-limiting examples include At 211 ,Ac 225 , 1 131 , 1 125 , Y 90 、Re 186 、Re 188 、Sm 153 、Bi 212 , P 32 , Pb 212 When the radioconjugate is used for detection, it may include a radioactive atom for scintigraphic studies, such as tc99m or I 123 , or spin labels for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0259] Conjugates of antibody fragments and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to antibodies. See WO 94 / 1 1026. The linker can be a "cleavable linker" that promotes the release of cytotoxic drugs in cells. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al., Cancer Res. 52: 127-131 (1992); U.S. Pat. No. 5,208,020). Non-limiting examples of linkers are disclosed above. The immunoconjugates disclosed herein expressly encompass, but are not limited to, such conjugates prepared with cross-linking agents including, but not limited to, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinyl sulfone) benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0260] f) Antibody Fusions

[0261] The subject matter disclosed herein also encompasses antibody fusions. For example, the proteins can be linked together by chemical or genetic manipulation using methods known in the art. See, for example, Gillies et al., Proc. Nat'l Acad. Sci. USA 89: 1428-1432 (1992) and U.S. Pat. No. 5,650,150.

[0262] In one example, the present disclosure encompasses anti-CTHRC1 antibody-cytokine fusion proteins. In principle, the anti-CTHRC1 antibodies disclosed herein are fused to any cytokine using recombinant molecular biology techniques. In a preferred embodiment, the anti-CTHRC1 antibody is fused to IL-2 (Gillies, S., Protein Engineering, Design and Selection 26 (10): 561-569 (2013); Klein, C. et al., OncoImmunology 6: 3 (2017).

[0263] In another example, the present disclosure encompasses anti-CTHRC1 antibody-T cell engager fusion proteins. The anti-CTHRC1 antibody-T cell engager fusion proteins discussed herein include a fusion between an anti-CTHRC1 antibody and a ligand of a receptor expressed on a T cell. Examples of such ligands include, but are not limited to, CD40L, OX40L, 4-1BBL, CD80 / 86, ICOSL, etc. In an embodiment, the ligand is fused to the Fc portion of an anti-CTHRC1 antibody. In an embodiment, the ligand is fused to the C-terminus of the light chain of an anti-CTHRC1 antibody. Such methods are described about 4-1BBL (Dafne M. et al., Journal of Immunotherapy 38 (8): 714-722 (2008)), and similar methods can be used to produce other antibody-T cell engager fusion proteins.

[0264] B. Certain methods of preparing antibodies

[0265] 1. Screening for anti-CTHRC1 antibodies with desired properties

[0266] Techniques for producing antibodies that bind to CTHRC1 polypeptides have been described above. Antibodies with certain biological properties may be further selected as desired.

[0267] The growth inhibition of the anti-CTHRC1 antibodies of the present invention can be evaluated by methods known in the art, such as using cells expressing CTHRC1 polypeptides endogenously or after transfection with the CTHRC1 gene. For example, appropriate tumor cell lines and CTHRC1 transfected cells can be treated for several days (e.g., 2-7 days) with various concentrations of the anti-CTHRC1 monoclonal antibodies of the present invention, and stained with crystal violet or MTT or analyzed by some other colorimetric assays. Another method of measuring proliferation is by comparing the 3H-thymidine uptake of treated cells in the presence or absence of the anti-CTHRC1 antibodies of the present invention. After treatment, cells are harvested and the amount of radioactivity incorporated into the DNA is quantified in a scintillation counter. Appropriate positive controls include treating the selected cell line with a growth inhibitory antibody known to inhibit the growth of the cell line. In vivo growth inhibition of tumor cells can be determined in various ways known in the art. Tumor cells can be cells that overexpress and / or display CTHRC1 polypeptides. In one embodiment, the anti-CTHRC1 antibody will inhibit cell proliferation of tumor cells expressing CTHRC1 by about 25-100%, more preferably about 30-100%, and even more preferably about 50-100% or 70-100% in vitro or in vivo at an antibody concentration of about 0.5 to 30 μg / mL, compared to untreated tumor cells. Growth inhibition can be measured at an antibody concentration of about 0.5 to 30 μg / mL or about 0.5 nM to 200 nM in cell culture, wherein growth inhibition is determined 1-10 days after the tumor cells are exposed to the antibody. If the administration of the anti-CTHRC1 antibody at about 1 μg / kg to about 100 mg / kg body weight results in a decrease in tumor size or a decrease in tumor cell proliferation within about 5 days to 3 months, preferably about 5 to 30 days from the first administration of the antibody, the antibody is growth inhibitory in vivo.

[0268] In order to select anti-CTHRC1 antibodies that induce cell death, the loss of membrane integrity as indicated by, for example, propidium iodide (PI), trypan blue or 7AAD uptake can be assessed relative to a control. PI uptake assays can be performed in the absence of complement and immune effector cells. Tumor cells expressing CTHRC1 polypeptides are incubated with culture medium alone or culture medium containing appropriate anti-CTHRC1 antibodies (e.g., about 10 μg / mL). The cells are incubated for a period of 3 days. After each treatment, the cells are washed and aliquoted into 35 mm 12×75 tubes with filter caps (1 mL per tube, 3 tubes per treatment group) to remove cell clumps. The tubes then receive PI (10 μg / mL). Can be used Flow cytometry and The samples were analyzed using CellQuest software (Becton Dickinson).Those anti-CTHRC1 antibodies that induce statistically significant levels of cell death as determined by PI uptake can be selected as anti-CTHRC1 antibodies that induce cell death.

[0269] In order to screen antibodies that bind to epitopes on CTHRC1 polypeptides that bind to the antibodies of interest, conventional cross-blocking assays can be performed, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Harlow and David Lane (1988). This assay can be used to determine whether the test antibody binds to the same site or epitope as a known anti-CTHRC1 antibody. Alternatively or in addition, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutated, such as by alanine scanning, to confirm contact residues. Initially, the mutant antibody is tested for binding to a polyclonal antibody to ensure correct folding. In different methods, peptides corresponding to different regions of the CTHRC1 polypeptide can be used for competitive assays with test antibodies or with test antibodies and antibodies with characterized or known epitopes.

[0270] In addition, the function of the candidate antibodies can also be screened by one or more of the following: in vivo screening for inhibition of metastasis, inhibition of chemotaxis by in vitro methods (e.g., US2010 / 0061978, incorporated herein by reference in its entirety), inhibition of angiogenesis, inhibition of tumor growth, and reduction of tumor size.

[0271] 2. Certain library screening methods

[0272] The anti-CTHRC1 antibodies of the present invention can be prepared by screening antibodies with one or more desired activities using combinatorial libraries. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding properties. Such methods are generally described in Hoogenboom et al. (2001), Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ), and in certain embodiments, described in Lee et al. (2004) J. Mol. Biol. 340: 1073-93.

[0273] In principle, synthetic antibody clones are selected by screening phage libraries containing phages displaying various fragments of antibody variable regions (Fv) fused to phage coat proteins. Such phage libraries are panned by affinity chromatography for the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed onto the antigen, thereby separating them from non-binding clones in the library. The binding clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles. Phage clones of interest are selected by designing a suitable antigen screening program, followed by using Fv sequences from phage clones of interest and Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), Vol. 1-3, suitable constant region (Fc) sequences described to construct full-length anti-CTHRC1 antibody clones, thereby any anti-CTHRC1 antibody of the present invention can be obtained.

[0274] In certain embodiments, the antigen-binding domain of an antibody is formed by two variable (V) regions of about 110 amino acids, each from a light (VL) chain and a heavy (VH) chain, each of which presents three hypervariable loops (HVRs) or complementary determining regions (CDRs). The variable domains can be functionally displayed on phage as single-chain Fv (scFv) fragments (wherein VH and VL are covalently linked by a short flexible peptide) or as Fab fragments (wherein each of them is fused to a constant domain and interacts non-covalently), as described in Winter et al., Ann. Rev. Immunol., 12: 433-55 (1994). As used herein, phage clones encoding scFv and phage clones encoding Fab are collectively referred to as "Fv phage clones" or "Fv clones".

[0275] The VH and VL gene libraries can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, and then antigen binding clones can be searched in phage libraries, as described in Winter et al., Ann. Rev. Immunol., 12: 433-55 (1994). Libraries from immune sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-34 (1993), the original library can be cloned to provide human antibodies from a single source for a variety of non-self antigens and self antigens without any immunization. Finally, the original library can also be prepared by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and complete rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-8 (1992).

[0276] In certain embodiments, filamentous phage is used to display antibody fragment by merging with minor coat protein pill.Antibody fragment can be displayed as single-chain Fv fragment, wherein VH and VL domains are connected on the same polypeptide chain by flexible polypeptide spacer, for example, such as Marks et al., J.Mol.Biol., 222:581-97 (1991) described, or displayed as Fab fragment, wherein one chain merges with pill, another chain is secreted into bacterial host cell periplasm, Fab-coat protein structure is assembled therein, this structure is displayed on phage surface by displacing some wild-type coat proteins, for example, such as Hoogenboom et al., Nucl.Acids Res., 19:4133-7 (1991) described.

[0277] Typically, nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from humans or animals. If a library biased toward anti-CTHRC1 clones is needed, CTHRC1 is used to immunize the subject to produce an antibody response, and spleen cells and / or circulating B cells and other peripheral blood lymphocytes (PBL) are recovered for library construction. In some embodiments, anti-CTHRC1 antibody responses are produced in transgenic mice carrying functional human immunoglobulin gene arrays (and lacking a functional endogenous antibody production system), so that CTHRC1 immunization produces B cells that produce human antibodies for CTHRC1, to obtain a human antibody gene fragment library biased toward anti-CTHRC1 clones. The generation of transgenic mice producing human antibodies is described below.

[0278] Additional enrichment of anti-CTHRC1 reactive cell populations can be obtained by isolating B cells expressing CTHRC1 specific membrane-bound antibodies using appropriate screening procedures, such as by cell separation using CTHRC1 affinity chromatography or adsorption of cells to fluorescent dye-labeled CTHRC1 followed by flow activated cell sorting (FACS).

[0279] Alternatively, the use of splenocytes and / or B cells or other PBLs from unimmunized donors can better represent the possible antibody repertoire and also allow the construction of antibody libraries using any animal (human or non-human) species for which CTHRC1 is not antigenic. For libraries constructed by incorporating antibody genes in vitro, stem cells are harvested from the subject to provide nucleic acids encoding unrearranged antibody gene segments. The immune cells of interest can be obtained from a variety of animal species, such as humans, mice, rats, rabbits, wolves, canines, felines, pigs, cattle, horses, and bird species, etc.

[0280] Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered from the cells of interest and amplified. In the case of rearranged VH and VL gene libraries, genomic DNA or mRNA can be isolated from lymphocytes, followed by polymerase chain reaction (PCR) with primers matching the 5' and 3' ends of the rearranged VH and VL genes to obtain the desired DNA, as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86: 3833-7 (1989), thereby generating different V gene libraries for expression.

[0281] V genes can be amplified from cDNA and genomic DNA, with the reverse primer at the 5' end of the exon encoding the mature V domain and the forward primer based on the J-segment as described in Orlandi et al. (1989) and Ward et al., Nature, 341:544-6 (1989). However, for amplification from cDNA, the reverse primer can also be based on the leading exon as described in Jones et al., Biotechnol., 9:88-9 (1991), and the forward primer is in the constant region as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86:5728-32 (1989). In order to maximize complementarity, degeneracy can be incorporated into the primers as described in Orlandi et al., (1989) or Sastry et al., (1989). In certain embodiments, library diversity is maximized by using PCR primers targeted to each V gene family to amplify all available VH and VL arrangements present in the immune cell nucleic acid sample, for example, as described in the method of Marks et al., J. Mol. Biol., 222: 581-97 (1991), or as described in the method of Orum et al., Nucleic Acids Res., 21: 4491-98 (1993). In order to clone the amplified DNA into an expression vector, a rare restriction site can be introduced into the PCR primer as a tag at one end, as described in Orlandi et al., (1989), or by further PCR amplification with a tagged primer, as described in Clackson et al., Nature, 352: 624-628 (1991).

[0282] Synthetically rearranged V gene repertoires can be derived in vitro from V gene segments. Most human VH gene segments have been cloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227: 776-98 (1992)), and mapped (reported in Matsuda et al., Nature Genet., 3: 88-94 (1993)); these cloned segments (including all major conformations of the H1 and H2 loops) can be used to generate different VH gene repertoires encoding H3 loops of different sequences and lengths using PCR primers, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). VH repertoires in which all sequence diversity is concentrated in a single length of long H3 loop can also be prepared, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-61 (1992). Human Vκ and Vλ segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23: 1456-61 (1993)) and can be used to prepare synthetic light chain libraries. Synthetic V gene libraries based on a range of VH and VL folds and L3 and H3 lengths will encode antibodies with considerable structural diversity. After amplifying the DNA encoding the V genes, the germline V gene segments can be rearranged in vitro according to the method of Hoogenboom and Winter, J. Mol. Biol., 227: 381-8 (1992).

[0283] The library of antibody fragments can be constructed by combining VH and VL gene libraries in several ways. Each library can be produced in a different vector, and the vector is recombined in vitro, for example, as described in Hogrefe et al., Gene, 128: 119-26 (1993), or in vivo by combinatorial infection, such as Waterhouse et al., Nucl. Acids Res., 21: 2265-66 (1993) described in the loxP system. The in vivo recombination method uses the double-stranded nature of the Fab fragment to overcome the limitation of the E. coli transformation efficiency on the library size. The original VH and VL libraries are cloned separately, one cloned into a phagemid, and the other cloned into a phage vector. The two libraries are then combined by phage infection of bacteria containing phagemids, so that each cell contains different combinations, and the library size is only limited by the number of cells present (about 1012 clones). Both vectors contain in vivo recombination signals, so that the VH and VL genes are recombined into a single replicon and packaged together into phage virus particles. These huge libraries provide a large number of different antibodies with good affinity (Kd-1 is about 10-8 M).

[0284] Alternatively, the libraries can be cloned sequentially into the same vector, e.g., as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or assembled together by PCR and then cloned, e.g., as described in Clackson et al., Nature, 352:624-628 (1991). PCR assembly can also be used to link VH and VL DNAs with DNA encoding flexible peptide spacers to form single-chain Fv (scFv) libraries. In another technique, "in-cell PCR assembly" is used to combine VH and VL genes in lymphocytes by PCR and then clone the library of linked genes, e.g., as described in Embleton et al., Nucl. Acids Res., 20:3831-3837 (1992).

[0285] Antibodies generated by the original library (natural or synthetic) may have moderate affinity (Kd-1 of about 10 6 Up to 10 7 M-1), but affinity maturation can also be simulated in vitro by construction and reselection from a secondary library, as described in Winter et al. (1994), supra. For example, in the method of Hawkins et al., J. Mol. Biol., 226: 889-96 (1992), or in the method of Gram et al., Proc. Natl. Acad. Sci USA, 89: 3576-80 (1992), mutations can be randomly introduced in vitro by using an error-prone polymerase (reported in Leung et al., Technique, 1: 11-5 (1989)). In addition, affinity maturation can be performed by randomly mutating one or more CDRs, for example, by performing PCR with primers carrying random sequences spanning the CDRs of interest in selected individual Fv clones, and screening for clones with higher affinity. WO 9607754 describes a method for inducing mutagenesis in the complementary determining region of an immunoglobulin light chain to create a library of light chain genes. Another effective method is to recombinant the VH or VL domain selected by phage display with a naturally occurring V domain variant library obtained from an unimmunized donor and screen for higher affinity in several rounds of chain shuffling, as described in Marks et al., Biotechnol., 10:779-83 (1992). This technology allows the production of antibodies and antibody fragments with an affinity of about 10-9 M or less.

[0286] Screening of the library can be accomplished by various techniques known in the art. For example, CTHRC1 can be used to coat the wells of an adsorption plate, expressed on host cells fixed to an adsorption plate or used for cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or any other method for panning phage display libraries.

[0287] The phage library sample is contacted with the fixed CTHRC1 under conditions suitable for binding at least a portion of the phage particles to the adsorbent. Typically, conditions including pH, ionic strength, temperature, etc. are selected to simulate physiological conditions. The phage bound to the solid phase is washed and then eluted with acid, for example, as described in Barbas et al., Proc. Natl. Acad. Sci USA, 88: 7978-82 (1991), or eluted with alkali, for example, as described in Marks et al., J. Mol. Biol., 222: 581-97 (1991), or eluted by CTHRC1 antigen competition, for example, with a procedure similar to the antigen competition method of Clackson et al., Nature, 352: 624-8 (1991). Phage can be enriched 20 to 1,000 times in a single round of selection. In addition, the enriched phage can be grown in bacterial culture and subjected to more rounds of selection.

[0288] The efficiency of selection depends on many factors, including the dissociation kinetics during washing, and whether multiple antibody fragments on a single phage can engage with the antigen simultaneously. Antibodies with fast dissociation kinetics (and weak binding affinity) can be retained by utilizing short-time washing, multivalent phage display, and high coating density of antigen in solid phase. High density not only stabilizes phage by multivalent interactions, but also helps the phage that has dissociated to re-bind. Antibodies with slow dissociation kinetics (and good binding affinity) can be promoted by utilizing long-time washing and monovalent phage display (such as Bass et al., Proteins, 8:309-314 (1990) and described in WO 92 / 09690) and low coating density of antigen (such as Marks et al., Biotechnol., 10:779-783 (1992)).

[0289] It is possible to select between phage antibodies with different affinities (even slightly different affinities) for CTHRC1. However, random mutations of selected antibodies (e.g., as performed in some affinity maturation techniques) may produce many mutants, most of which bind to the antigen and a few with higher affinity. Since CTHRC1 is restricted, rare high-affinity phages may be eliminated by competition. In order to retain all higher-affinity mutants, phages can be incubated with excess biotinylated CTHRC1, but the molar concentration of biotinylated CTHRC1 is lower than the target molar affinity constant of CTHRC1. High-affinity binding phages can then be captured by streptavidin-coated paramagnetic beads. This "equilibrium capture" allows antibodies to be selected based on the affinity of the binding, and its sensitivity allows mutant clones with only twice the high affinity to be separated from a large excess of lower affinity phages. The conditions for washing the phages bound to the solid phase can also be controlled to distinguish based on dissociation kinetics.

[0290] Anti-CTHRC1 clones can be selected based on activity. In certain embodiments, the present invention provides anti-CTHRC1 antibodies that bind to living cells that naturally express CTHRC1. In one embodiment, the present invention provides anti-CTHRC1 antibodies that block the binding between a CTHRC1 ligand and CTHRC1, but do not block the binding between a CTHRC1 ligand and a second protein. Fv clones corresponding to such anti-CTHRC1 antibodies can be selected by the following methods: (1) isolating anti-CTHRC1 clones from a phage library as described above, and optionally amplifying the population by culturing the isolated phage clone population in a suitable bacterial host; (2) selecting CTHRC1 and a second protein that need blocking and non-blocking activity, respectively; (3) adsorbing anti-CTHRC1 phage clones to fixed CTHRC1; (4) using an excess of a second protein to elute any unwanted clones that recognize CTHRC1 binding determinants that overlap or share binding determinants with the second protein; and (5) eluting clones that are still adsorbed after step (4). Optionally, clones with desired blocking / non-blocking properties can be further enriched by repeating the selection procedures described herein one or more times.

[0291] DNA encoding the hybridoma-derived monoclonal antibodies or phage-displayed Fv clones of the present invention is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from hybridoma or phage DNA templates). Once isolated, the DNA can be placed into an expression vector, which is then transfected into a host cell, such as an E. coli cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell that does not otherwise produce immunoglobulins, to obtain the synthesis of the desired monoclonal antibody in the recombinant host cell. Review articles on recombinant expression of DNA encoding antibodies in bacteria include Skerra et al., Curr. Opinion in Immunol. 5: 256 (1993) and Pluckthun, Immunol. Rev. 130: 151 (1992).

[0292] The DNA encoding the Fv clones of the present invention can be combined with known DNA sequences encoding heavy and / or light chain constant regions (e.g., appropriate DNA sequences can be obtained from Kabat et al., supra) to form clones encoding full-length or partial-length heavy and / or light chains. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be obtained from any human or animal species. The definitions of "chimeric" and "hybrid" antibodies as used herein include variable domain DNA derived from one animal (such as human) species, which is then fused with constant region DNA from another animal species to form a "hybrid" full-length heavy chain and / or light chain coding sequence Fv clones. In certain embodiments, Fv clones derived from human variable DNA are fused with human constant region DNA to form full-length or partial-length human heavy and / or light chain coding sequences.

[0293] The DNA encoding the anti-CTHRC1 antibody derived from the hybridoma may also be modified, for example, by replacing the homologous mouse sequences derived from the hybridoma clone with the coding sequences of the human heavy and light chain constant domains (e.g., as in the method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851-5 (1984)). The DNA encoding the antibody or fragment derived from the hybridoma or Fv clone may be further modified by covalently linking all or part of the coding sequence of the non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this way, a "chimeric" or "hybrid" antibody having the binding specificity of the antibody derived from the Fv clone or hybridoma clone of the present invention is prepared.

[0294] 3. Using CAR T cells to produce antibodies

[0295] The anti-CTHRC1 antibody of the present invention can be prepared by screening antibodies with one or more desired activities using a CAR T cell platform. Chimeric antigen receptor (CAR) consists of an extracellular antigen recognition domain (usually a single-chain variable fragment (scFv) antibody) connected to a transmembrane and cytoplasmic signaling domain. Alvarez-Vallina, L, Curr Gene Ther 1: 385–97 (2001). CAR-mediated recognition converts tumor-associated antigens (TAA) expressed on the cell surface into recruitment points for effector functions, thereby achieving the goal of independent activation of the major histocompatibility complex of effector cells. The first generation of CAR is constructed by fusing the scFv-based TAA binding domain with a cytoplasmic signaling domain that is usually derived from a ζ chain of a T cell receptor (TCR) / CD3 complex or from a γ chain associated with some Fc receptors (Gross, G. et al., Proc Natl Acad Sci USA 86: 10024-8 (1989)). A second generation CAR (CARv2) was also developed, which comprises a signaling region of TCRζ in series with a signaling domain derived from T cell co-stimulatory receptors CD28, 4-1BB (CD137) or OX40 (CD134) (Sanz, L. et al., Trends Immunol 25: 85-91 (2004)). The third generation CAR further combines the signaling potential of two co-stimulatory domains (e.g., CD28 and 4-1BB) (Subklewe, M. et al., Transfus Med Hemother 46 (1): 15-24 (2019)).

[0296] When encountering antigens, the interaction of the gene-transferred CAR triggers effector functions and can mediate cytolysis of tumor cells. The practicality and effectiveness of the CAR approach have been demonstrated in a variety of animal models, and ongoing clinical trials use CAR-based genetically engineered T lymphocytes to treat cancer patients. Lipowska-Bhalla, G. et al., Cancer Immunol Immunother 61: 953-62 (2012). CAR enables effector cells to target any natural extracellular antigens in which suitable antibodies exist. Engineered cells can target not only proteins, but also structures such as carbohydrate and glycolipid tumor antigens (Mezzanzanica, D. et al., Cancer Gene Ther 5: 401-7 (1998); Kershaw, MH. et al., Nat Rev Immunol 5: 928-40 (2005)).

[0297] Current methods for producing recombinant antibodies are mainly based on the use of purified proteins. Hoogenboom, HR et al., Nat Biotechnol 23:1105–1116 (2005). However, a mammalian cell-based antibody display platform has recently been described that exploits the functional capacity of T lymphocytes. Alonso-Camino et al., Molecular Therapy Nucleic Acids (2013) 2, e93. Due to the surface expression of activation markers, the display of antibodies on the surface of T lymphocytes as part of CAR-mediated signaling can ideally link antigen-antibody interactions with distinct changes in cell phenotype. Alonso-Camino, V. et al., PLoS ONE 4:e7174 (2009). By using scFv-based CARs that recognize TAAs, it has been demonstrated that combining CAR-mediated activation with fluorescence-activated cell sorting (FACS) of CD69+ T cells makes it possible to isolate binders to surface TAAs, with an enrichment factor of at least 10 after two rounds. 3 fold, thereby generating a homogeneous population of T cells expressing TAA-specific CARs. Alonso-Camino, V et al., PLoS ONE 4: e7174 (2009).

[0298] C. Preparation of anti-CTHRC1 antibodies

[0299] The following description mainly relates to the production of anti-CTHRC1 antibodies by culturing cells transformed or transfected with a vector containing a nucleic acid encoding an anti-CTHRC1 antibody. Of course, it is contemplated that alternative methods well known in the art can be used to prepare anti-CTHRC1 antibodies. For example, the appropriate amino acid sequence or part thereof can be produced by direct peptide synthesis using solid phase technology (e.g., Stewart et al., Solid-Phase Peptide Synthesis, WH Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc., 85: 2149-2154 (1963)). In vitro protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be performed, for example, using an Applied Biosystems peptide synthesizer (Foster City, CA) according to the manufacturer's instructions. The various parts of the anti-CTHRC1 antibody can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-CTHRC1 antibody.

[0300] 1. Isolation of DNA encoding anti-CTHRC1 antibodies

[0301] The DNA encoding anti-CTHRC1 antibody can be obtained from a cDNA library prepared from a tissue believed to have anti-CTHRC1 antibody mRNA and express it at a detectable level. Therefore, human anti-CTHRC1 antibody DNA can be conveniently obtained from a cDNA library prepared from human tissue. The gene encoding anti-CTHRC1 antibody can also be obtained from a genomic library or by a known synthesis procedure (e.g., automated nucleic acid synthesis).

[0302] The library can be screened with probes designed to identify the gene of interest or the protein encoded therein (e.g., oligonucleotides of at least about 20-80 bases). Screening of cDNA or genomic libraries with selected probes can be performed using standard procedures, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative method for isolating genes encoding anti-CTHRC1 antibodies is to use PCR methods (Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[0303] Techniques for screening cDNA libraries are well known in the art. The oligonucleotide sequence selected as a probe should have sufficient length and be sufficiently clear to minimize false positives. Preferably, the oligonucleotide is labeled so that it can be detected when hybridizing with the DNA in the screened library. Labeling methods are well known in the art and include the use of radioactive labels (such as 32P-labeled ATP), biotinylation or enzyme labels. Sambrook et al., supra, provide hybridization conditions including medium stringency and high stringency.

[0304] Sequences identified in such library screening methods can be compared and aligned with other known sequences stored and obtained in public databases (such as GenBank) or other dedicated sequence databases. Sequence identity (at the amino acid or nucleotide level) within a defined region of a molecule or throughout the full-length sequence can be determined using methods known in the art and as described herein.

[0305] Nucleic acids having protein coding sequences can be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequences disclosed herein for the first time, and, if necessary, detecting mRNA precursors and processing intermediates that may not have been reverse transcribed into cDNA using conventional primer extension procedures as described in Sambrook et al., supra.

[0306] 2. Selection and transformation of host cells

[0307] Host cells are transfected or transformed with expression or cloning vectors described herein for producing anti-CTHRC1 antibodies and cultured in conventional nutrient media modified to be suitable for inducing promoters, selecting transformants or amplifying genes encoding desired sequences. Culture conditions, such as culture media, temperature, pH, etc., can be selected by technicians without excessive experimentation. In general, the principles, protocols, and practical techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: a Practical Approach, edited by M. Butler (IRL Press, 1991) and Sambrook et al., supra.

[0308] Methods for eukaryotic cell transfection and prokaryotic cell transformation are known to those of ordinary skill in the art, such as CaCl2, CaPO4, liposome-mediated, polyethylene glycol / DMSO and electroporation, wherein eukaryotic cell transfection and prokaryotic cell transformation mean introducing DNA into a host so that the DNA can replicate as an extrachromosomal or through a chromosomal integrant. Depending on the host cell used, standard techniques suitable for such cells are used for transformation. Calcium treatment or electroporation using calcium chloride as described in Sambrook et al., supra, is commonly used for prokaryotic cells. Infection with Agrobacterium tumefaciens is used to transform certain plant cells, such as Shaw et al., Gene, 23:315 (1983) and WO 89 / 05859 published on June 29, 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be used. General aspects of mammalian cell host system transfection have been described in U.S. Pat. No. 4,399,216. Transformation into yeast is generally based on the methods of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods of introducing DNA into cells may also be used, such as by nuclear microinjection, electroporation, fusion of bacterial protoplasts with intact cells or polycations, such as polybrene, polyornithine. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

[0309] Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryotes, yeast, or higher eukaryotic cells.

[0310] a. Prokaryotic host cells

[0311] Suitable prokaryotes include, but are not limited to, archaea and eubacteria, such as gram-negative or gram-positive organisms, e.g., Enterobacteriaceae, such as Escherichia coli. Various Escherichia coli strains are publicly available, such as K12 strains MM294 (ATCC 31,446), X1776 (ATCC 31,537), W3110 (ATCC 27,325), and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae, such as Escherichia, e.g., Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, and Bacilli, such as B. subtilis and B. licheniformis (e.g., DD 12, April 1989). 266,710), Pseudomonas, such as Pseudomonas aeruginosa, Rhizobia, Vitreoscilla, Paracoccus and Streptomyces. These examples are illustrative and not limiting. Escherichia coli strain W3110 is a particularly preferred host or parent host because it is a common host strain for fermentation of recombinant DNA products. Preferably, the host cell secretes a minimal amount of proteolytic enzymes.For example, strain W3110 (Bachmann, Cellular and Molecular Biology, Vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) can be modified to achieve gene mutations in genes encoding endogenous proteins of the host, examples of such hosts include E. coli W3110 strain 1A2, which has a complete genotype tonA; E. coli W3110 strain 9E4, which has a complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has a complete genotype tonA ptr3 phoA E15 (argF-lac) 169degP ompT kanr; E. coli W3110 strain 37D6, which has a complete genotype tonA ptr3 phoA E15 (argF-lac) 169degP ompT rbs7 ilvG kanr; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistance degP deletion mutation; E. coli W3110 strain 33D3 (U.S. Pat. No. 5,639,635) with the genotype W3110ΔfhuA(ΔtonA)ptr3 lac Iq lacL8ΔompTΔ(nmpc-fepE)degP41 kanR and E. coli strains with mutant periplasmic proteases disclosed in U.S. Pat. No. 4,946,783, issued on August 7, 1990. Other strains and their derivatives such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ1776 (ATCC 31,537) and E. coli RV308 (ATCC 31,608) are also suitable. These examples are illustrative and non-limiting. Methods for constructing derivatives of any of the above-mentioned bacteria with a determined genotype are known in the art and are described in, for example, Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to consider the reproducibility of the replicon in the bacterial cell to select the appropriate bacteria. For example, when using well-known plasmids such as pBR322, pBR325, pACYC177 or pKN410 to provide the replicon, Escherichia coli, Serratia or Salmonella species can be suitably used as hosts. Generally, the host cell should secrete a minimum amount of proteolytic enzymes, and it may be desirable to incorporate additional protease inhibitors into the cell culture. Alternatively, in vitro cloning methods such as PCR or other nucleic acid polymerase reactions are suitable.

[0312] Full-length antibodies, antibody fragments and antibody fusion proteins can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. Full-length antibodies have a longer half-life in circulation. Production in E. coli is faster and more cost-effective. Regarding the expression of antibody fragments and polypeptides in bacteria, see, for example, US5,648,237, US5,789,199 and US5,840,523, which describe translation initiation regions (TIR) ​​and signal sequences for optimizing expression and secretion, which are incorporated herein by reference. After expression, the antibody is separated from the E. coli cell paste in the form of a soluble fraction and can be purified by, for example, protein A or G columns according to the isotype. Final purification can be performed similarly to the process of purifying antibodies expressed, for example, in CHO cells.

[0313] b. Eukaryotic host cells

[0314] In addition to prokaryotes, eukaryotic microorganisms (such as filamentous fungi or yeast) are also suitable cloning or expression hosts for vectors encoding anti-CTHRC1 antibodies. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 (1981); EP 139,383 published May 2, 1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al., Bio / Technology, 9: 968-75 (1991)), such as Kluyveromyces lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2): 737-742 (1983)), Kluyveromyces fragilis (ATCC 12,424), Kluyveromyces bulgaricus (ATCC 12,424). 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., Bio / Technology, 8:135 (1990)), K. thermotolerans and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 (1988)); Candida; Trichoderma reesia (EP 402,226); 244, 234); Neurosporacrassa (Case et al., Proc. Natl. Acad. Sci.USA, 76: 5259-5263 (1979); Schwanniomyces, such as Schwanniomyces occidentalis (EP 394,538 published on October 31, 1990); and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium (WO published on January 10, 1991). 91 / 00357), and Aspergillus hosts, such as A. nidulans (Ballance et al., Biochem. Biophys. Res. Commun., 112: 284-289 (1983); Tilburn et al., Gene, 26: 205-221 (1983); Yelton et al., Proc. Natl. Acad. Sci. USA, 81: 1470-1474 (1984)) and A. niger (Kelly and Hynes, EMBO J., 4: 475-479 (1985)). Methylotrophic yeasts are suitable herein and include, but are not limited to, yeasts capable of growing on methanol selected from the genera consisting of Hansenula, Candida, Kluyveromyces, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

[0315] Suitable host cells for expressing glycosylated anti-CTHRC1 antibodies are derived from multicellular organisms. Examples of invertebrate cells include insect cells, such as fruit flies S2 and Spodoptera litura Sf9, and plant cells, such as cell cultures of cotton, corn, potato, soybean, petunia, tomato and tobacco. Many baculovirus strains and variants and corresponding licensed insect host cells from hosts such as fall armyworm (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and silkworm have been identified. A variety of viral strains for transfection are available to the public, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as viruses of the present invention, particularly for transfecting fall armyworm cells.

[0316] However, of greatest interest are vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2).

[0317] Host cells are transformed with the above-mentioned expression or cloning vectors for producing anti-CTHRC1 antibodies and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0318] 3. Selection and use of replicable vectors

[0319] In order to recombinantly produce the antibodies of the present invention, the nucleic acid encoding it (e.g., cDNA or genomic DNA) is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. The DNA encoding the antibody is easily separated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). Many vectors are available. The selection of the vector depends in part on the host cell used. Generally, preferred host cells are prokaryotic or eukaryotic (usually mammalian) origins.

[0320] The vector can be in the form of, for example, a plasmid, a cosmid, a viral particle or a bacteriophage. The appropriate nucleic acid sequence can be inserted into the vector by a variety of procedures. In general, the DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art. The vector components typically include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence. Construction of a suitable vector containing one or more of these components uses standard ligation techniques known to the technician.

[0321] Anti-CTHRC1 antibodies can not only be directly recombinantly produced, but also produced as fusion polypeptides with heterologous polypeptides, which can be signal sequences or other polypeptides with specific cleavage sites at the N-terminus of mature proteins or polypeptides. Generally, the signal sequence can be a component of the vector, or it can be a part of the DNA encoding the anti-CTHRC1 antibody inserted into the vector. The signal sequence can be a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, Ipp or heat-stable enterotoxin II leader sequences. For yeast secretion, the signal sequence can be, for example, a yeast invertase leader sequence, an alpha factor leader sequence (including Saccharomyces and Kluyveromyces alpha-factor leaders, the latter described in U.S. Patent No. 5,010,182) or an acid phosphatase leader sequence, a Candida albicans glucoamylase leader sequence (EP362,179 published on April 4, 1990) or a signal described in WO 90 / 13646 published on November 15, 1990. In mammalian cell expression, mammalian signal sequences can be used to direct secretion of the protein, as can signal sequences of secreted polypeptides from the same or related species and viral secretory leaders.

[0322] a. Prokaryotic host cells

[0323] The polynucleotide sequence encoding the polypeptide assembly of the antibody of the present invention can be obtained by standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from cells such as hybridoma cells that produce antibodies. Alternatively, a nucleotide synthesizer or PCR technology can be used to synthesize polynucleotides. Once obtained, the sequence encoding the polypeptide is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of the present invention. The selection of appropriate vectors will depend primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cell in which it is located.

[0324] Typically, plasmid vectors containing replicons and control sequences derived from species compatible with the host cell are used in combination with these hosts. Expression vectors and cloning vectors all contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells and marker sequences that can provide phenotypic selection in transformed cells. Such sequences are well known for a variety of bacteria, yeasts, and viruses. The origin of replication from plasmid pBR322 contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance, thereby providing a simple method for confirming transformed cells, which is suitable for most Gram-negative bacteria, 2μ plasmid origins are suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) can be used to clone vectors in mammalian cells. pBR322, its derivatives, or other microbial plasmids or phages may also contain or be modified to contain promoters that can be used by microorganisms to express endogenous proteins. Carter et al., U.S. Patent No. 5,648,237 describes in detail examples of pBR322 derivatives for expressing specific antibodies.

[0325] In addition, phage vectors containing replicons and control sequences compatible with the host microorganisms can be used as transformation vectors in conjunction with these hosts. For example, phage, such as λGEM TM -11 can be used to prepare a recombinant vector, which can be used to transform susceptible host cells, such as Escherichia coli LE392.

[0326] The expression vector of the present invention may comprise two or more promoter-cistron pairs, encoding each of the polypeptide components. A promoter is a non-translated regulatory sequence located upstream (5') of a cistron that regulates its expression. Prokaryotic promoters are generally divided into two categories, inducible and constitutive. An inducible promoter is a promoter that initiates an increase in the transcription level of a cistron under its control in response to a change in culture conditions (e.g., the presence or absence of a nutrient or a change in temperature).

[0327] A large number of promoters recognized by a variety of potential host cells are well known. Remove the promoter from the source DNA via restriction enzyme digestion and insert the separated promoter sequence into the carrier of the present invention, thus the selected promoter can be operably connected to the cistron DNA encoding light chain or heavy chain. Natural promoter sequences and many heterologous promoters can be used to instruct the amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because compared with the natural target polypeptide promoter, they usually allow the target gene transcription of expression to be more and the yield is higher.

[0328] Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include PhoA promoter, β-galactosidase and lactose promoter systems (Chang et al., Nature, 275: 615 (1978); Goeddel et al., Nature, 281: 544 (1979)), alkaline phosphatase, tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8: 4057 (1980); EP 36,776) and hybrid promoters such as tac (deBoer et al., Proc. Natl. Acad. Sci. USA, 80: 21-25 (1983)) or trc promoter. Promoters for bacterial systems will also contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the anti-CTHRC1 antibody. However, other promoters that function in bacteria (such as other known bacterial or phage promoters) are also suitable. Their nucleotide sequences have been published, allowing the skilled artisan to operably link them to the cistrons encoding the target light and heavy chains using linkers or adapters (Siebenlist et al., (1980) Cell 20:269) to provide any desired restriction sites.

[0329] In one aspect of the invention, each cistron in the recombinant vector comprises a secretory signal sequence component for instructing the transmembrane translocation of the expressed polypeptide. In general, the signal sequence can be a component of the vector, or it can be a part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purpose of the present invention should be a signal sequence recognized and processed (i.e., cut by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the natural signal sequence for heterologous polypeptides, the signal sequence is replaced by the prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, Ipp or heat-stable enterotoxin II (STII) leader sequence, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequence used in the two cistrons of the expression system is an STII signal sequence or a variant thereof.

[0330] On the other hand, the production of immunoglobulins according to the present invention can occur in the cytoplasm of the host cell, and therefore does not require the presence of a secretion signal sequence in each cistron. In this regard, immunoglobulin light and heavy chains are expressed, folded and assembled in the cytoplasm to form functional immunoglobulins. Certain host strains (e.g., E. coli trxB-strains) provide cytoplasmic conditions that are conducive to disulfide bond formation, thereby allowing the correct folding and assembly of expressed protein subunits. Proba and Pluckthun Gene, 159: 203 (1995).

[0331] The present invention provides an expression system in which the quantitative ratio of expressed polypeptide components can be adjusted to maximize the yield of secreted and correctly assembled antibodies of the present invention. This regulation is at least partially achieved by simultaneously adjusting the translation strength of the polypeptide components.

[0332] Simmons et al., U.S. Pat. No. 5,840,523 discloses a technique for regulating translation strength. It utilizes variants of the translation initiation region (TIR) ​​within a cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants with a range of translation strengths can be generated, thereby providing a convenient method to adjust this factor to obtain the desired expression level of a particular chain. TIR variants can be generated by conventional mutagenesis techniques, which result in codon changes that can change the amino acid sequence, although silent changes in the nucleotide sequence are preferred. Changes in TIR can include, for example, changes in the number or spacing of Shine-Dalgarno sequences and changes in signal sequences. One method for generating mutant signal sequences is to generate a "codon library" at the beginning of the coding sequence that does not change the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be achieved by changing the third nucleotide position of each codon; in addition, some amino acids such as leucine, serine, and arginine have multiple first and second positions, which may increase the complexity of preparing the library. This mutagenesis method is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4: 151-158.

[0333] Preferably, a set of vectors with a range of TIR strengths is generated for each cistron therein. This limited set provides a comparison of the expression level of each chain and the yield of the desired antibody product at various TIR strength combinations. As detailed in Simmons et al., U.S. Pat. No. 5,840,523, TIR strength can be determined by quantifying the expression level of a reporter gene. Based on the translation strength comparison, the desired individual TIRs are selected for combination in the expression vector constructs of the present invention.

[0334] b. Eukaryotic host cells

[0335] Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0336] (1) Signal sequence components

[0337] Vectors for eukaryotic host cells may also contain a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders (e.g., herpes simplex gD signal) are available.

[0338] DNA for such precursor regions is ligated in reading frame to DNA encoding the antibody.

[0339] (2) Copy starting point

[0340] Typically, mammalian expression vectors do not require an origin of replication component. For example, the SV40 origin can often be used simply because it contains an early promoter.

[0341] (3) Selecting gene components

[0342] Expression vectors and cloning vectors usually contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins such as ampicillin, neomycin, methotrexate or tetracycline, (b) complement auxotrophic deficiencies, or (c) provide critical nutrients that cannot be obtained from complex culture media, such as the gene encoding D-alanine racemase of Bacillus.

[0343] An example of a selection scheme utilizes a drug to inhibit the growth of host cells. Those cells that are successfully transformed with the heterologous gene produce a protein that confers drug resistance and therefore survive the selection scheme. Examples of this type of dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0344] Examples of suitable selectable markers for mammalian cells are those capable of identifying cells that have the ability to take up nucleic acids encoding anti-CTHRC1 antibodies, such as DHFR or thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc. When wild-type DHFR is used, a suitable host cell is a CHO cell line deficient in DHFR activity (e.g., ATCC CRL-9096), which is prepared and propagated as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77: 4216 (1980). For example, cells transformed with a DHFR selection gene are first identified by culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Alternatively, host cells (particularly wild-type hosts containing endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, a wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH), can be selected by growing the cells in a medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

[0345] A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)). The trp1 gene provides a selection marker for yeast mutants lacking the ability to grow in tryptophan, such as ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)).

[0346] (4) Promoter Component

[0347] Expression vectors and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding the anti-CTHRC1 antibody to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known.

[0348] Almost all eukaryotic genes have the AT-rich zone that is positioned at about 25 to 30 bases upstream of the transcription start site.Another sequence that exists at 70-80 bases upstream of the transcription start site of many genes is the CNCAAT zone, in which N can be any Nucleotide.At the 3 ' end of most eukaryotic genes is the AATAAA sequence, which may be the signal that the poly-A tail is added to the 3 ' end of the encoding sequence.All these sequences are suitably inserted into the eukaryotic expression vector.

[0349] Examples of suitable promoter sequences for use with yeast hosts include the promoter for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0350] Other yeast promoters that are inducible promoters with the additional advantage that transcription is controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothioneins, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for yeast expression are further described in EP 73,657.

[0351] Transcription of the anti-CTHRC1 antibody from a vector in a mammalian host cell is controlled, for example, by a promoter derived from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published on July 5, 1989), adenovirus (e.g., adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40), from a heterologous mammalian promoter (e.g., an actin promoter or an immunoglobulin promoter), and from a heat shock promoter, as long as such promoter is compatible with the host cell systems.

[0352] The early and late promoters of the SV40 virus are conveniently available in the form of SV40 restriction fragments that also contain the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently available in the form of a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. Modifications to this system are described in U.S. Patent No. 4,601,978. Regarding the expression of human beta-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus, see also Reyes et al., Nature 297:598-601 (1982). Alternatively, the Rous sarcoma virus long terminal repeat sequence can be used as a promoter.

[0353] (5) Enhancer element assembly

[0354] The transcription of the DNA encoding anti-CTHRC1 antibodies by higher eukaryotic organisms can be increased by inserting an enhancer sequence into a vector. An enhancer is a cis-acting element of DNA, usually about 10 to 300 bp, that acts on a promoter to increase its transcription. Many enhancer sequences are now known to come from mammalian genes (globulins, elastase, albumin, alpha-fetoprotein, and insulin). However, enhancers from eukaryotic cell viruses are usually used. Examples include the SV40 enhancer (bp 100-270) on the late side of the replication origin, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and the adenovirus enhancer. See also Yaniv, Nature 297: 17-18 (1982) for enhancing elements that activate eukaryotic promoters. The enhancer can be spliced ​​into the vector at the position 5' or 3' of the anti-CTHRC1 antibody coding sequence, but is preferably located at the 5' site of the promoter.

[0355] (6) Transcription termination component

[0356] Expression vectors for eukaryotic host cells (yeast, fungi, insects, plants, animals, humans or nucleated cells from other multicellular organisms) also contain sequences necessary for terminating transcription and stabilizing mRNA. Such sequences are usually available from the 5' untranslated region of eukaryotic or viral DNA or cDNA, and sometimes from the 3' untranslated region. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-CTHRC1 antibody. A useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vectors disclosed therein.

[0357] Other methods, vectors, and host cells suitable for synthesizing anti-CTHRC1 antibodies in recombinant vertebrate cell culture are described in Gething et al., Nature, 293:620-625 (1981); Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

[0358] 4. Cultivation of host cells

[0359] Host cells used to produce the anti-CTHRC1 antibodies of the present invention can be cultured in a variety of culture media.

[0360] a. Prokaryotic host cells

[0361] Prokaryotic cells for producing the polypeptides of the present invention are grown in a culture medium known in the art and suitable for culturing the selected host cells. Examples of suitable culture medium include luria broth (LB) plus necessary nutritional supplements. In some embodiments, the culture medium also contains a selection agent selected based on the construction of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the culture medium for the growth of cells expressing an ampicillin resistance gene.

[0362] In addition to the carbon source, nitrogen source and inorganic phosphate source, any necessary supplements may also be included at appropriate concentrations, introduced alone or as a mixture with another supplement or medium (such as a complex nitrogen source). Optionally, the medium may contain one or more reducing agents selected from glutathione, cysteine, cystamine, thioglycolate, dithioerythritol and dithiothreitol.

[0363] Prokaryotic host cells are cultured at a suitable temperature. For example, for E. coli growth, the preferred temperature range is about 20°C to about 39°C, more preferably about 25°C to about 37°C, and even more preferably about 30°C. The pH of the culture medium can be any pH in the range of about 5 to about 9, depending primarily on the host organism. For E. coli, the pH is preferably about 6.8 to about 7.4, more preferably about 7.0.

[0364] If an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for promoter activation. In one aspect of the present invention, the PhoA promoter is used to control transcription of the polypeptide. Therefore, the transformed host cells are cultured in a phosphate-limited medium for induction. In some embodiments, the phosphate-limited medium is CRAP medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263: 133-47). As is known in the art, a variety of other inducers may be used depending on the vector construct employed.

[0365] In one embodiment, the expressed polypeptide of the present invention is secreted into the periplasm of the host cell and recovered therefrom. Protein recovery generally involves destroying the microorganism, generally by means such as osmotic shock, sonication or dissolution. Once the cells are destroyed, cell debris or whole cells can be removed by centrifugation or filtration. For example, the protein can be further purified by affinity resin chromatography. Alternatively, the protein can be transported to the culture medium and separated therein. Cells can be taken out from the culture, filtered and the culture supernatant is concentrated to further purify the protein produced. The polypeptide expressed can be further separated and confirmed by commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blotting.

[0366] In one aspect of the invention, antibody production is carried out in a large number by fermentation process. Various large-scale fed-batch fermentation procedures can be used for the production of recombinant proteins. Large-scale fermentation has a capacity of at least 1000 liters, preferably a capacity of about 1,000 to 100,000 liters. These fermentation tanks use a stirring impeller to distribute oxygen and nutrients, particularly glucose (preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in a fermentation tank that is no more than about 100 liters in volume capacity, and can be in the range of about 1 liter to about 100 liters.

[0367] In the fermentation process, the induction of protein expression usually begins after the cells are grown to a desired density (e.g., an OD550 of about 180-220) under suitable conditions, at which stage the cells are in an early stationary phase. As known in the art and described above, a variety of inducers can be used, depending on the vector construct used. The cells can be grown for a shorter period of time before induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction times can be used.

[0368] In order to improve the yield and quality of the polypeptide of the present invention, various fermentation conditions can be changed. For example, in order to improve the correct assembly and folding of secreted antibody polypeptides, other vectors that overexpress chaperone proteins such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (peptidyl prolyl cis- and trans-isomerases with chaperone activity) can be used to co-transform host prokaryotic cells. It has been shown that chaperone proteins promote the correct folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274:19601-5; U.S. Pat. No. 6,083,715; U.S. Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-5; Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-13; Arie et al. (2001) Mol. Microbiol. 39:199-210.

[0369] In order to minimize proteolysis of expressed heterologous proteins (particularly those that are proteolytically sensitive), the present invention can use certain host strains that lack proteolytic enzymes. For example, the host cell strain can be modified to achieve genetic mutations in genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI, and combinations thereof. Some E. coli protease-deficient strains are available and are described, for example, in Joly et al. (1998), supra; U.S. Pat. No. 5,264,365; U.S. Pat. No. 5,508,192; Hara et al., Microbial Drug Resistance, 2: 63-72 (1996).

[0370] In one embodiment, an E. coli strain lacking proteolytic enzymes and transformed with a plasmid overexpressing one or more chaperone proteins is used as a host cell in the expression system of the present invention.

[0371] b. Eukaryotic host cells

[0372] Commercially available culture media, such as Ham's F10 (Sigma), Minimum Essential Medium (MEM) (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle Medium (DMEM, Sigma), are suitable for culturing host cells. In addition, any of the culture media described in Ham et al., Meth. Enz. 58: 44 (1979); Barnes et al., Anal. Biochem. 102: 255 (1980); U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469; WO 90 / 03430, WO 87 / 00195; or U.S. Pat. No. 30,985 can be used as a culture medium for host cells. Any of these media may be supplemented as needed with hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN TM Drugs), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. Culture conditions such as temperature, pH, etc. are those previously employed by the host cell selected for expression and will be apparent to the skilled artisan.

[0373] 5. Detection of gene amplification / expression

[0374] For example, by conventional Southern blotting, Northern blotting for quantifying transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77: 5201-5 (1980)), dot blotting (DNA analysis) or in situ hybridization, using appropriately labeled probes, based on the sequences provided herein, gene amplification and / or expression in the sample can be directly measured. Alternatively, antibodies capable of recognizing specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes, can be used. The antibody can be labeled and can be measured in the case of duplex binding to the surface, so that when a duplex is formed on the surface, the presence of the antibody bound to the duplex can be detected.

[0375] Alternatively, gene expression can be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assays of cell cultures or body fluids to directly quantify the expression of gene products. Antibodies that can be used for immunohistochemical staining and / or sample fluid assays can be monoclonal or polyclonal and can be prepared in any mammal. Conveniently, antibodies can be prepared against native sequence CTHRC1 polypeptides or against synthetic peptides based on the DNA sequences provided herein or against exogenous sequences fused to CTHRC1 DNA and encoding specific antibody epitopes.

[0376] 6. Purification of anti-CTHRC1 antibodies

[0377] The anti-CTHRC1 antibody can be recovered from the culture medium or host cell lysate. If it is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells used to express anti-CTHRC1 antibodies can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents.

[0378] It may be necessary to purify the anti-CTHRC1 antibody from a recombinant cell protein or polypeptide. The following procedures are examples of suitable purification procedures: by fractionation on an ion exchange column; ethanol precipitation; reverse phase HPLC; silica gel or cation exchange resin such as DEAE chromatography; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A sepharose columns to remove contaminants such as IgG; and metal chelate columns to bind anti-CTHRC1 antibodies in epitope-tagged form. Various protein purification methods can be used, and such methods are known in the art and described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step selected will depend, for example, on the nature of the production method employed and the specific anti-CTHRC1 antibody produced.

[0379] When adopting recombinant technology, antibody can be produced in intracellular, periplasmic space, or directly secreted into culture medium.If antibody is produced in cell, as the first step, particle debris is removed, whether it is host cell or dissolved fragment, for example, by centrifugation or ultrafiltration.Carter et al., Bio / Technology 10:163-7 (1992) describes the procedure for separating the antibody secreted into the periplasmic space of Escherichia coli.In brief, cell paste is thawed more than about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA and phenylmethylsulfonyl fluoride (PMSF).Cell debris can be removed by centrifugation.In the case where antibody is secreted into culture medium, usually first use commercially available protein concentration filter (such as Amicon or MilliporePellicon ultrafiltration unit) to concentrate the supernatant from this type of expression system.Protease inhibitors can be included in any of the aforementioned steps, such as PMSF, to inhibit proteolysis, and antibiotics can be included to prevent the growth of foreign contaminants.

[0380] Antibody compositions prepared by cells can be purified by, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis and affinity chromatography, wherein affinity chromatography is a preferred purification technique. The suitability of protein A as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2 or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is usually agarose, but other matrices are also available. Mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene allow for faster flow rates and shorter processing times than with agarose. In the case where the antibody comprises a CH3 domain, Bakerbond ABX TM Resins (JT Baker, Phillipsburg, NJ) can be used for purification. Depending on the antibody to be recovered, other protein purification techniques are also available, such as ion exchange column fractionation, ethanol precipitation, reverse phase HPLC, silica gel chromatography, heparin SEPHAROSE TM Chromatography, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE and ammonium sulfate precipitation.

[0381] Following any preliminary purification steps, the mixture comprising the antibody of interest and contaminants can be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5 and typically at a low salt concentration (e.g., about 0-0.25 M salt).

[0382] D. Determination

[0383] The antibodies of the present invention can be used in any known assay method, such as ELISA, competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola, (1987) Monoclonal Antibodies: A Manual of Techniques, pp. 147-158, CRC Press, Inc.).

[0384] Detection labels can be used to locate, visualize and quantify binding or recognition events. The labeled antibodies of the present invention can detect cell surface receptors. Another use of detectably labeled antibodies is a bead-based immunocapture method, including conjugating beads to fluorescently labeled antibodies and detecting fluorescent signals when ligands bind. Similar binding detection methods utilize surface plasmon resonance (SPR) effects to measure and detect antibody-antigen interactions.

[0385] Detection labels (such as fluorescent dyes and chemiluminescent dyes) (Briggs et al. (1997) J. Chem. Soc., Perkin-Trans. 1: 1051-8) provide a detectable signal and are generally suitable for labeling antibodies, preferably having the following properties: (i) the labeled antibody should produce a very high signal with low background so that small amounts of the antibody can be sensitively detected in cell-free and cell-based assays; and (ii) the labeled antibody should be photostable so that the fluorescent signal can be observed, monitored and recorded without significant photobleaching. For applications involving cell surface binding of the labeled antibody to membranes or cell surfaces (particularly living cells), the label preferably (iii) has good water solubility to achieve effective conjugate concentration and detection sensitivity, and (iv) is nontoxic to living cells so as not to disrupt the normal metabolic processes of the cells or cause premature cell death.

[0386] Direct quantification of cellular fluorescence intensity and counting of fluorescent labeling events (e.g., cell surface binding of peptide-dye conjugates) can be performed in systems that automate the mixing and reading of non-radioactive assays performed with live cells or beads ( 8100HTS System, Applied Biosystems, Foster City, Calif.) (Miraglia, "Homogeneous cell-and bead-based assays for high throughput screening using fluorometric microvolume assay technology", (1999) J. of Biomolecular Screening 4: 193-204). Uses of labeled antibodies also include cell surface receptor binding assays, immunocapture assays, fluorescence-linked immunosorbent assays (FLISA), caspase cleavage (Zheng, "Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis invivo", (1998) Proc. Natl. Acad. Sci. USA 95: 618-23; US 6372907), apoptosis (Vermes, "A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V" (1995) J. Immunol. Methods 184: 39-51) and cytotoxicity assays. Fluorometric microvolume assay technology can be used to confirm upregulation or downregulation of molecules targeted to the cell surface (Swartzman, "A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolumeassay technology", (1999) Anal. Biochem. 271: 143-51).

[0387] The labeled antibodies of the present invention can be used as imaging biomarkers and probes by various methods and techniques of biomedical and molecular imaging, such as: (i) MRI (magnetic resonance imaging); (ii) MicroCT (computed tomography); (iii) SPECT (single photon emission computed tomography); (iv) PET (positron emission tomography) Chen et al. Bioconjugate Chem. 15: 41-9 (2004); (v) bioluminescence; (vi) fluorescence; and (vii) ultrasound. Immunoscintigraphy is an imaging procedure in which antibodies labeled with radioactive substances are administered to animals or human patients, and photographs are taken of the sites in the body where the antibodies are localized (US 6528624). Imaging biomarkers can be objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions.

[0388] Peptide labeling methods are well known (e.g., Haugland, 2003, Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, Molecular Probes, Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, (1997) Non-Radioactive Labelling: A Practical Approach, Academic Press, London; Means (1990) Bioconjugate Chem. 1:2; Glazer et al. (1975) Chemical Modification of Proteins. Laboratory Techniques in Biochemistry and Molecular Biology (TS Work and E. Work, eds.) American Elsevier Publishing Co., New York; Lundblad, RL and Noyes, CM (1984) Chemical Reagents for Protein Modification, Vols. I and II, CRC Press, New York, NY). De Leon-Rodriguez et al. (2004) Chem. Eur. J. 10: 1149-1155; Lewis et al. (2001) Bioconjugate Chem. 12: 320-324; Li et al. (2002) Bioconjugate Chem. 13: 110-115; Mier et al. (2005) Bioconjugate Chem. 16:240-237).

[0389] Peptides and proteins labeled with two parts (fluorescent reporter molecules and quenchers) undergo fluorescence resonance energy transfer (FRET) when they are close enough. The reporter group is usually a fluorescent dye, which is excited by light of a certain wavelength and transfers energy to an acceptor or quencher group, wherein there is an appropriate Stokes shift to emit at maximum brightness. Fluorescent dyes include molecules with extended aromaticity, such as fluorescein and rhodamine and their derivatives. The fluorescent reporter molecule can be partially or significantly quenched by the quencher part in the intact peptide. After the peptide is cleaved by a peptidase or protease, a detectable increase in fluorescence can be measured (Knight, C. (1995) "Fluorimetric Assays of Proteolytic Enzymes", Methods in Enzymology, Academic Press, 248: 18-34).

[0390] The labeled antibody of the present invention can also be used as an affinity purification agent. In this process, the labeled antibody is fixed on a solid phase such as a Sephadex resin or filter paper using methods well known in the art. The fixed antibody is contacted with a sample containing the antigen to be purified, and thereafter the support is washed with a suitable solvent, which will substantially remove all substances in the sample except the antigen to be purified, and the antigen to be purified is combined with the fixed polypeptide variant. Finally, the support is washed with another suitable solvent such as a glycine buffer (pH 5.0), which will release the antigen from the polypeptide variant.

[0391] 1. Activity assay

[0392] In one aspect, assays are provided for identifying anti-CTHRC1 antibodies that have biological activity. Biological activity can include, for example, the ability to inhibit cell growth or proliferation (e.g., "cell killing" activity) or the ability to induce cell death, including programmed cell death (apoptosis). Antibodies with such biological activity in vivo and / or in vitro are also provided.

[0393] In certain embodiments, the ability of anti-CTHRC1 antibodies to inhibit cell growth or proliferation in vitro is tested. Assays for inhibiting cell growth or proliferation are well known in the art. Certain cell proliferation assays exemplified by the "cell killing" assay described herein measure cell viability. One such assay is the CellTiter-GloTM luminescent cell viability assay, which can be obtained commercially from Promega (Madison, WI). The assay is based on the quantification of the ATP present (which is an indicator of metabolically active cells) to determine the number of viable cells in culture. See Crouch et al. (1993) J. Immunol. Meth. 160: 81-8, U.S. Patent No. 6602677. The assay can be performed in a 96-well or 384-well format, making it suitable for automated high-throughput screening (HTS) (see Cree et al. (1995) AntiCancer Drugs 6: 398-404). The assay procedure involves the addition of a single reagent ( The luciferase activity is measured by adding a luminescent reagent (e.g., a luciferase reagent) directly to the cultured cells. This results in cell lysis and the generation of a luminescent signal generated by the luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in the culture. Data can be recorded by a luminometer or CCD camera imaging device. Luminescent output is expressed in relative light units (RLU).

[0394] Another cell proliferation assay is the "MTT" assay, a colorimetric assay that measures the oxidation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by mitochondrial reductase. Similar to the CellTiter-Glo™ assay, this assay indicates the number of metabolically active cells present in a cell culture (see, e.g., Mosmann (1983) J. Immunol. Meth. 65:55-63, and Zhang et al. (2005) Cancer Res. 65:3877-82).

[0395] In one aspect, the ability of anti-CTHRC1 antibodies to induce cell death in vitro is tested. Assays for inducing cell death are well known in the art. In some embodiments, such assays measure, for example, the loss of membrane integrity, as indicated by the uptake of propidium iodide (PI), trypan blue (see Moore et al. Cytotechnology, 17: 1-11 (1995)) or 7AAD. In an exemplary PI uptake assay, cells are cultured in Dulbecco's modified Eagle's medium (D-MEM): Ham's F-12 (50: 50) supplemented with 10% heat-inactivated FBS (Hyclone) and 2mM L-glutamine. Therefore, the assay is performed in the absence of complement and immune effector cells. Cells are seeded in 100x 20mm culture dishes at a density of 3x 106 per dish and allowed to attach overnight. The culture medium is removed and replaced with a separate fresh culture medium or a culture medium containing various concentrations of antibodies. The cells are incubated for a period of 3 days. After treatment, the monolayer is washed with PBS and detached by trypsin digestion. The cells were then centrifuged at 1200 rpm for 5 minutes at 4°C, the pellet was resuspended in 3 mL of cold Ca2+ binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2), and aliquoted into 12 × 75 mm tubes with 35 mm filter caps (1 mL per tube, 3 tubes per treatment group) to remove cell clumps. The tubes then received PI (10 μg / mL). Using FACSCAN TM Flow Cytometry and FACSCONVERT TM Samples were analyzed using CellQuest software (Becton Dickinson). Antibodies that induced statistically significant levels of cell death as determined by PI uptake were thus identified.

[0396] In one aspect, the ability of anti-CTHRC1 antibodies to induce apoptosis (programmed cell death) in vitro is tested. An exemplary assay for antibodies that induce apoptosis is an annexin binding assay. In an exemplary annexin binding assay, cells are cultured and inoculated in a culture dish as discussed in the previous paragraph. The culture medium is removed and replaced with a separate fresh culture medium or a culture medium containing 0.001 to 10 μg / mL of the antibody. After a three-day incubation period, the monolayer is washed with PBS and detached by trypsin digestion. The cells are then centrifuged, resuspended in a Ca2+ binding buffer, and aliquoted into a test tube as discussed in the previous paragraph. The tube then receives labeled annexin (e.g., annexin V-FITC) (1 μg / mL). Using FACSCAN TM Flow Cytometry and FACSCONVERT TMCellQuest software (BD Biosciences) was used to analyze the samples. Antibodies that induced statistically significant levels of annexin binding relative to controls were thus identified. Another exemplary assay for antibodies that induce apoptosis is a histone DNA ELISA colorimetric assay for detecting internucleosomal degradation of genomic DNA. Such an assay can be performed using, for example, a cell death detection ELISA kit (Roche, Palo Alto, CA).

[0397] Cells used for any of the above in vitro assays include cells or cell lines that naturally express CTHRC1 or have been engineered to express CTHRC1. Such cells include tumor cells that overexpress CTHRC1 relative to normal cells of the same tissue origin. Such cells also include cell lines (including tumor cell lines) that express CTHRC1 and cell lines that do not normally express CTHRC1 but have been transfected with nucleic acids encoding CTHRC1.

[0398] In one aspect, the ability of its anti-CTHRC1 antibody to inhibit cell growth or proliferation in vivo is tested. In certain embodiments, the ability of its anti-CTHRC1 antibody to inhibit tumor growth in vivo is tested. This test can use an in vivo model system, such as a xenograft model. In an exemplary xenograft system, human tumor cells are introduced into a non-human animal (e.g., a SCID mouse) that is appropriately immunocompromised. The antibody of the present invention is administered to the animal. The ability of the antibody to inhibit or reduce tumor growth is measured. In certain embodiments of the above-mentioned xenograft system, human tumor cells are tumor cells from human patients. In certain embodiments, human tumor cells are introduced into a non-human animal that is appropriately immunocompromised by subcutaneous injection or by transplantation to a suitable site (e.g., mammary fat pad).

[0399] 2. Binding and other assays

[0400] In one aspect, the antigen binding activity of the anti-CTHRC1 antibody is tested. For example, in certain embodiments, the ability of the anti-CTHRC1 antibody to bind to CTHRC1 expressed on the surface of a cell is tested. FACS assays can be used for such testing.

[0401] In one aspect, competition assays can be used to identify monoclonal antibodies that compete with the variable domains of any one of SEQ ID NOs: 1-10 or chimeric antibodies comprising the variable domains of monoclonal antibodies containing sequences of Tables 3 and 4 and the constant domains from IgG1 or IgG4 for binding to CTHRC1. In certain embodiments, such competing antibodies bind to the same epitope (e.g., linear or conformational epitopes) that is bound by a monoclonal antibody comprising the variable domains of any one of SEQ ID NOs: 1-10 or a chimeric antibody comprising the variable domains of monoclonal antibodies containing sequences of Tables 3 and 4 and the constant domains from IgG1 or IgG4. Exemplary competition assays include, but are not limited to, conventional assays such as those provided in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed exemplary methods for locating epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology Vol. 66 (Humana Press, Totowa, NJ). Two antibodies are considered to bind to the same epitope if they block each other's binding by 50% or more.

[0402] In an exemplary competitive assay, fixed CTHRC1 is incubated in a solution containing a first labeled antibody (e.g., a monoclonal antibody containing the variable domain of any one of SEQ ID NOs: 1-10 or a chimeric antibody containing the variable domain of a monoclonal antibody containing the sequence of Tables 3 and 4 and the constant domain from IgG1 or IgG4) that binds to CTHRC1 and a second unlabeled antibody that is tested for its ability to compete with the first antibody for binding to CTHRC1. The second antibody may be present in a hybridoma supernatant. As a control, fixed CTHRC1 is incubated in a solution containing the first labeled antibody but not containing the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to CTHRC1, excess unbound antibody is removed, and the amount of label associated with fixed CTHRC1 is measured. If the amount of label associated with fixed CTHRC1 in the test sample is significantly reduced relative to the control sample, it indicates that the second antibody competes with the first antibody for binding to CTHRC1. In certain embodiments, the immobilized CTHRC1 is present on the surface of a cell or in a membrane preparation obtained from a cell expressing CTHRC1 on its surface.

[0403] In one aspect, the purified anti-CTHRC1 antibodies can be further characterized by a battery of assays including, but not limited to, N-terminal sequencing, amino acid analysis, native size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

[0404] E. CAR-modified immune cells

[0405] In certain embodiments, the present invention relates to compositions and methods for treating cancer, including but not limited to hematological malignancies and solid tumors. In certain embodiments, CAR-modified immune cells are used. CAR-T cells can be therapeutically used for patients suffering from non-hematological tumors, such as solid tumors caused by, for example, breast, CNS and skin malignancies. In certain embodiments, CAR-NK cells can be therapeutically used for patients suffering from any of a variety of malignancies. In certain embodiments, CAR-macrophages can be therapeutically used for patients suffering from any of a variety of malignancies.

[0406] In certain embodiments, the present invention relates to an adoptive cell transfer strategy of a T cell or NK cell or macrophage transduced to express a chimeric antigen receptor (CAR). CAR is a molecule that combines antibody-based specificity for a desired antigen (e.g., a tumor antigen) with, for example, a T cell receptor activating intracellular domain to produce a chimeric protein that exhibits specific anti-tumor cell immune activity.

[0407] In one aspect, the present invention relates to the use of NK cells that are genetically modified to stably express the desired CAR. NK cells expressing CAR are referred to herein as CAR-NK cells or CAR-modified NK cells. Preferably, the cells can be genetically modified to stably express antibody binding domains on their surface, thereby conferring new antigen specificity. Methods for producing CAR-NK cells are known in the art. For example, see Glienke et al., Front Pharmacol. 2015; 6: 21. Services for producing CAR-NK cells are commercially available. See, for example, Creative Biolabs Inc., 45-1 Ramsey Road, Shirley, NY 11967, USA.

[0408] In one aspect, the present invention relates to the use of genetically modified T cells that stably express the desired CAR. T cells expressing CAR are referred to herein as CAR-T cells or CAR-modified T cells. Preferably, the cells can be genetically modified to stably express antibody binding domains on their surface, thereby conferring new antigen specificity that is independent of MHC. In some cases, T cells are genetically modified to stably express CAR, which combines the antigen recognition domain of a specific antibody with the intracellular domain of a CD3-ζ chain or FcγRI protein into a single chimeric protein.

[0409] In one embodiment, the CAR of the present invention comprises an extracellular domain, a transmembrane domain and a cytoplasmic domain having an antigen recognition domain. The intracellular domain or the cytoplasmic domain comprises at least one co-stimulatory signaling region and a ζ chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain of the CAR comprising a co-stimulatory molecule. Co-stimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for lymphocytes to effectively respond to antigens. In one embodiment, a transmembrane domain naturally associated with one of the domains in the CAR is used. In another embodiment, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins, thereby minimizing interactions with other members of the receptor complex. In one embodiment, the transmembrane domain is a CD8α hinge domain.

[0410] Between the extracellular domain and the transmembrane domain of CAR, or between the cytoplasmic domain and the transmembrane domain of CAR, a spacer domain can be incorporated. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide, the function of which is to connect the transmembrane domain to the extracellular domain or cytoplasmic domain in the polypeptide chain. The spacer domain may include up to 300 amino acids, 10 to 100 amino acids, and typically 25 to 50 amino acids.

[0411] Regarding the cytoplasmic domain, the CAR of the present invention can be designed to include CD28 and / or 4-1BB signaling domains themselves or in combination with any other desired cytoplasmic domains useful in the context of the CAR of the present invention. In one embodiment, the cytoplasmic domain of CAR can be designed to further include a signaling domain of CD3-ζ. For example, the cytoplasmic domain of CAR may include, but is not limited to, CD3-ζ, 4-1BB and CD28 signaling modules and combinations thereof. Therefore, the present invention provides CAR T cells and methods thereof for adoptive therapy.

[0412] In one embodiment, the CAR T cells of the present invention can be produced by introducing a lentiviral vector comprising a desired CAR into a cell, such as a CAR comprising anti-CTHRC1, CD8α hinge and transmembrane domains, and human 4-1BB and CD3ζ signaling domains. The CAR T cells of the present invention are capable of replicating in vivo, thereby producing long-term persistence, which can lead to sustained tumor control.

[0413] In one embodiment, the present invention relates to administering genetically modified T cells expressing CAR for use in lymphocyte infusion treatment of patients with cancer or at risk of cancer. Preferably, autologous lymphocyte infusion is used in treatment. Autologous PBMCs from patients in need of treatment are collected, and T cells are activated and amplified using methods described herein and known in the art, and then infused back into the patient. The present invention also includes treating malignant tumors or autoimmune diseases, wherein the patient's chemotherapy and / or immunotherapy causes significant immunosuppression in the patient, thereby increasing the risk of developing malignant tumors (eg, CLL) in the patient.

[0414] The present invention includes T cells (also referred to as CARTPODO T cells) expressing anti-CTHRC1 antibody-derived CARs, the CARs including CD3-ζ and 4-1BB or CD28 co-stimulatory domains. The CARTPODO T cells of the present invention can be robustly expanded in vivo for T cells, and memory cells specific for cells displaying CTHRC1 tumor epitopes can be established, which last for an extended amount of time at high levels in the blood and bone marrow.

[0415] 1. Antigen binding part

[0416] In one embodiment, the CAR of the present invention comprises a target-specific binding element, also referred to elsewhere as an antigen binding portion or a targeting arm. The antigen binding portion used in the present invention is capable of binding to a CTHRC1 epitope, such as a CTHRC1 tumor epitope. Therefore, the antigen binding portion is selected to recognize a ligand that acts as a cell surface marker on a target cell associated with a specific disease state.

[0417] The CAR of the present invention is engineered to target cells displaying the CTHRC1 epitope by engineering an appropriate antigen binding moiety that specifically binds to the CTHRC1 epitope.

[0418] Preferably, the antigen binding portion in the CAR of the present invention is scFv or scFab, wherein the nucleic acid sequence of scFv comprises a nucleic acid sequence encoding one or more light chain CDRs and one or more heavy chain CDRs disclosed herein for anti-CTHRC1 antibodies, and wherein the nucleic acid sequence of scFab comprises a nucleic acid sequence encoding one or more light chain CDRs and one or more heavy chain CDRs disclosed herein for anti-CTHRC1 antibodies.

[0419] Preferably, the antigen binding portion in the CAR of the present invention is a scFv or scFab, which comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1-10.

[0420] Preferably, the antigen binding portion in the CAR of the present invention is an scFv or scFab comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1, 3, 5, 7 and 9, more preferably an scFv or scFab comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 and 9.

[0421] Preferably, the antigen binding portion in the CAR of the present invention is an scFv or scFab comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 2, 4, 6, 8 and 10, more preferably an scFv or scFab comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 10.

[0422] Preferably, the antigen binding portion in the CAR of the present invention is a scFv or scFab comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1, 3, 5, 7 and 9 and any one of SEQ ID NOs: 2, 4, 6, 8 and 10. More preferably, the antigen binding portion in the CAR of the present invention is a scFv or scFab comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 3 and 9 and any one of SEQ ID NOs: 4 and 10.

[0423] In an embodiment, the antigen binding portion in the CAR of the present invention is a scFv or scFab comprising amino acids encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 100-109. In an embodiment, the antigen binding portion in the CAR of the present invention is a scFv or scFab comprising amino acids encoded by a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 100, 102, 104, 106 and 108.

[0424] In an embodiment, the antigen binding portion in the CAR of the present invention is a scFv or scFab, which comprises amino acids encoded by a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 101, 103, 105, 107 and 109.

[0425] In an embodiment, the antigen binding portion in the CAR of the present invention is a scFv or scFab, which comprises amino acids encoded by a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 100, 102, 104, 106 and 108 and the group consisting of any one of SEQ ID NOs: 101, 103, 105, 107 and 109.

[0426] In an embodiment, the antigen binding portion in the CAR of the present invention is a scFv or scFab, which comprises an amino acid sequence selected from the group consisting of any one CDR sequence in Table 3 and Table 4.

[0427] Preferably, the antigen binding portion in the CAR of the present invention is a scFv or scFab comprising an amino acid sequence selected from the group consisting of any one CDR sequence in Table 3 and Table 4; and further comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1-10. More preferably, the antigen binding portion of the CAR of the present invention is a scFv or scFab, which comprises: a heavy chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 150-154, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 180-184, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 210-214; and a light chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 240-244, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 270-274, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 300-304; and the antigen binding portion further comprises an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1-10.

[0428] In one embodiment, the antigen binding portion in the CAR of the invention comprises a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 2.

[0429] In one embodiment, the antigen binding portion in the CAR of the invention comprises a heavy chain variable region comprising SEQ ID NO:3 and a light chain variable region comprising SEQ ID NO:4.

[0430] In one embodiment, the antigen binding portion in the CAR of the invention comprises a heavy chain variable region comprising SEQ ID NO:5 and a light chain variable region comprising SEQ ID NO:6.

[0431] In one embodiment, the antigen binding portion in the CAR of the invention comprises a heavy chain variable region comprising SEQ ID NO:7 and a light chain variable region comprising SEQ ID NO:8.

[0432] In one embodiment, the antigen binding portion in the CAR of the present invention comprises a heavy chain variable region comprising SEQ ID NO:9 and a light chain variable region comprising SEQ ID NO:10.

[0433] In one embodiment, the antigen binding portion in the CAR of the present invention is a scFv or scFab comprising an amino acid sequence having about 80%, 85%, 90% or 95% identity with the above SEQ ID NO.

[0434] 2. Transmembrane domain

[0435] About transmembrane domain, CAR can be designed to include transmembrane domain fused with the extracellular domain of CAR.In one embodiment, a transmembrane domain naturally associated with one of the domains in CAR is used.In some cases, the transmembrane domain can be selected or modified by amino acid substitution to avoid such domains from binding to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing the interaction with other members of the receptor complex.

[0436] The transmembrane domain may be derived from a natural or synthetic source. In the case of a natural source, the domain may be derived from any membrane-bound protein or transmembrane protein. Particularly useful transmembrane regions in the present invention may be derived from α, β or ζ chains (i.e., comprising at least the above-mentioned transmembrane region) of T cell receptors, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will mainly comprise hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be present at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, preferably 2 to 10 amino acids in length, may form a connection between the transmembrane domain of CAR and the cytoplasmic signaling domain. Glycine-serine doublets provide particularly suitable linkers.

[0437] Preferably, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence of SEQ ID NO: 16 of U.S. Patent No. 9,102,760. In one embodiment, the CD8 transmembrane domain comprises a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 22 of U.S. Patent No. 9,102,760. In another embodiment, the CD8 transmembrane domain comprises an amino acid sequence of SEQ ID NO: 22 of U.S. Patent No. 9,102,760.

[0438] In some cases, the transmembrane domain of the CAR of the present invention comprises a CD8 alpha hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 15 of U.S. Patent No. 9,102,760. In one embodiment, the CD8 hinge domain comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 21 of U.S. Patent No. 9,102,760. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 21 of U.S. Patent No. 9,102,760.

[0439] 3. Cytoplasmic domain

[0440] The cytoplasmic domain or intracellular signaling domain of the CAR of the present invention is responsible for activating at least one normal effector function of the immune cell in which the CAR is placed. The term "effector function" refers to a specialized function of a cell. The effector function of a T cell can be, for example, a cytolytic activity or an auxiliary activity, including the secretion of cytokines. Therefore, the term "intracellular signaling domain" refers to a protein portion that transduces an effector function signal and instructs the cell to perform a specialized function. Although the entire intracellular signaling domain can usually be used, it is not necessary to use the entire chain in many cases. In terms of using a truncated portion of an intracellular signaling domain, such a truncated portion can be used to replace the complete chain as long as it transduces an effector function signal. Therefore, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transduce an effector function signal.

[0441] Preferred examples of intracellular signaling domains for use in the CAR of the present invention include cytoplasmic sequences of T cell receptors (TCRs) and co-receptors, which act synergistically to initiate signal transduction following antigen receptor engagement, as well as any derivatives or variants of these sequences and any synthetic sequences with the same functional capabilities.

[0442] It is known that the signal generated by TCR alone is not sufficient to fully activate T cells, and secondary or co-stimulatory signals are also required. Therefore, T cell activation can be said to be mediated by two different types of cytoplasmic signaling sequences: sequences that initiate antigen-dependent primary activation through TCR (primary cytoplasmic signaling sequences) and sequences that act in an antigen-independent manner to provide secondary or co-stimulatory signals (secondary cytoplasmic signaling sequences).

[0443] The primary cytoplasmic signaling sequence regulates the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner. The primary cytoplasmic signaling sequence that acts in a stimulatory manner may contain a signaling motif known as an immunoreceptor tyrosine-based activation motif or ITAM.

[0444] Examples of ITAMs containing primary cytoplasmic signaling sequences particularly used in the present invention include those derived from TCRζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CAR of the present invention comprises a cytoplasmic signaling sequence derived from CD3ζ.

[0445] In some embodiments, the cytoplasmic domain of CAR can be designed to include the CD3-ζ signaling domain itself or in combination with any other desired cytoplasmic domain useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of CAR may include a CD3ζ chain portion and at least one costimulatory signaling region. The costimulatory signaling region refers to a portion of the intracellular domain of CAR that includes a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands required for lymphocytes to effectively respond to antigens. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83, etc.

[0446] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the present invention can be connected to each other in a random or specified order. Optionally, a short oligopeptide or polypeptide linker (preferably between 2 and 10 amino acids in length) can form a connection. Glycine-serine doublets provide particularly suitable linkers.

[0447] In one embodiment, the cytoplasmic domain is designed to include the signaling domain of CD3-ζ and the signaling domain of CD28. In another embodiment, the cytoplasmic domain is designed to include the signaling domain of CD3-ζ and the signaling domain of 4-1BB. In yet another embodiment, the cytoplasmic domain is designed to include the signaling domain of CD3-ζ and the signaling domains of CD28 and 4-1BB.

[0448] In one embodiment, the cytoplasmic domain in the CAR of the present invention is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB includes the nucleic acid sequence shown in SEQ ID NO: 17 of U.S. Patent No. 9,102,760, and the signaling domain of CD3-ζ includes the nucleic acid sequence shown in SEQ ID NO: 18 of U.S. Patent No. 9,102,760.

[0449] In one embodiment, the cytoplasmic domain in the CAR of the present invention is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 23 of U.S. Patent No. 9,102,760, and the signaling domain of CD3-ζ includes a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 of U.S. Patent No. 9,102,760.

[0450] In one embodiment, the cytoplasmic domain in the CAR of the present invention is designed to include the signaling domain of 4-1BB and the signaling domain of CD3-ζ, wherein the signaling domain of 4-1BB includes the amino acid sequence shown in SEQ ID NO: 23 of U.S. Patent No. 9,102,760, and the signaling domain of CD3-ζ includes the amino acid sequence shown in SEQ ID NO: 24 of U.S. Patent No. 9,102,760.

[0451] 4. Alternative builds

[0452] In alternative embodiments, the CAR can be engineered to include an antigen binding domain that can bind to an engineered ligand fusion protein that includes a ligand that binds to the antigen binding domain instead of CTHRC1, wherein the engineered ligand fusion protein can include a ligand fused to an anti-CTHRC1 antibody of the present disclosure, thereby providing orthogonal binding to CTHRC1. For example, but not limited to, the antigen binding domain can be an engineered receptor, such as a modified NKG2D receptor that does not bind its natural ligand but binds a non-natural ligand, wherein the non-natural ligand is the ligand portion of an engineered ligand fusion protein, such as those described in U.S. Pat. No. 10,259,858, U.S. Pat. No. 10,259,858, U.S. Patent Application Publication No. 2019 / 0300594, U.S. Patent Application Publication No. 2020 / 0138866, WO 2017 / 222556, and U.S. Patent Application Publication No. 2016 / 0304578, each of which is incorporated herein by reference.

[0453] 5. Carrier

[0454] The present invention encompasses a DNA construct comprising a CAR sequence, wherein the sequence comprises a nucleic acid sequence of an antigen binding moiety operably connected to a nucleic acid sequence of an intracellular domain. Exemplary intracellular domains that can be used in the CAR of the present invention include, but are not limited to, intracellular domains of CD3-ζ, CD28, 4-1BB, etc. In some cases, CAR may include any combination of CD3-ζ, CD28, 4-1BB, etc.

[0455] In one embodiment, the CAR of the present invention comprises an anti-CTHRC1 antibody-derived scFv, human CD8 hinge and transmembrane domains, and human 4-1BB and CD3ζ signaling domains.

[0456] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, for example, by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the gene, or by isolating directly from cells and tissues containing the gene using standard techniques. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0457] The present invention also provides vectors into which the DNA of the present invention is inserted. Vectors derived from retroviruses (such as lentiviruses) are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of transgenes and their propagation in daughter cells. Lentiviral vectors have increased advantages over vectors derived from tumor retroviruses (such as murine leukemia viruses) because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of increased low immunogenicity.

[0458] In short, the expression of natural or synthetic nucleic acids encoding CAR is generally achieved by operably connecting the nucleic acid encoding the CAR polypeptide or part thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration of eukaryotic cells. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

[0459] In addition to the above method, the following method can be used.

[0460] The expression construct of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery schemes. Methods for gene delivery are known in the art (e.g., U.S. Patents 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety). In another embodiment, the present invention provides gene therapy vectors.

[0461] Nucleic acids can be cloned into various types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0462] Further, the expression vector can be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and is described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. The viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses and slow viruses. In general, suitable vectors contain a functional origin of replication, a promoter sequence, a convenient restriction endonuclease site and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Patent No. 6,326,193).

[0463] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene transfer systems. The selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to a subject's cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0464] Other promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110bp upstream of the start site, although recently many promoters have been shown to also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible so that when the elements are inverted or moved relative to each other, promoter function is retained. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50bp before activity begins to decline. Depending on the promoter, it seems that individual elements can work collaboratively or independently to activate transcription.

[0465] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Further, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered to be part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on the expression of an operably linked polynucleotide sequence when such expression is desired, or turn off such expression when such expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0466] In order to evaluate the expression of CAR polypeptides or parts thereof, the expression vector to be introduced into the cell may also contain a selectable marker gene or a reporter gene or both, so as to confirm and select expressing cells from a cell population attempted to be transfected or infected by a viral vector. In other aspects, selectable markers can be carried on separate DNA fragments and used for co-transfection procedures. Both selectable markers and reporter genes may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes, such as neo, etc.

[0467] Reporter gene is used to identify potential transfected cells and for evaluating the functionality of regulatory sequences. In general, reporter gene is a gene that is not present in or expressed by a recipient organism or tissue, and it encodes a polypeptide, the expression of which is expressed by some easily detectable properties (e.g., enzymatic activity). The expression of reporter gene is measured at the appropriate time after DNA has been introduced into the recipient cell. Suitable reporter gene may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein gene (e.g., Ui-Tei et al., 2000FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared or commercially obtained using known techniques. In general, the construct with the minimum 5' flanking region showing the highest expression level of reporter gene is confirmed as a promoter. This type of promoter region can be connected to a reporter gene and used to assess the ability of a medicament to regulate promoter-driven transcription.

[0468] Methods for introducing genes into cells and expressing them are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast or insect cells, by any method in the art. For example, the expression vectors can be transferred to host cells by physical, chemical or biological means.

[0469] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0470] Biological methods for introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used methods for inserting genes into mammals (e.g., human cells). Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patents Nos. 5,350,674 and 5,585,362.

[0471] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). In the case of utilizing a non-viral delivery system, an exemplary delivery vehicle is a liposome. Consider using lipid formulations to introduce nucleic acids into host cells (in vitro, in vitro, or in vivo). On the other hand, nucleic acids can be associated with lipids. Nucleic acids associated with lipids can be encapsulated in the aqueous interior of liposomes, dispersed in the lipid bilayer of liposomes, connected to liposomes via connecting molecules associated with liposomes and oligonucleotides, trapped in liposomes, compounded with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, included in lipids as a suspension, included in micelles, or compounded with micelles or otherwise associated with lipids. Lipid, lipid / DNA or lipid / expression vector related compositions are not limited to any specific structure in solution. For example, they may exist in a bilayer structure, as micelles, or have a "collapsed" structure. They may also simply be dispersed in a solution, possibly forming aggregates of uneven size or shape. Lipids are fatty substances that may be naturally occurring or synthetic lipids. For example, lipids include fat droplets naturally present in the cytoplasm and a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives (such as fatty acids, alcohols, amines, amino alcohols and aldehydes).

[0472] Suitable lipids are available from commercial sources. For example, dimyristoylphosphatidylcholine ("DMPC") is available from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") is available from K&K Laboratories (Plainview, NY); cholesterol ("Choi") is available from Calbiochem-Behring; dimyristoylphosphatidylglycerol ("DMPG") and other lipids are available from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at about -20°C. Chloroform is used as the only solvent because it evaporates more easily than methanol. "Liposome" is a general term covering various monolayer and multilayer lipid vehicles formed by producing closed lipid bilayers or aggregates. Liposomes can be characterized by having a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. When phospholipids are suspended in an excess of aqueous solution, they form spontaneously. The lipid assembly undergoes self-rearrangement before forming a closed structure, and water and dissolved solutes are trapped between lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions having structures different from normal vesicle structures in solution are also contemplated. For example, lipids can present micellar structures, or exist only as heterogeneous aggregates of lipid molecules. Cationic liposomes (lipofectamine)-nucleic acid complexes are also contemplated.

[0473] Regardless of the method used to introduce exogenous nucleic acid into host cells or expose cells to the inhibitors of the present invention, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biology" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, such as by immunological means (ELISA and Western blotting) or by assays described herein, to identify agents falling within the scope of the present invention.

[0474] 6. Source of T cells

[0475] Prior to T cell amplification and genetic modification of the present invention, a T cell source is obtained from a subject. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, infection site tissue, ascites, pleural effusion, spleen tissue and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art can be used. In certain embodiments of the present invention, any number of techniques known to the skilled person can be used, such as Ficoll. TMSeparation, T cells are obtained from a blood unit collected from a subject. In a preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma portion, and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps. In one embodiment of the present invention, cells are washed with phosphate buffered saline (PBS). In alternative embodiments, the washing solution lacks calcium, and may lack magnesium, or may lack many (if not all) divalent cations. Surprisingly, again, the initial activation step in the absence of calcium leads to amplified activation. As will be readily appreciated by those of ordinary skill in the art, the washing step can be accomplished by methods known to those skilled in the art according to the manufacturer's instructions, such as by using a semi-automatic "flow-through" centrifuge (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics cell recycler 5). After washing, the cells can be resuspended in a variety of biocompatible buffers, such as Ca-free 2+ , Mg-free 2+ Alternatively, the undesired components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0476] In another embodiment, the monocytes are depleted by lysing the red blood cells, for example by PERCOLL TM T cells are isolated from peripheral blood lymphocytes by gradient centrifugation or by counterflow centrifugal elutriation. Specific T cell subsets, such as CD3 + 、CD28 + 、CD4 + 、CD8 + 、CD45RA + and CD45RO + T cells. For example, in one embodiment, by conjugating beads (such as anti-CD3 / anti-CD28 (i.e., 3×28) In one embodiment, the time period is about 30 minutes. In another embodiment, the range of the time period is 30 minutes to 36 hours or longer and all integer values ​​therebetween. In another embodiment, the time period is at least 1, 2, 3, 4, 5 or 6 hours. In another preferred embodiment, the time period is 10 to 24 hours. In a preferred embodiment, the incubation time period is 24 hours. For separating T cells from leukemia patients, using a longer incubation time (such as 24 hours) can increase cell yield. Compared with other cell types, longer incubation times can be used to separate T cells in any case where T cells are less, such as separating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immunocompromised individuals. Further, using a longer incubation time can improve the capture efficiency of CD8+T cells. Therefore, by simply shortening or extending the time for T cells to bind to CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as further described herein), T cell subsets can be preferentially selected or not selected at the beginning of culture or at other time points during the culture process. In addition, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on beads or other surfaces, T cell subsets can be preferentially selected or not selected at the beginning of culture or at other desired time points. The technician will recognize that multiple rounds of selection can also be used in the context of the present invention. In certain embodiments, it may be desirable to perform a selection procedure and use "unselected" cells in activation and amplification processes. "Unselected" cells can also be subjected to more rounds of selection.

[0477] Enrichment of T cell populations by negative selection can be accomplished with a combination of antibodies against surface markers unique to the negatively selected cells. One approach is to perform cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies against cell surface markers present on the negatively selected cells. For example, to enrich CD4 + For cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich or positively select for cells that normally express CD4 + 、CD25 + 、CD62L hi GITR + and FoxP3 + Alternatively, in certain embodiments, T regulatory cells are depleted by anti-CD25 conjugated beads or other similar selection methods.

[0478] In order to separate the desired cell colony by positive or negative selection, the concentration of cells and surfaces (e.g., particles, such as beads) can be changed. In certain embodiments, it may be necessary to significantly reduce the volume (i.e., increase the concentration of cells) that beads and cells are mixed together to ensure the maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells / mL is used. In one embodiment, a concentration of 1 billion cells / mL is used. In another embodiment, greater than 100 million cells / mL are used. In another embodiment, a cell concentration of 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 50 million cells / mL is used. In yet another embodiment, a cell concentration of 7500, 8000, 8500, 9000, 9500 or 100 million cells / mL is used. In a further embodiment, a concentration of 125 or 150 million cells / mL can be used. Cell yield, cell activation and cell expansion can be increased using high concentrations. Further, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28 negative T cells, or cells from samples where many tumor cells are present (i.e., leukemic blood, tumor tissue, etc.). Such cell populations may have therapeutic value and are desirable to obtain. For example, the use of high concentrations of cells allows for more efficient selection of CD8 T cells that typically have weak CD28 expression. + T cells.

[0479] In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells are minimized. This selects for cells that express large amounts of the desired antigen to be bound to the particles. For example, CD4 + T cells expressed higher levels of CD28 and at dilute concentrations than CD8 + T cells are captured more efficiently. In one embodiment, the concentration of cells used is 5x10 6 In other embodiments, the concentration used may be about 1x10 5 / mL to 1x10 6 / mL, and any integer values ​​therebetween.

[0480] In other embodiments, cells can be incubated on a rotator at 2-10°C or at room temperature for different lengths of time at different speeds.

[0481] T cells for stimulation can also be frozen after the washing step. Without wishing to be bound by theory, freezing and subsequent thawing steps provide a more uniform product by removing the granulocytes in the cell mass and removing the monocytes in the cell mass to a certain extent. After the washing step of removing plasma and platelets, the cells can be suspended in a freezing solution. Although many freezing solutions and parameters are known in the art and can be used herein, a method involves using PBS containing 20% ​​DMSO and 8% human serum albumin, or a culture medium containing 10% dextran 40 and 5% glucose, 20% human serum albumin and 7.5% DMSO, or a culture medium containing 31.25% β-glucan A, 5% glucose, 0.45% NaCl, 10% dextran 40 and 5% glucose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing culture medium containing, for example, Hespan and β-glucan A, then the cells are frozen to -80°C at a rate of 1°C / minute and stored in the gas phase of a liquid nitrogen storage tank. Other controlled freezing methods may be used as well as immediate uncontrolled freezing at -20°C or in liquid nitrogen.

[0482] In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to stand at room temperature for one hour prior to activation using the methods of the invention.

[0483] It is also contemplated in the context of the present invention that a blood sample or a single blood component product is collected from a subject in a time period before the amplified cells as described herein may be needed. Therefore, the cell source to be amplified can be collected at any necessary time point, and the required cells, such as T cells, can be separated and frozen for later use in T cell therapy for many diseases or conditions that will benefit from T cell therapy as described herein. In one embodiment, a blood sample or a single blood component is taken from a generally healthy subject. In certain embodiments, a blood sample or a single blood component is taken from a generally healthy subject who is at risk of illness but not yet ill, and the cells of interest are separated and frozen for later use. In certain embodiments, T cells can be amplified, frozen, and used at a later time. In certain embodiments, samples are collected from patients shortly after a specific disease as described herein is diagnosed but before any treatment. In another embodiment, cells are isolated from a blood sample or apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressants (e.g., cyclosporine, azathioprine, methotrexate, mycophenolate mofetil, and FK506), antibodies or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cyclophosphamide, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation. These drugs inhibit the calcium-dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit p70S6 kinase (rapamycin), which is important for growth factor-induced signaling (Liu et al., Cell 66:807-15, 1991; Henderson et al., Immun 73:316-21, 1991; Bierer et al., Curr. Opin. Immun 5:763-73, 1993). In another embodiment, cells are isolated and frozen for later use in conjunction with bone marrow or stem cell transplantation, T cell ablation therapy using chemotherapeutic agents such as fludarabine, external particle beam radiation therapy (XRT), cyclophosphamide or antibodies such as OKT3 or CAMPATH (e.g., before, at the same time or after). In another embodiment, cells are isolated before and can be frozen for later use in treatment after B cell ablation therapy (e.g., an agent reactive with CD20, e.g., rituximab).

[0484] In another embodiment of the present invention, T cells are obtained directly from the patient after treatment. In this regard, it has been observed that after certain cancer treatments, particularly with drugs that damage the immune system, shortly after treatment, when the patient normally recovers from treatment, the quality of the T cells obtained may be optimal or improved because they can be expanded in vitro. Similarly, after the in vitro operation using the methods described herein, these cells may be in a preferred state of enhanced transplantation and in vivo expansion. Therefore, it is considered within the context of the present invention to collect blood cells during this recovery phase, including T cells, dendritic cells or other cells of the hematopoietic lineage. Further, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to produce conditions that are conducive to the re-proliferation, recirculation, regeneration and / or expansion of specific cell types in the subject, particularly during the time window determined after treatment. Illustrative cell types include T cells, B cells, dendritic cells and other cells of the immune system.

[0485] 7. T cell activation and expansion

[0486] Whether before or after genetically modifying the T cells to express the desired CAR, the T cells can generally be activated and expanded using methods as described in, e.g., U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[0487] Typically, the T cells of the present invention are amplified by contacting with a surface of a ligand attached with an agent that stimulates a CD3 / TCR complex-related signal and a co-stimulatory molecule on the T cell surface. In particular, a T cell population can be stimulated as described herein, such as by contacting with an anti-CD3 antibody or its antigen-binding fragment or an anti-CD2 antibody fixed on a surface, or by contacting with a protein kinase C activator (e.g., bryostatin) together with a calcium ion carrier. In order to costimulate the auxiliary molecules on the surface of the T cells, a ligand that binds the auxiliary molecule is used. For example, a T cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating the proliferation of T cells. In order to stimulate CD4 + T cells or CD8 +Proliferation of T cells using anti-CD3 antibodies and anti-CD28 antibodies. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), which can be used in the same manner as other methods generally known in the art (Berg et al., Transplant Proc. 30 (8): 3975-7, 1998; Haanen et al., J. Exp. Med. 190 (9): 13191328, 1999; Garland et al., J. Immunol Meth. 227 (1-2): 53-63, (1999)).

[0488] In certain embodiments, the primary stimulation signal and the costimulatory signal of T cell can be provided by different schemes.For example, the medicament providing each signal can be in solution or coupled to the surface.When coupled to the surface, the medicament can be coupled to the same surface (that is, in "cis" form) or to the surface coupled to separate (that is, in "trans" form). Alternatively, a medicament can be coupled to the surface, and another medicament is in solution.In one embodiment, the medicament providing the costimulatory signal is combined with the cell surface, and the medicament providing the primary activation signal is in solution or coupled to the surface.In certain embodiments, both medicaments can be in solution.In another embodiment, the medicament can be in soluble form, then cross-linked with the surface, such as cells or antibodies expressing Fc receptors or other binding agents to be combined with the medicament.In this regard, see, for example, U.S. Patent Application Publication No. 20040101519 and No. 20060034810 on artificial antigen presenting cells (aAPC), which are intended to be used for activating and amplifying T cells in the present invention.

[0489] In one embodiment, the two agents are immobilized on the beads, either on the same bead, i.e., "cis", or on separate beads, i.e., "trans". For example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the co-stimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof; and the two agents are co-immobilized on the same bead in equal molecular numbers. In one embodiment, a 1:1 ratio of each antibody bound to the bead is used for CD4 +T cell expansion and T cell growth. In certain aspects of the invention, a certain ratio of anti-CD3:CD28 antibody to bead binding is used so that an increase in T cell expansion is observed compared to the expansion observed using a ratio of 1:1. In a specific embodiment, an increase of about 1 to about 3 times is observed compared to the expansion observed using a ratio of 1:1. In one embodiment, the ratio range of CD3:CD28 antibody to bead binding is 100:1 to 1:100 and all integer values ​​therebetween. In one aspect of the invention, more anti-CD28 antibodies are bound to the particles than anti-CD3 antibodies, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the invention, the ratio of anti-CD28 antibodies to bead binding is greater than 2:1. In a specific embodiment, a 1:100 CD3:CD28 ratio of antibody to bead binding is used. In another embodiment, a 1:75 CD3:CD28 ratio of antibody to bead binding is used. In another embodiment, a 1:50 CD3:CD28 ratio of antibody to bead binding is used. In another embodiment, a 1:30 CD3:CD28 ratio of antibody to bead binding is used. In a preferred embodiment, a 1:10 CD3:CD28 ratio of antibody to bead binding is used. In another embodiment, a 1:3 CD3:CD28 ratio of antibody to bead binding is used. In yet another embodiment, a 3:1 CD3:CD28 ratio of antibody to bead binding is used.

[0490] Particle to cell ratios of 1:500 to 500:1 and any integer value therebetween can be used to stimulate T cells or other target cells. One of ordinary skill in the art can readily appreciate that the ratio of particles to cells can depend on the particle size relative to the target cells. For example, beads of small size can only bind to a few cells, while larger beads can bind to many cells. In certain embodiments, the ratio of cells to particles ranges from 1:100 to 100:1 and any integer value therebetween, and in another embodiment, the ratio includes 1:9 to 9:1 and any integer value therebetween, which can also be used to stimulate T cells. The ratio of anti-CD3 and anti-CD28 conjugated particles to T cells resulting in T cell stimulation can vary as described above, however certain preferred values ​​include 1:100, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1 and 15:1, with a preferred ratio of at least 1:1 particles per T cell. In one embodiment, a particle to cell ratio of 1:1 or less is used. In a specific embodiment, a preferred particle:cell ratio is 1:5. In another embodiment, the particle to cell ratio can vary depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is 1:1 to 10:1 on the first day, and additional particles are added to the cells every day or every other day thereafter for up to 10 days, with a final ratio of 1:1 to 1:10 (based on the cell count on the day of addition). In a specific embodiment, the ratio of particles to cells is 1:1 on the first day of stimulation, and is adjusted to 1:5 on the third and fifth days of stimulation. In another embodiment, particles are added every day or every other day, reaching a final ratio of 1:1 on the first day, and 1:5 on the third and fifth days of stimulation. In another embodiment, the ratio of particles to cells is 2:1 on the first day of stimulation, and is adjusted to 1:10 on the third and fifth days of stimulation. In another embodiment, particles are added every day or every other day, reaching a final ratio of 1:1 on the first day, and 1:10 on the third and fifth days of stimulation. It will be appreciated by those skilled in the art that a variety of other ratios may also be applicable to the present invention. In particular, the ratio will vary according to the particle size and cell size and type.

[0491] In a further embodiment of the present invention, cells (such as T cells) are combined with agent-coated beads, beads and cells are subsequently separated, and cells are then cultured. In an alternative embodiment, the agent-coated beads and cells are not separated before culture, but cultured together. In another embodiment, beads and cells are first concentrated by applying a force (such as a magnetic force), resulting in an increase in the connection of cell surface markers, thereby inducing cell stimulation.

[0492] For example, cell surface proteins can be linked by contacting T cells with paramagnetic beads (3×28 beads) attached with anti-CD3 and anti-CD28. 4 Up to 10 9 T cells) and beads (e.g., M-450 CD3 / CD28 T paramagnetic beads, ratio 1:1) are combined in a buffer, preferably PBS (without divalent cations such as calcium and magnesium). Again, one of ordinary skill in the art will readily appreciate that any cell concentration may be used.

[0493] For example, target cells can be very rare in a sample and only account for 0.01% of the sample, or the entire sample (i.e., 100%) may contain target cells of interest. Therefore, any cell number is within the scope of the present invention. In certain embodiments, it may be necessary to significantly reduce the volume in which particles and cells are mixed together (i.e., increase cell concentration) to ensure maximum contact between cells and particles. For example, in one embodiment, a concentration of about 2 billion cells / mL is used. In another embodiment, greater than 100 million cells / mL is used. In another embodiment, a cell concentration of 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500 or 50 million cells / mL is used. In yet another embodiment, a cell concentration of 7500, 8000, 8500, 9000, 9500 or 100 million cells / mL is used. In a further embodiment, a concentration of 125 or 150 million cells / mL may be used. Using high concentrations can increase cell yield, cell activation and cell expansion. Further, the use of high cell concentrations allows for more efficient capture of cells that may weakly express the target antigen of interest, such as CD28 negative T cells. Such cell populations may have therapeutic value in certain embodiments and are desirable to obtain. For example, the use of high concentrations of cells allows for more efficient selection of CD8+ T cells that typically have weak CD28 expression.

[0494] In one embodiment of the invention, the mixture can be cultured for several hours (about 3 hours) to about 14 days or any hour integer value therebetween. In another embodiment, the mixture can be cultured for 21 days. In one embodiment of the invention, beads and T cells are cultured together for about eight days. In another embodiment, beads and T cells are cultured together for 2-3 days. Several stimulation cycles may also be required so that the culture time of T cells can be 60 days or longer. Conditions suitable for T cell culture include suitable culture media (e.g., minimum essential medium or RPMI culture media 1640 or X-vivo 15 (Lonza)), which may contain factors necessary for proliferation and survival, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α, or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, plasma protein solutions (plasmanate) and reducing agents, such as N-acetyl-cysteine ​​and 2-mercaptoethanol. Culture medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo15 and X-Vivo 20, Optimizer, to which amino acids, sodium pyruvate and vitamins are added, serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines sufficient to grow and amplify T cells. Antibiotics (e.g., penicillin and streptomycin) are only included in experimental cultures, not in cell cultures to be infused into subjects. Target cells are maintained under conditions necessary to support growth, such as appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[0495] T cells that have been exposed to different stimulation times can exhibit different characteristics. For example, a typical helper T cell population (T H 、CD4 + ) is larger than the cytotoxic or suppressive T cell population (T C 、CD8 + ). Ex vivo expansion of T cells by stimulation of CD3 and CD28 receptors generates a T cell population that consists primarily of T cells before about day 8-9. H cells, and after about day 8-9, the T cell population contains an increasing number of T C Therefore, according to the purpose of treatment, the subject is infused with a cell population mainly containing T H Similarly, if a T cell population has been isolated CIf an antigen-specific subset of cells is expressed, it may be beneficial to expand that subset to a greater extent.

[0496] Furthermore, with the exception of CD4 and CD8 markers, other phenotypic markers varied significantly during cell expansion but were largely reproducible. Thus, this reproducibility enables customization of activated T cell products for specific purposes.

[0497] 8. Therapeutic applications

[0498] The present invention encompasses cells (e.g., T cells) transduced with lentiviral vectors (LV). For example, LV encodes a CAR that combines the antigen recognition domain of a specific antibody with the intracellular domain of CD3-ζ, CD28, 4-1BB, or any combination thereof. Therefore, in some cases, transduced T cells can elicit CAR-mediated T cell responses.

[0499] The present invention provides the use of CAR for redirecting the specificity of primary T cells to tumor antigens. Therefore, the present invention also provides a method for stimulating a T cell-mediated immune response to a target cell population or tissue in a mammal, the method comprising the step of administering to a mammal a T cell expressing CAR, wherein the CAR comprises a binding portion that specifically interacts with a predetermined target, a ζ chain portion comprising, for example, an intracellular domain of human CD3ζ, and a co-stimulatory signaling region.

[0500] In one embodiment, the present invention includes a type of cell therapy in which T cells are genetically modified to express CAR, and the CAR T cells are infused into a recipient in need. The infused cells are able to kill tumor cells in the recipient. Unlike antibody therapy, CAR T cells are able to replicate in vivo, resulting in long-term persistence, which can sustainably control tumors.

[0501] In one embodiment, the CAR T cells of the present invention can undergo robust in vivo T cell expansion and can last for an extended amount of time. In another embodiment, the CAR T cells of the present invention evolve into specific memory T cells that can be reactivated to inhibit any additional tumor formation or growth.

[0502] Without wishing to be bound by any particular theory, the anti-tumor immune response elicited by CAR-modified T cells can be an active or passive immune response. In addition, the CAR-mediated immune response can be part of an adoptive immunotherapy approach, in which CAR-modified T cells induce an immune response specific for the antigen-binding portion in the CAR.

[0503] Treatable cancers include tumors that are not vascularized or not yet fully vascularized and vascularized tumors. Cancer may include non-solid tumors (e.g., blood tumors, such as leukemia and lymphoma) or may include solid tumors. The types of cancers to be treated with the CAR of the present invention include, but are not limited to, cancer, blastoma and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Also included are adult tumors / cancers and childhood tumors / cancers. In certain embodiments, CAR-T cells can be therapeutically used for patients suffering from non-hematological tumors, such as solid tumors caused by breast, CNS, and skin malignancies.

[0504] Hematological cancer is a cancer of the blood or bone marrow. Examples of hematological (or blood-borne) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia and myeloblastic leukemia, promyelocytic leukemia, myelomonocytic leukemia, monocytic leukemia and erythroleukemia), chronic leukemias (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and advanced forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[0505] Solid tumors are abnormal tissue masses that usually do not contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors (such as sarcomas and carcinomas) include fibrosarcomas, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovial tumors, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, uterine fibroids ... carcinoma, testicular tumors, seminoma, bladder cancer, melanoma, and CNS tumors such as gliomas (e.g., brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, schwannomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendrogliomas, polyangiomas, neuroblastomas, retinoblastomas, and brain metastases.

[0506] In one aspect, CAR T cells can be used for ex vivo immunization. Regarding ex vivo immunization, at least one of the following occurs in vitro before the cells are administered to a mammal: i) expansion of the cells, ii) introduction of a nucleic acid encoding a CAR into the cells, and / or iii) cryopreservation of the cells.

[0507] Ex vivo procedures are well known in the art and are discussed in more detail below. In short, cells are isolated from mammals (e.g., humans) and genetically modified (i.e., in vitro transduction or transfection) with vectors expressing CAR disclosed herein. CAR-modified cells can be administered to mammalian recipients to provide therapeutic benefits. The mammalian recipient can be a human, and the CAR-modified cells can be autologous for the recipient. Alternatively, the cell can be allogeneic, isogenic or xenogeneic for the recipient.

[0508] Procedures for ex vivo expansion of hematopoietic stem and progenitor cells are described in U.S. Pat. No. 5,199,942, which is incorporated herein by reference and can be applied to the cells of the present invention. Other suitable methods are known in the art, and thus the present invention is not limited to any particular method for ex vivo expansion of cells. Briefly, the ex vivo culture and expansion of T cells include: (1) collecting CD34+ hematopoietic stem and progenitor cells from a peripheral blood harvest or bone marrow explant of a mammal; (2) expanding such cells ex vivo. In addition to the cell growth factors described in U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3, and c-kit ligand can also be used for the culture and expansion of cells.

[0509] In addition to the use of cell-based vaccines for ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response to an antigen in a patient.

[0510] The CAR-modified T cells of the present invention can be administered alone, or as a pharmaceutical composition with a diluent and / or in combination with other components (such as IL-2 or other cytokines or cell colonies). In short, the pharmaceutical composition of the present invention may include a target cell group as described herein in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers, such as neutral buffered saline, phosphate buffered saline, etc.; carbohydrates, such as glucose, mannose, sucrose or dextran, mannitol; protein; polypeptides or amino acids, such as glycine; antioxidants; chelating agents, such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. The CAR composition of the present invention is preferably formulated for intravenous administration.

[0511] The pharmaceutical composition of the present invention can be administered in a manner suitable for the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such as the patient's condition and the type and severity of the patient's disease, although the appropriate dosage can be determined by clinical trials.

[0512] When an "immunologically effective amount", "anti-tumor effective amount", "tumor suppressive effective amount" or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by the doctor taking into account individual differences in the patient's (subject's) age, weight, tumor size, degree and condition of infection or metastasis. In general, it can be said that the pharmaceutical composition comprising the T cells described herein can be administered in an amount of 10 4 Up to 10 9 cells / kg body weight, preferably 10 5 Up to 10 6The T cell composition can be administered at a dose of 1 cell / kg body weight, including all integer values ​​within those ranges. The T cell composition can also be administered multiple times at these doses. The cells can be administered using infusion techniques commonly known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988)). Those skilled in the art of medicine can easily determine the optim...

Claims

1. An anti-CTHRC1 antibody that binds to human CTHRC1. 2 . The anti-CTHRC1 antibody according to claim 1 , which (i) selectively binds to CTHRC1, (ii) blocks cell adhesion to CTHRC1, and / or (iii) is internalized when bound to a cell expressing CTHRC1. 3 . The anti-CTHRC1 antibody according to claim 1 or claim 2 , comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5, 7 and 9. 4 . The anti-CTHRC1 antibody according to any one of claims 1 to 2, comprising a heavy chain variable region comprising an amino acid sequence selected from Table 3. 5 . The anti-CTHRC1 antibody according to claim 2 , comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8 and 10. The anti-CTHRC1 antibody according to claim 2 , comprising a light chain variable region comprising an amino acid sequence selected from Table 4.

7. The anti-CTHRC1 antibody according to claim 2, comprising a heavy chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 150-154; a CDR2 sequence selected from the group consisting of SEQ ID NOs: 180-184; and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 210-214.

8. The anti-CTHRC1 antibody according to claim 2, comprising a light chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 240-244; a CDR2 sequence selected from the group consisting of SEQ ID NOs: 270-274; and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 300-304.

9. The anti-CTHRC1 antibody according to claim 2, comprising a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 150; a CDR2 sequence comprising SEQ ID NO: 180; and a CDR3 sequence comprising SEQ ID NO: 210; and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 240; a CDR2 sequence comprising SEQ ID NO: 270; and a CDR3 sequence comprising SEQ ID NO:

300.

10. The anti-CTHRC1 antibody according to any one of claims 1 to 9, wherein the binding affinity (K D ) is less than 10 nM, preferably less than 5 nM, more preferably less than 1 nM. The anti-CTHRC1 antibody according to any one of claims 1 to 9, comprising a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO:

2. 12 . The anti-CTHRC1 antibody according to any one of claims 1 to 9 , comprising a heavy chain variable region comprising SEQ ID NO: 3 and a light chain variable region comprising SEQ ID NO:

4. 13 . The anti-CTHRC1 antibody according to any one of claims 1 to 9 , comprising a heavy chain variable region comprising SEQ ID NO: 5 and a light chain variable region comprising SEQ ID NO:

6. The anti-CTHRC1 antibody according to any one of claims 1 to 9, comprising a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO:

8. The anti-CTHRC1 antibody according to any one of claims 1 to 9, comprising a heavy chain variable region comprising SEQ ID NO: 9 and a light chain variable region comprising SEQ ID NO:

10. The anti-CTHRC1 antibody according to any one of claims 1 to 15, wherein the anti-CTHRC1 antibody is a chimeric antibody, a humanized antibody or a human antibody. 17 . The anti-CTHRC1 antibody according to any one of claims 1 to 15 , wherein the anti-CTHRC1 antibody is a monoclonal antibody. 18 . The anti-CTHRC1 antibody according to any one of claims 1 to 15 , wherein the anti-CTHRC1 antibody is an antibody fragment. 19 . The anti-CTHRC1 antibody according to any one of claims 1 to 15 , wherein the anti-CTHRC1 antibody comprises a single-chain antibody. 20 . The anti-CTHRC1 antibody according to any one of claims 3 to 4 or 7 , wherein the anti-CTHRC1 antibody is a heavy chain-only antibody (single domain antibody).

21. A modified immune cell comprising a chimeric antigen receptor (CAR), wherein the CAR comprises the anti-CTHRC1 antibody according to claim 19.

22. The modified immune cell of claim 21, wherein the modified immune cell is a modified T cell.

23. The modified immune cell of claim 21, wherein the modified immune cell is a modified NK cell.

24. The modified immune cell of claim 21, wherein the modified immune cell is a modified macrophage.

25. An antibody-drug conjugate (ADC) comprising the antibody according to any one of claims 1 to 15.

26. The ADC of claim 25, wherein the ADC is a radioconjugate. 27 . A method of inhibiting the growth of a cell displaying a CTHRC1 epitope, comprising contacting the cell with an anti-CTHRC1 antibody according to any one of claims 1 to 15 , a modified immune cell according to claim 21 , or an ADC according to claim 25 or claim 26 .

28. A method of treating a subject having cancer, comprising administering to the subject an anti-CTHRC1 antibody according to any one of claims 1 to 15, a modified immune cell according to claim 21, or an ADC according to claim 25 or claim 26.

29. The method of claim 28, wherein the subject is a human.

30. The method of claim 28, wherein the cancer is selected from the group consisting of adrenocortical carcinoma, bladder cancer, breast cancer, cervical cancer, bile duct cancer, colon adenocarcinoma, B-cell lymphoma, esophageal cancer, glioblastoma multiforme, head and neck cancer, renal clear cell carcinoma, renal papillary cell carcinoma, myeloid leukemia, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, pancreatic adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, melanoma, gastric adenocarcinoma, testicular germ cell cancer, thymoma, uterine corpus cancer, and uterine carcinosarcoma. 31 . A method of treating fibrosis and / or a fibrotic disease in a subject in need thereof, comprising administering to the subject an anti-CTHRC1 antibody according to any one of claims 1 to 15, a modified immune cell according to claim 21 , or an ADC according to claim 25 or claim 26.

32. The method of claim 31 , wherein the subject is a human.

33. A pharmaceutical composition comprising the antibody according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

34. A pharmaceutical composition comprising the modified immune cell according to claim 21 and a pharmaceutically acceptable carrier.

35. A pharmaceutical composition comprising the ADC according to claim 25 or claim 26 and a pharmaceutically acceptable carrier.

36. Use of the antibody according to any one of claims 1 to 15, the modified immune cell according to claim 21, or the ADC according to claim 25 or claim 26 in the preparation of a medicament for treating cancer.

37. Use of the antibody according to any one of claims 1 to 15, the modified immune cell according to claim 21, or the ADC according to claim 25 or claim 26 in the preparation of a medicament for treating fibrosis and / or fibrotic diseases.

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