Combination therapy including anti-CTHRC1 antibody and method of use thereof

Abstract

The present invention relates to anti-CTHRC1 antibodies, compositions comprising same, and methods of using such antibodies and compositions for the prevention, diagnosis, and treatment of diseases or disorders such as cancer, bone disease, fibrotic disease, arthritis, and osteoporosis.

Description

[Technical Field]

[0001] The present invention relates to antibodies and antibody domains that specifically bind to CTHCR1, compositions thereof, and methods of their use in combination therapies for the treatment of cancer and fibrosis. [Background technology]

[0002] Collagen triple helix repeat-containing 1 (CTHRC1) was identified from a subtractive hybridization cDNA library searching for genes involved in arterial injury repair (Lindner, V., et al., Journal of Bone and Mineral Research, 2004). Additionally, CTHRC1 was found to be involved in bone development. Notably, overexpression of CTHRC1 in vivo leads to disorganization of epiphyseal growth plate chondrocytes and incomplete formation of proteoglycan complexes on collagen fibrils, resulting in collagen disorganization and severe deformation. Studies have further linked CTHRC1 to fibroblast activation and proper collagen organization in arterial and cardiac repair (LeClair, Renee J., et al., Circulation research, 2007; Ruiz-Villalba, Adrian, et al., Circulation, 2020), as well as wound healing more generally (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 have been observed on smooth muscle cells, but significant CTHRC1 expression requires injury (Leclair et al., Arterioscler. Thromb. Vasc. Biol., 2008).

[0003] CTHRC1 contains a short motif (12 Gly-XY repeats) common to collagen-related proteins and is conserved across species (Mei et al., Mediators Inflamm., 2020). At the molecular level, a series of distinct reports suggest that CTHRC1 regulates several signaling pathways, including TGF-β (J. Li et al., EBioMedicine, 2019; Ni et al., Cancer Med., 2018; Zhang et al., Oncogene, 2021), the canonical Wnt / β-catenin pathway (Hou et al., Oncotarget., 2015), the non-canonical Wnt / PCP pathway (Yamamoto et al., Dev. Cell, 2008), and the integrin / FAK pathway (Y.-L. Chen et al., J. Ovarian Res., 2013; Guo et al., PLoS One, 2017). However, no consensus has yet emerged regarding its precise molecular mechanism of action. Thus, while early reports indicate that CTHRC1 is important for bone growth during development and wound healing in adults, and likely acts downstream of TGF-β / Wnt signaling, it remains a poorly characterized protein, and there are clear gaps in our understanding of its precise function in humans.

[0004] In cancer, CTHRC1 overexpression has been reported in colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC), where CTHRC1 correlates with disease 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 lines suggest that CTHRC1 promotes migration and invasive behaviors 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 implicate CTHRC1 in angiogenesis; specifically, xenograft growth was impaired in CTHRC1 knockout mice, coupled with the observation that vascular organization was significantly disrupted (Lee et al., Exp. & Mol. Med., 2016). Overall, several reports have linked CTHRC1 to various pro-tumorigenic roles in cancer, although again, mechanistic details are scarce.

[0005] In addition to promoting cancer, CTHRC1 has also been implicated in fibrosis. Notably, a recent study linked CTHRC1 expression to a pathological subset of fibroblasts in a mouse model of pulmonary fibrosis, which can also be found 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, depletion of CTHRC1 by genetic knockout suppressed the development of fibrosis in a rodent model of chemical-induced liver fibrosis (J. Li et al., EBioMedicine, 2019). Thus, collectively, this data also implicates CTHRC1 in fibrosis and fibroblast biology, suggesting that its pro-tumorigenic role in the tumor microenvironment may also depend on fibroblasts. Outside of cancer, studies have also linked CTHRC1 to both protective anti-inflammatory activity in rheumatoid arthritis (Jin et al., Bone, 2017), where blood CTHRC1 levels can also distinguish healthy individuals from rheumatoid arthritis patients (Myngbay et al., Frontiers in Immunology, 2019). CTHRC1 has also been implicated as a positive regulator of bone formation and is therefore protective in osteoporosis (Chen et al., Bone Research, 2019; Kimura et al., PloS One, 2008).

[0006] Overall, research on CTHRC1 to date indicates that it plays a role in bone development, wound repair, cancer, fibrosis, arthritis, and osteoporosis. However, despite these findings, the lack of any published inhibitors of CTHRC1 precludes further investigation of the causal role CTHRC1 plays in disease, let alone providing a basis for the development of therapeutic inhibitors against CTHRC1 and / or mAbs based on CTHRC1-binding mAbs to target payloads to the tumor microenvironment. Thus, there is currently an unmet need for antibodies and antibody domains that specifically bind to CTHRC1. Summary of the Invention

[0007] The present invention addresses the aforementioned shortcomings in the prior art by providing and characterizing various antibodies and antibody domains against CTHRC1 and methods for their use in the prevention, diagnosis, and treatment of cancer, fibrosis, and / or fibrotic diseases. The anti-CTHRC1 antibodies of the present invention have been demonstrated to (i) selectively bind to CTHRC1, (ii) block cell adhesion to CTHRC1, (iii) be internalized into cells expressing CTHRC1 upon binding to the cells, and / or iv) recruit CD8+ T cells to the tumor microenvironment. Thus, 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 conditions in which CTHRC1 is upregulated. In some embodiments, the subject anti-CTHRC1 antibodies are administered to a subject with cancer, preferably in conjunction with chemotherapy, radiation therapy, or immunotherapy.

[0008] In one aspect, the invention provides anti-CTHRC1 antibodies that bind to human CTHRC1. In embodiments, the anti-CTHRC1 antibodies (i) selectively bind to CTHRC1, (ii) block cell adhesion to CTHRC1, and / or (iii) are internalized upon binding to cells expressing CTHRC1.

[0009] 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 NOs: 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.

[0010] 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 NOs: 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.

[0011] 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 to 154, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 180 to 184, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 210 to 214.

[0012] In one embodiment, the 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.

[0013] In one embodiment, the 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.

[0014] In one embodiment, the 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.

[0015] In one embodiment, the 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.

[0016] In one embodiment, the 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.

[0017] 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 to 244, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 270 to 274, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 300 to 304.

[0018] In one embodiment, the 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.

[0019] In one embodiment, the 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.

[0020] In one embodiment, the 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.

[0021] In one embodiment, the 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.

[0022] In one embodiment, the 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.

[0023] In one embodiment, the 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.

[0024] In one embodiment, the 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.

[0025] In one embodiment, the 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.

[0026] In one embodiment, the 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.

[0027] In one embodiment, the 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.

[0028] In one embodiment, the anti-CTHRC1 antibody has a binding affinity (K) for CTHRC1 of less than 10 nM, preferably less than 5 nM, and more preferably less than 1 nM. D )

[0029] 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.

[0030] In one embodiment, the invention provides an anti-CTHRC1 antibody that competes for binding to a 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.

[0031] 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 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 to a CTHRC1 epitope.

[0032] 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.

[0033] In one embodiment, the anti-CTHRC1 antibody inhibits or neutralizes one or more functions of human CTHRC1.

[0034] In one embodiment, the anti-CTHRC1 antibody is a chimeric, humanized, or 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.

[0035] In some embodiments, the anti-CTHRC1 antibody, or fragment thereof, elicits little to no immunogenic response against the anti-CTHRC1 antibody, or fragment thereof, in a subject, e.g., a human subject. In some embodiments, the invention provides humanized antibodies that elicit and / or are expected to elicit minimal or no human anti-mouse antibody response (HAMA). In one example, the antibodies of the invention elicit an anti-mouse antibody response that is below a clinically acceptable level.

[0036] In some aspects, the present invention provides a nucleic acid comprising DNA encoding any of the anti-CTHRC1 antibodies or portions thereof, or CARs or portions thereof described herein. In some embodiments, the nucleic acid comprises any one or more of SEQ ID NOs: 100-109. In several embodiments, the present invention provides a vector comprising a nucleic acid encoding any of the anti-CTHRC1 antibodies or portions thereof, or CARs or portions thereof described herein. In some embodiments, the vector comprises any one or more of SEQ ID NOs: 100-109. In several embodiments, the present invention provides a host cell comprising any such vector. By way of example, the host cell can be a CHO cell, an E. coli cell, or a yeast cell. Further provided is a process for producing any of the polypeptides described herein, comprising culturing a host cell under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.

[0037] In one aspect, the invention provides methods for producing antibodies of the invention. In embodiments, the invention provides methods for producing a CTHRC1 antibody (which includes full-length and fragments thereof, as defined herein), the method comprising expressing a recombinant vector of the invention encoding the antibody (or fragment thereof) in a suitable host cell, and recovering the antibody.

[0038] 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 capable of binding to a CTHRC1 epitope.

[0039] 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 a heavy chain variable region of an anti-CTHRC1 antibody as disclosed herein and a light chain variable region of an anti-CTHRC1 antibody as disclosed herein.

[0040] 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 an scFv.

[0041] In one aspect, the invention provides a method for activating T cells in a tumor microenvironment, the method comprising contacting a tumor with a humanized anti-CTHRC1 antibody of the disclosure.

[0042] In one aspect, the present invention provides a method for inhibiting the growth of cells that present a CTHRC1 epitope, e.g., a CTHRC1 tumor epitope, either directly or in a complex, comprising contacting the cells 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 radioconjugate. In several embodiments, contacting the cells comprises administering to a patient a therapeutically effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell.

[0043] In one aspect, the present invention provides a method for inhibiting metastasis of a tumor that presents a CTHRC1 epitope, e.g., a CTHRC1 tumor epitope, either directly or in a complex, comprising contacting cells of the tumor 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 radioconjugate. In several embodiments, contacting the cells comprises administering to the patient a therapeutically effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell.

[0044] In one aspect, the present invention provides a method for inducing death of cells that display a CTHRC1 epitope, e.g., a CTHRC1 tumor epitope, comprising contacting the cells 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 radioconjugate. In several embodiments, contacting the cells comprises administering to a patient a therapeutically effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell.

[0045] In one aspect, the invention provides a method for reducing the size of a tumor composed of cells that display a CTHRC1 epitope, e.g., a CTHRC1 tumor epitope, comprising contacting the cells with an anti-CTHRC1 antibody of the 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 radioconjugate. In several embodiments, contacting the cells comprises administering to the patient a therapeutically effective amount of an anti-CTHRC1 antibody of the invention or a CAR-modified immune cell.

[0046] In one aspect, the present invention provides a method for inhibiting angiogenesis of a tumor comprising cells presenting a CTHRC1 tumor epitope, the method comprising contacting the cells 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 radioconjugate. In several embodiments, contacting the cells comprises administering to a patient a therapeutically effective amount of an anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell.

[0047] In one aspect, the present invention provides a method for exerting cytostatic activity on tumor cells or cancer-associated fibroblasts presenting CTHRC1, the method comprising contacting the cells / cancer-associated fibroblasts 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 radioconjugate. In several embodiments, contacting the cells comprises administering to a patient a therapeutically effective amount of the anti-CTHRC1 antibody of the present invention or the CAR-modified immune cell.

[0048] In one aspect, the present invention provides a method for preventing immune cell suppression in a tumor microenvironment, the method comprising contacting at least one cell in 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 radioconjugate. In several embodiments, contacting the cell comprises administering to a patient a therapeutically effective amount of the anti-CTHRC1 antibody of the present invention or a CAR-modified immune cell.

[0049] In one aspect, the present invention provides a method for enhancing the infiltration of anti-tumor immune cells in an in vivo tumor microenvironment, 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 a cellular immunotherapy, such as an allogeneic or autologous T or NK cell therapy. In some embodiments, contacting the cell comprises administering a therapeutically effective amount of an anti-CTHRC1 antibody to a patient in conjunction with administration of the cellular immunotherapy, such as a CAR-T or CAR-NK cell therapy.

[0050] In embodiments of the invention, the cells presenting the CTHRC1 tumor epitope are cancer cells.

[0051] In one aspect, the present invention provides a method for treating or preventing a cell proliferative disorder associated with increased expression and / or presentation 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 radioconjugate. In one embodiment, the cell proliferative disorder is cancer.

[0052] 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 radioconjugate.

[0053] In one aspect, the present invention provides a method for reducing tumor size 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 radioconjugate.

[0054] 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, cholangiocarcinoma, colon adenocarcinoma, B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck cancer, renal clear cell carcinoma, papillary renal cell carcinoma, myeloid leukemia, hepatocellular carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, ovarian cancer, pancreatic adenocarcinoma, prostate cancer, rectal adenocarcinoma, sarcoma, melanoma, gastric adenocarcinoma, testicular germ cell carcinoma, thymoma, uterine corpus, and uterine carcinosarcoma.

[0055] In one aspect, the 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 invention, or a CAR-modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage. In embodiments, the anti-CTHRC1 antibody is administered in conjunction with radiation therapy.

[0056] In one embodiment, the present invention provides a method for treating a subject suffering from 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 radioconjugate. In several embodiments, the subject is a human subject.

[0057] In embodiments, the fibrotic disease may be selected from the group consisting of idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, pulmonary arterial hypertension, renal fibrosis, hyperkeratosis, non-alcoholic fatty liver disease (NASH), scleroderma, rheumatoid arthritis, Crohn's disease, ulcerative colitis, myelofibrosis, and systemic lupus erythematosus.

[0058] 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 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 radioconjugate.

[0059] In one aspect, the present invention provides a method for producing an anti-CTHRC1 antibody. In one aspect, the present invention provides a method for producing a CAR-modified immune cell disclosed herein. In one embodiment, the present invention provides a method for producing an ADC comprising an anti-CTHRC1 antibody.

[0060] In one aspect, the present invention provides a method for determining the presence of CTHRC1, e.g., a CTHRC1 epitope, e.g., a CTHRC1 tumor epitope, in a biological sample in a subject or from a subject. In one embodiment, the method includes contacting the sample with an anti-CTHRC1 antibody and determining binding of the anti-CTHRC1 antibody to the sample, wherein binding of the anti-CTHRC1 antibody to the sample indicates the presence of the CTHRC1 epitope in the sample.

[0061] In one aspect, the present invention provides a method for diagnosing a cell proliferative disorder (e.g., cancer) in a subject associated with (i) an increase in cells expressing CTHRC1, such as, for example, breast cancer cells, ovarian cancer cells, melanoma cells, liver, kidney, pancreas, or glioblastoma cells, or (ii) increased CTHRC1 expression in a tumor. In embodiments, the method includes detecting the presence of a CTHRC1 epitope, e.g., a CTHRC1 tumor epitope, in the subject or in a biological sample derived from the subject.

[0062] In one aspect, the present invention provides a method for determining a prognosis for 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 in a biological sample from the subject. In one embodiment, the method involves detecting the presence of a CTHRC1 epitope in a subject or in a biological sample from the subject after the subject has received a therapeutic agent for the treatment of cancer.

[0063] In one aspect, the invention provides use of a CTHRC1 antibody, or a CAR-modified immune cell, preferably a CAR-T or CAR-NK cell, or a CAR-macrophage, of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease or disorder, such as a cancer, a tumor and / or a cell proliferative disorder, fibrosis, and / or a fibrotic disease.

[0064] In one aspect, the invention provides use of a nucleic acid of the invention in the preparation of a medicament for the therapeutic and / or prophylactic treatment of a disease or disorder, such as a cancer, a tumor and / or a cell proliferative disorder, fibrosis, and / or a fibrotic disease.

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

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

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

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

[0069] In one aspect, a method for treating a subject with cancer is provided, the method comprising administering an anti-CTHRC1 antibody that binds to human CTHRC1 in conjunction with chemotherapy, radiation therapy or immunotherapy, wherein the anti-CTHRC1 antibody selectively binds to soluble CTHRC1 without binding to cells presenting the CTHRC1 epitope.

[0070] In another aspect, a humanized anti-CTHRC1 antibody is provided, comprising heavy chain complementarity determining regions (HCDR) 1, HCDR2 and HCDR3 comprising the sequences of SEQ ID NO: 150, SEQ ID NO: 180 and SEQ ID NO: 210, respectively, and / or light chain complementarity determining regions (LCDR) 1, LCDR2 and LCDR3 comprising the sequences of SEQ ID NO: 240, SEQ ID NO: 270 and SEQ ID NO: 300, respectively.

[0071] In another embodiment, a method for activating T cells in a tumor microenvironment comprises contacting the tumor with an anti-CTHRC1 antibody of the present disclosure.

[0072] In another embodiment, a method of inhibiting the growth of cancer cells that display a CTHRC1 epitope comprises contacting the cells with an anti-CTHRC1 antibody of the disclosure.

[0073] In another aspect, there is provided a use of a pharmaceutical composition comprising an anti-CTHRC1 antibody of the present disclosure for the preparation of a medicament for the treatment of a cell proliferative disorder, preferably cancer, or a fibrotic disease.

[0074] Also provided herein are kits and methods for their use. [Brief explanation of the drawings]

[0075] [Figure 1] 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. [Figure 2] 1 is a bar graph illustrating the assessment 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, colon). [Figure 3] 1 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. [Figure 4] 1 is a bar graph illustrating the selective blocking of cell adhesion by CTHRC1S-M5 (AB987) and CTHRC1S-M23 (AB988) in ovarian cancer cells. [Figure 5A] We show that CTHRC1 mRNA is a top marker for cancer-associated fibroblasts (CAFs) in cancer-rich, immune-cold tumor samples. Aggregate tumor samples were profiled across cancer scRNA studies to create a large scRNA atlas. Samples were then grouped into T cell-rich, cancer-poor samples (immune-hot) and cancer-rich, T cell-poor samples (immune-cold). [Figure 5B] Figure 1 illustrates that CTHRC1 mRNA is a top marker of cancer-associated fibroblasts (CAFs) in cancer-enriched and immunocold tumor samples. Genes expressed by CAFs were compared between the two groups to determine which were most associated with cancer-enriched samples (Wilcoxon rank). These genes were then narrowed down to genes specifically expressed by CAFs in all samples (top 500; Wilcoxon rank). CTHRC1 was the 11th top gene in this analysis. [Figure 6] This figure illustrates that CTHRC1 is highly upregulated in cancer versus normal adjacent tissue, as determined in bulk RNA measurements analyzed from The Cancer Genome Atlas. Across the set of indications profiled, the highest levels of CTHRC1 expression were found in solid tumors, particularly breast, lung, ovarian, and pancreatic cancers, sarcoma, melanoma, and uterine carcinosarcoma (P<0.001 in all cases tested). Together, this indicates that CTHRC1 expression is highest in cancers rich in desmoplastic stroma, consistent with the notion that it is a target primarily secreted by CAFs. [Figure 7]1 illustrates a series of survival plots showing CTHRC1 survival curves for several solid tumors in which CTHRC1 is associated with poor survival. Values ​​were obtained from The Cancer Genome Atlas. Survival curves were calculated and plotted using the online tool GEPIA. [Figure 8] Figure 1 illustrates that CTHRC1 levels increase with disease stage in liver cancer (left) and colon cancer (right), alongside 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. [Figure 9A] Figure 9A shows histograms showing bulk-RNA expression levels of known stromal targets FAP (Figure 9A) and LRRC15 (Figure 9B) alongside CTHRC1 (Figure 9C) in pancreatic cancer samples (The Cancer Genome Atlas) versus all normal tissue samples (GTEX). The histograms highlight that, based on bulk-RNA measurements, there is a significant therapeutic window for targeting CTHRC1 in pancreatic cancer. This window is similar, if not larger, than that for known / explored stromal targets FAP and LRRC15. [Figure 9B] Figure 9A shows histograms showing bulk-RNA expression levels of known stromal targets FAP (Figure 9A) and LRRC15 (Figure 9B) alongside CTHRC1 (Figure 9C) in pancreatic cancer samples (The Cancer Genome Atlas) versus all normal tissue samples (GTEX). The histograms highlight that, based on bulk-RNA measurements, there is a significant therapeutic window for targeting CTHRC1 in pancreatic cancer. This window is similar, if not larger, than that for known / explored stromal targets FAP and LRRC15. [Figure 9C]Figure 9A shows histograms showing bulk-RNA expression levels of known stromal targets FAP (Figure 9A) and LRRC15 (Figure 9B) alongside CTHRC1 (Figure 9C) in pancreatic cancer samples (The Cancer Genome Atlas) versus all normal tissue samples (GTEX). The histograms highlight that, based on bulk-RNA measurements, there is a significant therapeutic window for targeting CTHRC1 in pancreatic cancer. This window is similar, if not larger, than that for known / explored stromal targets FAP and LRRC15. [Figure 10] This dataset illustrates the extremely high levels of CTHRC1 expression in CAFs across many solid tumors, as well as in cancer epithelia in breast, pancreatic, lung, ovarian, and skin cancers. In contrast, minimal CTHRC1 expression is observed in normal tissues. The data were obtained based on a previously generated large-scale integrated single-cell RNA-sequencing atlas to enable probing gene expression at the single-cell level across multiple cancer and normal tissue samples (Swechha, 2021). The data highlight the potentially large therapeutic window for blocking CTHRC1 and the value of using mAbs against CTHRC1 to target payloads, such as ADCs, to the tumor microenvironment. [Figure 11] This dataset, using the same atlas discussed in Figure 10, was performed on LRRC15, a known non-toxic stromal target. Here, an antibody ADC has been engineered and shown to be safe in clinical settings. Low levels of LRRC15 were observed in normal tissues compared to CTHRC1. In a cancer single-cell RNA (scRNA) dataset, LRRC15 expression, distinct from CTHRC1, was observed to be preferentially localized by specific CAFs in certain breast cancers, with low levels also observed on sarcoma cancer cells. [Figure 12]1 is a graph illustrating quantitative ELISA of CTHRC1 in monocultures and cocultures. Supernatants from different sets of monocultures (fibroblasts or cancer cells) and cocultures (fibroblasts and cancer cells) were profiled for CTHRC1 levels. CTHRC1 was expressed at low levels in fibroblast monocultures (BJ, CCD18-Co) and was upregulated in cocultures, indicating that interactions between fibroblasts and cancer cells drive CTHRC1 expression. [Figure 13] A series of images of tissues from three mouse models probed with CTHRC1-specific mAb are shown. Extensive staining was observed within the tumor area, indicating that CTHRC1 protein is localized to cancerous areas in vivo. [Figure 14] A series of images of tissues from three human cancers show variations in CTHRC1 expression patterns at the protein level, indicating different expression kinetics. In head and neck and melanoma cancer samples, CTHRC1 is localized at the boundary between cancer and stromal tissue. In pancreatic cancer, CTHRC1 is seen to be widely expressed over areas of high CAF / stromal density. [Figure 15A] 1 illustrates CTHRC1 expression in human cancer cell lines. [Figure 15B] Figure 13 illustrates that CTHRC1 mAbs M14 and M23 bind to the surface of various human cancer cell lines that express CTHRC1. Binding of the same mAbs was observed to EMT6 cancer cells that express CTHRC1 in vivo, as shown in Figure 13. As shown, the higher affinity mAb M14 exhibits higher levels of cell surface binding than the lower affinity mAb M23. [Figure 16A]Figures 16A and 16B show that CTHRC1 mAb is rapidly internalized by cancer cells, regardless of the presence or absence of 50 nM exogenous CTHRC1. Internalization was much faster in SKOV3 ovarian cells, which express high levels of CTHRC1, compared with KP4 pancreatic cells (Figures 16C-16D), and the effect of exogenous CTHRC1 on internalization rate was also less (Figures 16A-16B). Consistent with higher levels of surface binding, CTHRC1S-M14 (Figures 16A and 16C) was internalized more rapidly than CTHRC1S-M23 (Figures 16B and 16D). [Figure 16B] Figures 16A and 16B show that CTHRC1 mAb is rapidly internalized by cancer cells, regardless of the presence or absence of 50 nM exogenous CTHRC1. Internalization was much faster in SKOV3 ovarian cells, which express high levels of CTHRC1, compared with KP4 pancreatic cells (Figures 16C-16D), and the effect of exogenous CTHRC1 on internalization rate was also less (Figures 16A-16B). Consistent with higher levels of surface binding, CTHRC1S-M14 (Figures 16A and 16C) was internalized more rapidly than CTHRC1S-M23 (Figures 16B and 16D). [Figure 16C] Figures 16A and 16B show that CTHRC1 mAb is rapidly internalized by cancer cells, regardless of the presence or absence of 50 nM exogenous CTHRC1. Internalization was much faster in SKOV3 ovarian cells, which express high levels of CTHRC1, compared with KP4 pancreatic cells (Figures 16C-16D), and the effect of exogenous CTHRC1 on internalization rate was also less (Figures 16A-16B). Consistent with higher levels of surface binding, CTHRC1S-M14 (Figures 16A and 16C) was internalized more rapidly than CTHRC1S-M23 (Figures 16B and 16D). [Figure 16D]Figures 16A and 16B show that CTHRC1 mAb is rapidly internalized by cancer cells, regardless of the presence or absence of 50 nM exogenous CTHRC1. Internalization was much faster in SKOV3 ovarian cells, which express high levels of CTHRC1, compared with KP4 pancreatic cells (Figures 16C-16D), and the effect of exogenous CTHRC1 on internalization rate was also less (Figures 16A-16B). Consistent with higher levels of surface binding, CTHRC1S-M14 (Figures 16A and 16C) was internalized more rapidly than CTHRC1S-M23 (Figures 16B and 16D). [Figure 17] This graph shows that CTHRC1 mAb is internalized by murine EMT6 and 4T1 cancer cells without the addition of exogenous CTHRC1. Internalization was observed to occur much more rapidly in EMT6 cancer cells compared to 4T1 cells, consistent with the cancer cells in this model having a more mesenchymal appearance. Similar to human cell lines, a higher internalization rate was observed in CTHRC1S-M14 (left) compared to CTHRC1S-M23 (right). [Figure 18] 1 shows a graph illustrating that CTHRC1 mAb conjugated to MMAE (vedotin) results in selective killing of SKOV3 cells. [Figure 19A] 1 illustrates the efficacy of CTHRC1 tested in the syngeneic mouse breast tumor model EMT6. Results are shown for anti-CTHRC1 antibodies, specifically M5. [Figure 19B] 1 illustrates the efficacy of CTHRC1 tested in the syngeneic mouse breast tumor model EMT6. Results are shown for anti-CTHRC1 antibodies, specifically M23. [Figure 19C] 1 illustrates the efficacy of CTHRC1 tested in the syngeneic mouse breast tumor model EMT6. Results are shown for anti-CTHRC1 antibodies, specifically M14. [Figure 20A] 1 illustrates the efficacy of anti-CTHRC1 (clone M5) in the PD-1-resistant Pan02 pancreatic cancer model. [Figure 20B]1 illustrates the efficacy of anti-CTHRC1 (clone M5) in the PD-1-resistant Pan02 pancreatic cancer model. [Figure 21] 1 illustrates the effect of preconditioning cells with anti-CTHRC1 antibody on CD8 T cell infiltration. [Figure 22A] Illustrates FACS data for staining of cancer cell lines with anti-CTHRC1 antibodies. [Figure 22B] Illustrates FACS data for staining of cancer cell lines with anti-CTHRC1 antibodies. [Figure 22C] Illustrates FACS data for staining of cancer cell lines with anti-CTHRC1 antibodies. [Figure 23] Illustrates heat maps for the various light chains of the M5 antibody. [Figure 24] 1 illustrates binding to CTHRC1 for humanized variants of the M5 antibody. [Figure 25] Illustrates antibody aggregation for humanized variants of the M5 antibody. [Figure 26] Illustrates the sequences of humanized variants of the M5 antibody. [Figure 27] Illustrates the sequences of humanized variants of the M5 antibody. [Figure 28] Illustrates the potency and quality for humanized variants of the M5 antibody. [Figure 29A] ELISA binding data for humanized variants of the M5 antibody are depicted. [Figure 29B] ELISA binding data for humanized variants of the M5 antibody are depicted. [Figure 29C] ELISA binding data for humanized variants of the M5 antibody are depicted. [Figure 29D] 1 depicts a heat map of ELISA binding data for humanized variants of the M5 antibody. [Figure 30A] 1 depicts CTHRC1 binding data for AB1234. [Figure 30B] 1 depicts CTHRC1 binding data for AB1241. [Figure 30C] 1 depicts CTHRC1 binding data for AB1083 (parent antibody of the M5 humanized variant). DETAILED DESCRIPTION OF THE INVENTION

[0076] general technique The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of a person skilled in the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989), "Oligonucleotide Synthesis" (M.J. Gait, ed., 1984), "Animal Cell Culture" (R.I. Freshney, ed., 1987), "Methods in Enzymology" (Academic Press, Inc.), "Current Protocols in Molecular Biology" (F.M. Usubel et al., eds., 1987, and regularly updated editions), "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994), "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988), and "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

[0077] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, 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 below.

[0078] II. Definition For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0079] As used herein, the term "collagen triple helix repeat containing 1 (CTHRC1)," unless otherwise indicated, refers to any naturally occurring 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 the nucleotide sequence corresponding to GenBank Accession No. NG031985.

[0080] The term "collagen triple helix repeat-containing 1" encompasses "full-length" unprocessed CTHRC1 as well as any form of CTHRC1 resulting from processing in cells. This term encompasses naturally occurring variants of CTHRC1, such as splice variants, allelic variants, and isoforms. The CTHRC1 polypeptides described herein may be isolated from various sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. A "native-sequence CTHRC1 polypeptide" includes a polypeptide having the same amino acid sequence as a corresponding CTHRC1 polypeptide derived from nature. Such native-sequence CTHRC1 polypeptides can be isolated from nature or produced by recombinant or synthetic means. The term "native-sequence CTHRC1 polypeptide" specifically encompasses naturally occurring truncated or secreted forms of a particular CTHRC1 polypeptide (e.g., extracellular domain sequences), naturally occurring variant forms of the polypeptide (e.g., alternatively spliced ​​forms), and naturally occurring allelic variants. In certain embodiments of the invention, the native sequence CTHRC1 polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acid sequences set forth in the accompanying disclosure.

[0081] As used herein, a "modification" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, where the modification results from a change in the sequence containing the amino acid residue / position. For example, typical modifications include substitution of the residue (or at the position) with another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more (usually less than five or three) amino acids adjacent to the residue / position, and deletion of the residue / position. An "amino acid substitution" or variant thereof refers to the replacement of an existing amino acid residue in a predetermined (starting) amino acid sequence with a different amino acid residue. Generally, the modification results in a change in at least one physico-biochemical activity of the variant polypeptide compared to the polypeptide containing the starting (or "wild-type") amino acid sequence. For example, in the case of an antibody, the altered physico-biochemical activity can be binding affinity, binding ability, and / or binding effect for a target molecule.

[0082] The term "antibody" is used in the broadest sense and specifically encompasses, for example, single anti-CTHRC1 monoclonal antibodies (including agonist, antagonist, neutralizing, full-length or intact monoclonal antibodies), anti-CTHRC1 antibody compositions with polyepitopic specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain anti-CTHRC1 antibodies, and fragments of anti-CTHRC1 antibodies, including Fab, Fab', F(ab')2, and Fv fragments (see below), diabodies, and single domain antibodies (sdAbs), so long as they exhibit the desired biological or immunological activity. Also included among the anti-CTHRC1 antibodies, and particularly among the fragments, are portions of anti-CTHRC1 antibodies (and combinations of anti-CTHRC1 antibody portions, e.g., scFvs) that can be used as targeting arms, e.g., directed against the CTHRC1 tumor epitope, in the chimeric antigen receptors of CAR-T cells, CAR-NK cells, or CAR-macrophages. Such fragments are not necessarily proteolytic fragments, but rather portions of a polypeptide sequence that can confer affinity for a target. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein. An antibody can be, for example, a human antibody, a humanized antibody, and / or an affinity matured antibody.

[0083] The terms "anti-CTHRC1 antibody," "CTHRC1 antibody," and "antibody that binds to CTHRC1" are used interchangeably. The anti-CTHRC1 antibody, whether isolated or as part of a fusion protein, cell, or cell composition, is preferably capable of binding with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent.

[0084] In one embodiment, a CTHRC1 antibody is used herein to specifically refer to an anti-CTHRC1 monoclonal antibody that (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 set forth in Table 3 or Table 4.

[0085] An "isolated antibody" is an antibody that has been identified, separated, and / or recovered from components of its natural environment. Contaminant components of its natural environment are materials that would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0086] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, the four-chain unit is generally approximately 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced interchain disulfide bridges. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Particular amino acid residues are believed to form an interface between the light-chain variable domain and the heavy-chain variable domain. The pairing of VH and VL together forms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, page 71 and Chapter 6 of "Basic and Clinical Immunology," 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994.

[0087] Light chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The 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.

[0088] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain is designated "VH" or "VH". H The variable domain of the light chain is sometimes referred to as a "VL" or "V L These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[0089] The term "variable" refers to the fact that certain segments of variable domains differ significantly in sequence among antibodies. V domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, V regions consist of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions," each 9-12 amino acids long. Native heavy and light chain variable domains each contain four FRs that adopt a primarily β-sheet configuration, connected by three hypervariable regions that form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0090] An "intact" antibody is one that comprises an antigen-binding site as well as a 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 intact antibody has one or more effector functions.

[0091] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding region or one or more variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-62(1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment comprises the antigen-binding site of the intact antibody and thus retains the ability to bind to an antigen. Anti-CTHRC1 antibody fragments also include portions of anti-CTHRC1 antibodies (and combinations of anti-CTHRC1 antibody portions, e.g., scFvs) that can be used as targeting arms, e.g., directed against the CTHRC1 tumor epitope, 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 a polypeptide sequence that may confer affinity for a target.

[0092] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments and a residual "Fc" fragment, a designation reflecting the ability to crystallize readily. The Fab fragment consists of an entire L chain plus the variable region domain (VH) of the H chain 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 bivalent antigen-binding activity and is still capable of cross-linking antigen. Fab' fragments differ from Fab fragments by having a few additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0093] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of an antibody are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells.

[0094] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy-chain variable region domain and one light-chain variable region domain in tight, non-covalent association. In single-chain Fv (scFv) species, one heavy-chain variable domain and one light-chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to that in two-chain Fv species. The folding of these two domains generates six hypervariable loops (three loops each from the H and L chains) that provide the amino acid residues for antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind antigen, albeit with lower affinity than the complete binding site.

[0095] A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment comprising the 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 scFv to form the desired structure for antigen binding. For a review of sFvs, see, e.g., Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995 (infra). In one embodiment, an scFv derived from an anti-CTHRC1 antibody is used as the targeting arm of a CAR-T cell, CAR-NK cell, or CAR-macrophage disclosed herein.

[0096] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic, or plant cells (e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using, for example, the techniques described in Clackson et al., Nature, 352:624-8 (1991) and Marks et al., J. Mol. Biol., 222:581-97 (1991).

[0097] As used herein, the terms "hypervariable region," "HVR," or "HV" refer to regions of an antibody variable domain that are highly variable in sequence and / or form structurally defined loops. Antibodies generally contain six hypervariable regions, three in the VH (H1, H2, and H3) and three in the VL (L1, L2, and L3). Several hypervariable region boundary demarcations are in use and are encompassed herein. Kabat complementarity-determining regions (CDRs) are the most commonly used, based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia instead refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The terminus of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, varies between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32; if only 35A is present, the loop ends at 33; and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on an analysis of available complex crystal structures. Residues from each of these hypervariable regions are listed below. [Table 1]

[0098] The hypervariable regions may also include "extended hypervariable regions" as follows: 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. The variable domain residues are numbered according to Kabat et al. (supra) for each of these definitions.

[0099] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0100] The terms "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 the heavy or light chain variable domains of the antibody collection in Kabat et al. (supra). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or CDR of the variable domain. For example, a heavy chain variable domain may contain 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 according to Kabat, etc.). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat numbered sequence at the regions of homology.

[0101] The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is generally used when referring to residues in the immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise specified herein, references to residue numbers in the variable domain of an antibody refer to residue numbering according to the Kabat numbering system.

[0102] A "blocking" or "antagonist" antibody is an antibody that inhibits or reduces the biological activity of the antigen to which it binds. Preferred blocking 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.

[0103] An antibody that "binds" to an antigen or epitope of interest is one that binds to the antigen or epitope with sufficient affinity that it differs measurably from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule (which is generally a molecule of similar structure that has no binding activity).

[0104] An antibody that inhibits tumor cell growth is an antibody that results in measurable growth inhibition of cancer cells. In one embodiment, an anti-CTHRC1 antibody can inhibit the growth of cancer cells that display a CTHRC1 tumor epitope. As referred to herein, a CTHRC1 tumor epitope includes a CTHRC1 epitope that can be bound by an anti-CTHRC1 antibody as disclosed herein, or a fragment thereof, or that can be at least partially bound by an antibody or other molecule that competes with an anti-CTHRC1 antibody as disclosed herein for binding to the epitope. Preferred growth-inhibitory anti-CTHRC1 antibodies inhibit the growth of CTHRC1-expressing tumor cells by more than 20%, preferably about 20% to about 50%, and even more preferably more than 50% (e.g., about 50% to about 100%) compared to a suitable control, which is typically tumor cells not treated with the antibody being tested.

[0105] Anti-CTHRC1 antibodies may (i) inhibit tumor metastasis in vivo, (ii) inhibit tumor growth in vivo, (iii) reduce tumor size in vivo, (iv) inhibit tumor angiogenesis in vivo, (v) exhibit cytotoxic activity against CTHRC1-expressing tumor cells and cancer-associated fibroblasts in vivo, (vi) exhibit cytostatic activity against CTHRC1-expressing tumor cells or cancer-associated fibroblasts in vivo, or (vii) prevent immune cell suppression in the tumor microenvironment in vivo.

[0106] 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 an antigen. Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native CTHRC1 polypeptides, peptides, antisense oligonucleotides, small organic molecules, and the like. A method for identifying an antagonist of a CTHRC1 polypeptide may include contacting the CTHRC1 polypeptide with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the CTHRC1 polypeptide.

[0107] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), skin cancer, melanoma, small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung cancer, including adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, 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, hepatocellular carcinoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial cancer, or uterine cancer, salivary gland cancer, kidney cancer, or renal cancer. Cancers include, but are not limited to, adrenocortical carcinoma, cholangiocarcinoma, colon adenocarcinoma, B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, renal clear cell carcinoma, papillary renal cell carcinoma, myeloid leukemia, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, rectal adenocarcinoma, sarcoma, gastric adenocarcinoma, thymoma, uterine and uterine carcinosarcoma.

[0108] The term "metastatic cancer" refers to a cancerous condition in which cancer cells from a primary tissue spread by blood or lymphatic vessels from the primary site to one or more other sites in the body, forming one or more secondary tumors in one or more organs in addition to the primary tissue. A notable example is metastatic breast cancer.

[0109] As used herein, a "CTHRC1-associated cancer" is a cancer associated with overexpression of the CTHRC1 gene or gene product and / or associated with presentation of the CTHRC1 tumor epitope. Suitable control cells can be, for example, cells from an individual not afflicted with cancer, or non-cancerous cells from a subject with cancer.

[0110] The methods include methods for treating subjects with cancer, particularly cancers associated with expression of the CTHRC1 tumor epitope. The methods also include methods for modulating the behavior of certain cells, particularly cancer cells, particularly cancer cells that display the CTHRC1 tumor epitope.

[0111] 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.

[0112] As used herein, "tumor" refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[0113] As used herein, the term "fibrotic disease" broadly refers to several different diseases characterized by the development of organ fibrosis, including, but not limited to, idiopathic pulmonary fibrosis (IPF) and scleroderma. The term "fibrosis" refers to the development of fibrous connective tissue as a typical response to injury or damage. Fibrosis can occur as part of normal healing or in response to excessive tissue deposition as part of a pathological process.

[0114] The terms "prediction" and "prognosis" as used herein are also interchangeable. In one sense, a method for prediction or prognosis allows one practicing the prediction / prognosis method of the invention to select patients (usually, but not necessarily, prior to treatment) who are deemed more likely to respond to treatment with an anti-cancer agent, preferably an anti-CTHRC1 antibody or CAR-engineered cell of the invention.

[0115] III. Compositions and Methods of the Invention A. Anti-CTHRC1 antibody In one embodiment, the present invention provides anti-CTHRC1 antibodies that may be utilized as therapeutic agents herein. Exemplary antibodies include polyclonal, monoclonal, chimeric, humanized, and human antibodies.

[0116] 1. Polyclonal antibodies Polyclonal antibodies can be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species being immunized. For example, antigens 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 (conjugation via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R'N=C=NR (where R and R are different alkyl groups).

[0117] 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 serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Conjugates can also be produced in recombinant cell culture as protein fusions. Aggregating agents such as alum are also preferably used to enhance the immune response.

[0118] 2. Monoclonal antibodies Monoclonal antibodies (mAbs) to an antigen of interest can be prepared using any technique known in the art, including, but not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Selected 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 USA, 1996. 93(15):7843-8.) and (McLean G et al., 2005, J Immunol. 174(8):4768-78). Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. Hybridomas for producing mAbs useful in the present invention can be cultivated in vitro or in vivo.

[0119] 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 (U.S. Pat. No. 4,816,567).

[0120] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce, or are capable of producing, antibodies that 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, pp. 59-103 (Academic Press, 1986)).

[0121] The hybridoma cells thus prepared are seeded and grown in a suitable medium which 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 phosphoribosyltransferase (HGPRT or HPRT), the selective medium for hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibit the growth of HGPRT-deficient cells.

[0122] Preferred fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to selective media that select against unfused parental cells. Preferred myeloma cell lines are mouse myeloma lines, such as those derived from the MOPC-21 and MPC-11 mouse tumors 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)).

[0123] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0124] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem. 107:220 (1980).

[0125] Once hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in animals, for example, by intraperitoneal injection of the cells into mice.

[0126] 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, for example, affinity chromatography (e.g., using Protein A or Protein G-Sepharose) or ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, or dialysis.

[0127] DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into host cells that do not otherwise produce antibody protein, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol. 5:256-62 (1993) and Pluckthun, Immunol. Rev. 130:151-88 (1992).

[0128] In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated 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 isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), and combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (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.

[0129] DNA encoding an antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy and light chain constant domains (CH and CO sequences) for the homologous murine sequences (U.S. Pat. No. 4,816,567, and Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing immunoglobulin coding sequences with all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). Non-immunoglobulin polypeptide sequences can be substituted for the constant domains of an antibody, or they can be substituted for the variable domains of one antigen-binding site of an antibody, creating a chimeric, bivalent antibody with one antigen-binding site with specificity for one antigen and another antigen-binding site with specificity for a different antigen.

[0130] 3. Chimeric, Humanized, and Human Antibodies In some embodiments, the anti-CTHRC1 antibody is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent 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 a further example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0131] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the non-human parent antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and the FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0132] The anti-CTHRC1 antibodies of the present invention may include humanized or human antibodies. Humanized forms of non-human (e.g., mouse or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies) that contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from a complementarity-determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. 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)).

[0133] A humanized antibody of the invention may comprise one or more human and / or human consensus non-hypervariable region (e.g., framework) sequences in its heavy and / or light chain variable domains. In some embodiments, one or more additional modifications are present within the human and / or human consensus non-hypervariable region sequences. In one embodiment, the heavy chain variable domain of an antibody of the invention comprises a human consensus framework sequence, which in one embodiment is a subgroup III consensus framework sequence. In one embodiment, an antibody of the invention comprises a variant subgroup III consensus framework sequence modified at at least one amino acid position.

[0134] In embodiments, an anti-CTHRC1 antibody may comprise a heavy chain variable region (HCVR) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 305, 308, and 309, and a light chain variable region (LCVR) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 306-307 and 310-332. In some embodiments, an anti-CTHRC1 antibody may comprise an HCVR of any one of SEQ ID NOs: 305, 308, and 309, and a light chain variable region (LCVR) of any one of SEQ ID NOs: 306-307 and 310-322. In some embodiments, an anti-CTHRC1 antibody may comprise an HCVR having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 305, 308, and 309. In some embodiments, an anti-CTHRC1 antibody may be a humanized antibody comprising an LCVR having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 306-307 and 310-332. In some embodiments, an anti-CTHRC1 antibody may be a humanized antibody comprising an HCVR of any one of SEQ ID NOs: 305, 308, and 309. In some embodiments, an anti-CTHRC1 antibody may be a humanized antibody comprising an LCVR of any one of SEQ ID NOs: 306-307 and 310-332. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising an HCVR of SEQ ID NO: 305. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising an LCVR of SEQ ID NO: 306. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising an LCVR of SEQ ID NO: 307. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising an HCVR of SEQ ID NO: 305 and an LCVR of SEQ ID NO: 306 or SEQ ID NO: 307. In some embodiments, the anti-CTHRC1 antibody comprises an HCVR of SEQ ID NO: 305 and an LCVR of SEQ ID NO: 306.In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising the HCVR of SEQ ID NO:305 and the LCVR of SEQ ID NO:307.

[0135] In any of the above-described embodiments, the anti-CTHRC1 antibody may comprise 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.

[0136] In embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising a heavy chain variable region (HCVR) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 305, 308, and 309, and a light chain variable region (LCVR) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 306-307 and 310-332. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising a HCVR of any one of SEQ ID NOs: 305, 308, and 309, and a light chain variable region (LCVR) of any one of SEQ ID NOs: 306-307 and 310-322. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising an HCVR having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 305, 308, and 309. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising an LCVR having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 306-307 and 310-332. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising an HCVR of any one of SEQ ID NOs: 305, 308, and 309. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising an LCVR of any one of SEQ ID NOs: 306-307 and 310-332. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising an HCVR of SEQ ID NO: 305. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising an LCVR of SEQ ID NO: 306. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising an LCVR of SEQ ID NO: 307. In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising an HCVR of SEQ ID NO: 305 and an LCVR of SEQ ID NO: 306 or SEQ ID NO: 307. In some embodiments, the anti-CTHRC1 antibody comprises an HCVR of SEQ ID NO: 305 and an LCVR of SEQ ID NO: 306.In some embodiments, the anti-CTHRC1 antibody may be a humanized antibody comprising the HCVR of SEQ ID NO:305 and the LCVR of SEQ ID NO:307.

[0137] In any of the above-described embodiments, the anti-CTHRC1 antibody may be a humanized antibody 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.

[0138] As known in the art and described in more detail herein, the amino acid positions / boundaries defining an antibody hypervariable region can vary depending on the context and various definitions known in the art (described below). Some positions within a variable domain can be considered hybrid hypervariable positions, in that these positions can be considered within a hypervariable region under one set of criteria, while being considered outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in an extended hypervariable region (defined further below). The present invention provides antibodies containing modifications at these hybrid hypervariable positions. In one embodiment, these hypervariable positions include one or more of 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, an antibody of the invention comprises a human variant human subgroup consensus framework sequence altered at one or more hybrid hypervariable positions.

[0139] The antibodies of the invention can 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.

[0140] Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, and these residues typically come from an "import" variable domain. Humanization is essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). Such "humanized" antibodies are thus essentially chimeric antibodies (U.S. Pat. No. 4,816,567), in which less than an intact human variable domain has been substituted by the 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.

[0141] If an antibody is intended for human therapeutic use, the selection of human variable domains, both light and heavy, used in making the humanized antibody is very important to reduce antigenicity and the 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 antibodies that have been humanized to reduce or eliminate HAMA responses. Further variants of these antibodies can be 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 against the entire library of known human variable domain sequences. The human V-domain sequence that is closest to the rodent V-domain sequence is identified, and the human framework regions (FRs) therein are accepted for the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses specific framework regions derived from the consensus sequence of all human antibodies of a particular light 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)).

[0142] For example, an amino acid sequence from an antibody as described herein can serve as the starting (parent) sequence for diversifying the framework and / or hypervariable sequence(s). The selected framework sequence to which the starting hypervariable sequence is linked is referred to herein as the acceptor human framework. The acceptor human framework may be from or derived from a human immunoglobulin (its VL and / or VH regions), but preferably the acceptor human framework is from or derived from a human consensus framework sequence, as such frameworks have been demonstrated to have minimal or no immunogenicity in human patients.

[0143] If the acceptor is derived from a human immunoglobulin, optionally, the human framework sequence may be selected based on its homology with the donor framework sequence by aligning the donor framework sequence with various human framework sequences in a population of human framework sequences and selecting the framework sequence that is most homologous to the acceptor.

[0144] 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.

[0145] Although the acceptor may be identical in sequence to the selected human framework sequence, whether derived from a human immunoglobulin or a human consensus framework, the present invention contemplates that the acceptor sequence may contain pre-existing amino acid substitutions relative to the human immunoglobulin sequence or human consensus framework sequence. These pre-existing substitutions are preferably minimal, typically no more than four, three, two, or one amino acid difference relative to the human immunoglobulin sequence or consensus framework sequence.

[0146] Hypervariable region residues of a 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 may be introduced. Optionally, extended hypervariable region residues such as 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) are incorporated.

[0147] While the "incorporation" of hypervariable region residues is discussed herein, it will be understood that this can be achieved in a variety of ways. For example, a nucleic acid encoding a desired amino acid sequence can be generated by mutating a nucleic acid encoding a murine variable domain sequence so that its framework residues are changed to acceptor human framework residues, or by mutating a nucleic acid encoding a human variable domain sequence so that hypervariable domain residues are changed to non-human residues, or by synthesizing a nucleic acid encoding the desired sequence, etc.

[0148] Hypervariable region-grafted variants may be generated by Kunkel mutagenesis of nucleic acids encoding human acceptor sequences, using separate oligonucleotides for each hypervariable region, as described herein (Kunkel et al., Methods Enzymol. 154:367-382 (1987)). Appropriate changes can be introduced into the framework and / or hypervariable regions using conventional techniques to modify and re-establish proper hypervariable region-antigen interactions.

[0149] Phage(mid) display (also referred to herein in some contexts as phage display) can be used as a convenient and rapid method for generating and screening many different potential variant antibodies in libraries generated by sequence randomization. However, other methods for generating and screening altered antibodies are available to those skilled in the art.

[0150] Phage(mid) display technology has provided a powerful tool for generating and selecting novel proteins that bind to ligands such as antigens. Phage(mid) display techniques enable the generation of large libraries of protein variants that can be rapidly screened for sequences that bind to target molecules with high affinity. Nucleic acids encoding variant polypeptides are generally fused to nucleic acid sequences encoding viral coat proteins, such as gene III or gene VIII proteins. Monovalent phagemid display systems have been developed in which nucleic acid sequences encoding proteins or polypeptides are fused to nucleic acid sequences encoding portions of gene III proteins (Bass, S., Proteins, 8:309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3:205 (1991)). In monovalent phagemid display systems, gene fusions are expressed at low levels, and wild-type gene III proteins are also expressed to maintain particle infectivity. Methods for generating peptide libraries and screening those libraries are disclosed in many patents (e.g., U.S. Patent No. 5,723,286, U.S. Patent No. 5,432,018, U.S. Patent No. 5,580,717, U.S. Patent No. 5,427,908, and U.S. Patent No. 5,498,530).

[0151] Libraries of antibodies or antigen-binding polypeptides have been prepared in several ways, including by altering a single gene, inserting random DNA sequences, or cloning a family of related genes. Methods for displaying antibodies or antigen-binding fragments using phage(mid) display are described in U.S. Patent Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727. The libraries are then screened for the expression of antibodies or antigen-binding proteins with the desired properties.

[0152] Methods for substituting selected amino acids into a template nucleic acid are well established in the art, and some of these 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)).

[0153] The sequence of the oligonucleotide contains 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 used to encode the desired variant amino acids. Codon sets can be represented using symbols that designate specific nucleotides or equimolar mixtures of nucleotides, as shown below in the IUB code: IUB Code G Guanine A Adenine T thymine C cytosine R (A or G) Y (C or T) M (A or C) K (G or T) S (C or G) W (A or T) H (A or C or T) B (C ​​or G or T) V (A or C or G) D (A or G or T) H N (A or C or G or T)

[0154] 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 represent 18 different codons and can encode the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

[0155] Oligonucleotide or primer sets can be synthesized using standard methods. A set of oligonucleotides containing sequences that represent all possible combinations of nucleotide triplets provided by a codon set and will encode the desired amino acids can be synthesized, for example, by solid-phase synthesis. The synthesis of oligonucleotides with selected nucleotide "degeneracy" at specific positions is well known in the art. Such a set of oligonucleotides with a specific codon set can be synthesized using a commercial nucleic acid synthesizer (e.g., available from Applied Biosystems, Foster City, Calif.) or can be obtained commercially (e.g., from Life Technologies, Rockville, Md.). Thus, a set of synthesized oligonucleotides with a specific codon set typically includes multiple oligonucleotides with different sequences, with the difference being established by the codon set within the overall sequence. Oligonucleotides used in the present invention have sequences that allow hybridization to a variable domain nucleic acid template and may also include restriction enzyme sites for cloning purposes.

[0156] In one method, the nucleic acid sequence encoding variant amino acid can be generated by oligonucleotide-mediated mutagenesis.This technique is well known in the art, as described by Zoller et al.Nucleic Acids Res.10:6487-6504(1987).In brief, the nucleic acid sequence encoding variant amino acid is generated by hybridizing the oligonucleotide set encoding desired codon set to a DNA template, where the template is a single-stranded plasmid containing the nucleic acid template sequence of variable region.After hybridization, DNA polymerase is used to synthesize the entire second complementary strand of the template, which thus incorporates the oligonucleotide primer and contains the codon set provided by the oligonucleotide set.

[0157] Generally, oligonucleotides of at least 25 nucleotides in length are used. Optimal oligonucleotides will have 12-15 nucleotides that are perfectly complementary to the template on each side of the nucleotide(s) encoding the mutation(s). This ensures that the oligonucleotide hybridizes properly to the single-stranded DNA template molecule. Oligonucleotides can be easily synthesized using techniques known in the art, such as those described by Crea et al., Proc. Nat'l. Acad. Sci. USA, 75:5765 (1978).

[0158] The DNA template is generated either by a vector derived from a bacteriophage M13 vector (commercially available M13 mp 18 and M13 mp 19 vectors are suitable) or by a vector containing a single-stranded phage origin of replication as described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus, to generate a single-stranded template, the DNA to be mutated can be inserted into one of these vectors. Production of single-stranded template is described in sections 4.21-4.41 of Sambrook et al., supra.

[0159] To alter the native DNA sequence, an oligonucleotide is hybridized to a single-stranded template under suitable hybridization conditions. A DNA polymerizing enzyme, usually T7 DNA polymerase or the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis. This results in a heteroduplex molecule in which one DNA strand encodes the mutant version of gene 1 and the other strand (the original template) encodes the unchanged native sequence of gene 1. This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. coli JM101. After the cells are grown, they are plated on agarose plates and screened using 32-phosphate radiolabeled oligonucleotide primers to identify bacterial colonies containing the mutant DNA.

[0160] The method just described may be modified to create a homoduplex molecule in which both strands of the plasmid contain the mutation(s). The modification is as follows: a single-stranded oligonucleotide is annealed to a single-stranded template as described above. A mixture of the three deoxyribonucleotides deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTT) is combined with a modified thiodeoxyribocytosine called dCTP-(aS), available from Amersham. This mixture is added to the template-oligonucleotide complex. Addition of DNA polymerase to this mixture produces a DNA strand identical to the template except for the mutated base. In addition, this new DNA strand will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion. After the double-stranded heteroduplex template strand is nicked with an appropriate restriction enzyme, the template strand can be digested with ExoIII nuclease or another appropriate nuclease beyond the region containing the site(s) to be mutagenized. The reaction is then stopped, leaving a molecule that is only partially single-stranded. A complete double-stranded DNA homoduplex is then formed using 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.

[0161] As noted above, the sequences of the oligonucleotide sets are of sufficient length to hybridize to the template nucleic acid and may, but need not, contain restriction sites. DNA templates can be generated either by vectors derived from bacteriophage M13 vectors or by vectors containing a single-stranded phage origin of replication as described by Viera et al., Meth. Enzymol., 153:3 (1987). Therefore, to generate single-stranded templates, the DNA to be mutated must be inserted into one of these vectors. Production of single-stranded templates is described in sections 4.21-4.41 of Sambrook et al. (supra).

[0162] According to another method, antigen binding can be restored during antibody humanization by selecting restored hypervariable regions (see, e.g., U.S. Application No. 11 / 061,841, filed February 18, 2005). This method involves incorporating non-human hypervariable regions 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 can be accomplished by modification in the acceptor framework sequence.

[0163] According to another method, a library can be generated by providing upstream and downstream oligonucleotide sets, each set having a plurality of oligonucleotides with different sequences, the different sequences being established by the codon sets provided within the sequences of the oligonucleotides. The upstream and downstream oligonucleotide sets can be used in a polymerase chain reaction with a variable domain template nucleic acid sequence to generate a "library" of PCR products. The PCR products can be referred to as "nucleic acid cassettes" because they can be fused to other related or unrelated nucleic acid sequences, such as viral coat proteins and dimerization domains, using established molecular biology techniques.

[0164] The sequences of the PCR primers contain one or more of the codon sets designed for solvent-accessible and highly diverse positions in the hypervariable regions. As noted above, a codon set is a set of different nucleotide triplet sequences used to encode desired variant amino acids.

[0165] Antibody selectants that meet the desired criteria as selected by appropriate screening / selection steps can be isolated and cloned using standard recombinant techniques.

[0166] It is further important that antibodies be humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences so that the desired antibody characteristic, such as increased affinity for the target antigen(s), is achieved. In general, hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[0167] Various forms of humanized anti-CTHRC1 antibodies are contemplated. For example, the humanized antibody may be an antibody fragment such as a Fab. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

[0168] As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice has been described, resulting in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigenic stimulation (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. Patent Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807, and WO 97 / 17852).

[0169] 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 repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in frame into either the major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in a variety of formats, as reviewed, for example, in Johnson, Kevin S., and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a wide variety of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the pancreases of immunized mice. Essentially following 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), a repertoire of V genes from unimmunized human donors can be constructed, and antibodies against a wide variety of antigens (including self-antigens) can be isolated.

[0170] As discussed above, human antibodies may also be generated by in vitro activated B cells (see, eg, US Pat. Nos. 5,567,610 and 5,229,275).

[0171] In another embodiment, the antibodies of the present disclosure are human monoclonal antibodies. Such human monoclonal antibodies directed against CTHRC1 can be generated using transgenic or transchromosomic mice carrying parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include those referred to herein as HuMAb Mice™ and KM Mice™, respectively, and are collectively referred to herein as "human Ig mice."

[0172] HuMAb Mice™ (Medarex, Inc.) contain human immunoglobulin gene minilocuses encoding unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences, along with 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 mouse IgM or κ expression, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate 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). The preparation and use of HuMAb Mice™ and the genomic modifications carried by such mice are 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, the entire contents of which are expressly incorporated herein by reference in their entirety.Further, U.S. Patent 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 No. 5,545,807, PCT Publication Nos. WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884, and WO 99 / 45962, and PCT Publication No. WO 01 / 14424.

[0173] In another embodiment, the human antibodies of the disclosure can be produced using a mouse carrying human immunoglobulin sequences on a transgene and transchromosome, such as a mouse carrying a human heavy chain transgene and a human light chain transchomosome, referred to herein as a "KM Mouse™," and described in detail in PCT Publication No. WO 02 / 43478.

[0174] Still further, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-CTHRC1 antibodies of the present disclosure. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used; such mice are described, for example, in U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963.

[0175] Furthermore, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-CTHRC1 antibodies of the present disclosure. For example, mice carrying both a human heavy chain transchromosome and a human light chain transchromosome, 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, cattle 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. WO2002 / 092812) and can be used to produce the anti-CTHRC1 antibodies of the present disclosure. Additional examples of transgenic animals that can be used to produce anti-CTHCR1 antibodies include OmniRat™ and OmniMouse™ (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. Patent No. 8,907,157; ​​European Patent No. 2152880B1; European Patent No. 2336329B1). Yet another example is the use of VELOCIMMUNE™ technology (see, e.g., U.S. Patent No. 6,596,541; Regeneron Pharmaceuticals, VELOCIMMUNE™). Briefly, VELOCIMMUNE® technology involves the generation of transgenic mice whose genomes comprise human heavy and light chain variable regions operably linked to endogenous mouse constant region loci, such that the mice produce antigen-binding proteins, e.g., antibodies, comprising human variable regions and mouse constant regions in response to antigenic challenge. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding the human heavy and light chain constant regions.This DNA is then expressed in cells capable of expressing fully human antibodies.

[0176] 4. Antibody fragment In certain circumstances, there are advantages to using antibody fragments rather than whole antibodies: the smaller size of the fragments allows for rapid clearance and may lead to improved access to solid tumors.

[0177] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were 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, allowing for the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries described 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 approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-lives containing salvage receptor-binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv) (see WO 93 / 16185, U.S. Patent No. 5,571,894, and U.S. Patent No. 5,587,458). Fv and sFv are the only species with intact binding sites that are devoid of constant regions and are therefore suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins can be constructed to generate fusion of effector proteins at either the amino or carboxy terminus of the sFv (see Antibody Engineering, ed. Borrebaeck, supra). An antibody fragment may also be a "linear antibody," eg, as described in US Pat. No. 5,641,870.

[0178] In one embodiment, an scFv derived from an anti-CTHRC1 antibody is used in 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 (and combinations of anti-CTHRC1 antibody portions, e.g., scFvs) that can be used as targeting arms directed against the CTHRC1 tumor epitope in the chimeric antigen receptors of CAR-T cells or CAR-NK cells or CAR-macrophages. Such fragments are not necessarily proteolytic fragments, but rather portions of a polypeptide sequence that can confer affinity for a target.

[0179] 5. Bispecific antibodies Bispecific antibodies are antibodies with binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the CTHRC1 protein described herein. Other such antibodies may combine a CTHRC1-binding site with a binding site for another protein. Alternatively, the anti-CTHRC1 arm may be combined with an arm that binds to a triggering molecule on leukocytes, such as a T cell receptor molecule (e.g., CD3), or an Fc receptor for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), to focus and localize cellular defense mechanisms to CTHRC1-expressing cells. Bispecific antibodies may also be used to localize cytotoxic agents to CTHRC1-expressing cells. These antibodies possess a CTHRC1-binding arm and an arm that binds a cytotoxic agent (e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain, methotrexate, or radioactive isotope hapten). Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0180] WO96 / 16673 describes a bispecific anti-ErbB2 / anti-FcγRIII antibody, and U.S. Patent No. 5,837,234 discloses a bispecific anti-ErbB2 / anti-FcγRI antibody. A bispecific anti-ErbB2 / Fcα antibody is shown in WO98 / 02463. U.S. Patent No. 5,821,337 teaches a bispecific anti-ErbB2 / anti-CD3 antibody.

[0181] Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy-light chain pairs, with these two chains having different specificities (Millstein et al., Nature 305:537-9 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, usually performed by affinity chromatography steps, is somewhat 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).

[0182] 6. Antibody Variants and Modifications a) Substitution, insertion, and deletion variants In addition to the anti-CTHRC1 antibodies described herein, it is contemplated that variant anti-CTHRC1 antibodies can be prepared. Variant anti-CTHRC1 antibodies can be prepared by introducing appropriate nucleotide changes into the coding DNA and / or by synthesizing the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid changes can alter post-translational processes of anti-CTHRC1 antibodies, such as altering the number or position of glycosylation sites or altering membrane-tethering properties.

[0183] Alterations in the anti-CTHRC1 antibodies described herein can be made using any of the techniques and guidelines for conservative and non-conservative mutations described, for example, in U.S. Patent No. 5,364,934. Alterations can be substitutions, deletions, or insertions of one or more codons encoding the antibody or polypeptide, resulting in a change in the amino acid sequence compared to the native sequence antibody or polypeptide. Optionally, the alteration is through the substitution of at least one amino acid with any other amino acid in one or more domains of the anti-CTHRC1 antibody. Guidance for determining which amino acid residues can be inserted, substituted, or deleted without adversely affecting the desired activity can be found by comparing the sequence of the anti-CTHRC1 antibody with that of a known protein molecule of the same species and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can result from replacing one amino acid with another amino acid having similar structural and / or chemical properties, e.g., replacing leucine with serine, i.e., conservative amino acid substitutions. Insertions or deletions can optionally range from about 1 to 5 amino acids. Permissible changes may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

[0184] Anti-CTHRC1 antibody fragments are provided herein. Such fragments may be, for example, N-terminally or C-terminally shortened or lack internal residues when compared with the full-length native antibody or protein. Certain fragments lack amino acid residues that are not essential for the desired biological activity of anti-CTHRC1 antibodies.

[0185] Anti-CTHRC1 antibody fragments may be prepared by any of several conventional techniques. The desired peptide fragment may be chemically synthesized. An alternative approach involves producing antibody or polypeptide fragments by enzymatic digestion, for example, by treating the protein with an enzyme known to cleave the protein at sites defined by specific amino acid residues, or by digesting DNA with a suitable restriction enzyme and isolating the desired fragment. Yet another suitable technique involves isolating a DNA fragment encoding the desired antibody or polypeptide fragment and amplifying it by polymerase chain reaction (PCR). Oligonucleotides defining the desired termini of the DNA fragment are used at the 5' and 3' primers for PCR. Preferably, the anti-CTHRC1 antibody fragment shares at least one biological and / or immunological activity with the native anti-CTHRC1 antibody disclosed herein.

[0186] In certain 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, such as those referred to as exemplary substitutions in Table 1 or further described below with reference to amino acid classes, are introduced and the products screened. [Table 2]

[0187] Substantial alterations in the function or immunological properties of anti-CTHRC1 antibodies are achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​substitution, e.g., as a sheet or helix structure, (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 the following groups based on common side chain properties: (1) Hydrophobic: norleucine, met, ala, val, leu, ile, (2) Neutral hydrophilicity: cys, ser, thr, (3) Acidic: asp, glu, (4) Basic: asn, gln, his, lys, arg, (5) Residues that affect chain orientation: gly, pro, and (6) Aromatic: trp, tyr, phe

[0188] Non-conservative substitutions involve exchanging a member of one of these classes for another. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

[0189] Changes can be made using 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 enzyme selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)), or other known techniques can be performed on cloned DNA to produce anti-CTHRC1 antibody variant DNA.

[0190] Scanning amino acid analysis can also be used to identify one or more amino acids along a contiguous sequence. Among preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically the preferred scanning amino acid within this group because it eliminates the side chain beyond the beta-carbon and is unlikely to alter the main-chain conformation of the variant (Cunningham and Wells, Science, 244:1081-5 (1989)). Alanine is also typically preferred because it is the most common amino acid. Furthermore, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (WH Freeman & Co., NY); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not yield a sufficient amount of variant, an isosteric amino acid can be used.

[0191] Any cysteine ​​residue not involved in maintaining the proper conformation of the anti-CTHRC1 antibody may also be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) may be added to an anti-CTHRC1 antibody to improve its stability, particularly where the antibody is an antibody fragment such as an Fv fragment.

[0192] A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of an antigen-antibody complex to identify contact points between the antibody and CTHRC1 polypeptide. Such contact and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants can be subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0193] Nucleic acid molecules encoding amino acid sequence variants of anti-CTHRC1 antibodies are prepared by a variety of methods known in the art, including, but not limited to, isolation 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 pre-prepared variant or non-variant versions of anti-CTHRC1 antibodies.

[0194] b) Qualification Covalent modification of anti-CTHRC1 antibodies is included within the scope of the present invention. One type of covalent modification involves reacting targeted amino acid residues of anti-CTHRC1 antibodies with organic derivatizing agents that can react with selected side chains or N- or C-terminal residues of anti-CTHRC1 antibodies. For example, derivatization with bifunctional agents is useful for cross-linking 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., with 4-azidosalicylic acid, homobifunctional imidoesters including disuccinimidyl esters such as 3,3′-dithiobis(succinimidyl propionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane, and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0195] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0196] Another type of covalent modification of an anti-CTHRC1 antibody within the scope of the present invention involves altering the native glycosylation pattern of the antibody or polypeptide. For purposes herein, "altering the native glycosylation pattern" is intended to mean deleting one or more carbohydrate moieties found in a native-sequence anti-CTHRC1 antibody (either by removing the underlying glycosylation site or by deleting glycosylation by chemical and / or enzymatic means) and / or adding one or more glycosylation sites that are not present in the native-sequence anti-CTHRC1 antibody. In addition, this phrase includes qualitative alterations in the glycosylation of the native protein, involving changes in the nature of the various carbohydrate moieties present and their proportions.

[0197] Glycosylation of antibodies and other polypeptides is typically either 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 the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either 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 can also be used.

[0198] Addition of glycosylation sites to an anti-CTHRC1 antibody is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above-mentioned tripeptide sequences (in the case of N-linked glycosylation sites). Alterations may also be made by adding or substituting one or more serine or threonine residues to the original anti-CTHRC1 antibody sequence (in the case of O-linked glycosylation sites). Optionally, the anti-CTHRC1 antibody amino acid sequence may be altered at the DNA level, particularly by mutating the DNA encoding the anti-CTHRC1 antibody at preselected bases to generate codons that translate into the desired amino acids.

[0199] Another means of increasing the number of carbohydrate moieties on an 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).

[0200] Removal of carbohydrate moieties present on anti-CTHRC1 antibodies may be accomplished chemically or enzymatically, or by mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and are described, for example, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of various endo- and exo-glycosidases, as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

[0201] c) Fc region variants It may be desirable to modify the antibody of the invention with respect to effector function, for example, to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Alternatively, or additionally, cysteine ​​residue(s) may be introduced in the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated 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, such as those described in Wolff et al., Cancer Research 53:2560-5 (1993). Alternatively, antibodies can be engineered to have dual Fc regions, thereby enhancing complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-30 (1989). To increase the serum half-life of an antibody, a salvage receptor binding epitope may be incorporated into the antibody (e.g., antibody fragment), as described, for example, in U.S. Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in 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 the IgG molecule.

[0202] In some embodiments, the Fc portion of an anti-CTHRC1 antibody, including a humanized antibody, of the present disclosure may comprise a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the following Fc sequences: [Table 3-1] [Table 3-2] Alternatively, it may comprise a sequence pair having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the following Fc sequence pairs: [Table 4]

[0203] In some embodiments, the Fc portion of an anti-CTHRC1 antibody, including a humanized antibody, of the present disclosure can optionally comprise a sequence having the sequence of any one of the following Fc sequences: [Table 5-1] [Table 5-2] Alternatively, it may comprise a pair of sequences, each having the sequence of any one of the following Fc sequence pairs: [Table 6]

[0204] d) Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody, which may 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 generated, for example, as described in U.S. Pat. No. 7,521,541.

[0205] e) Immunoconjugates The presently disclosed inventive subject matter also provides immunoconjugates comprising an antibody disclosed herein conjugated to one or more cytotoxic agents, e.g., chemotherapeutic agents or drugs, growth inhibitory agents, proteins, peptides, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes. For example, an antibody of the disclosed inventive subject matter can be operatively linked (e.g., by chemical coupling, genetic fusion, noncovalent association, or other methods) to one or more other binding molecules, e.g., another antibody, antibody fragment, peptide, or binding mimetic.

[0206] 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 maytansinoids (see U.S. Pat. Nos. 5,208,020, 5,416,064, and European Patent EP 0425235 B1), auristatins, such as monomethyl auristatin drug moieties DE and DF (MMAE and MMAF), and the like. ) (see U.S. Patent Nos. 5,635,483, 5,780,588, and 7,498,298), dolastatins, calicheamicin or derivatives thereof (see U.S. Patent 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 (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. Patent No. 6,630,579), methotrexate, vindesine, taxanes (e.g., docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel), trichothecenes, and CC1065.In certain embodiments, the immunoconjugate comprises an antibody 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, modeccin A chain, alpha-sarcin, Aleurites fordii protein, diansin protein, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0207] In certain embodiments, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of 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 a radioactive conjugate is used for detection, it can be a radioactive atom, such as tc99m or I, for scintigraphy studies. 123 , or spin labels for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, MRI), such as iodine-123, iodine-131, indium-111, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron.

[0208] 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 (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins 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 WO94 / 11026. The linker can be a "cleavable linker" that facilitates the release of the cytotoxic drug inside the cell. For example, an acid-labile linker, a peptidase-sensitive linker, a photolabile linker, a dimethyl linker, or a disulfide-containing linker (Chari et al., Cancer Res. 52:127-131 (1992), U.S. Patent No. 5,208,020) can be used. Non-limiting examples of linkers are disclosed above.The immunoconjugates disclosed herein expressly contemplate, but are not limited to, such conjugates prepared with cross-linker reagents 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-vinylsulfone)benzoate), which are commercially available (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0209] f) Antibody fusion The inventive subject matter of the present disclosure also encompasses antibody fusions. For example, proteins can be linked together by either chemical or genetic engineering using methods known in the art. See, e.g., Gillies et al., Proc. Nat'l Acad. Sci. USA 89:1428-1432 (1992) and U.S. Patent No. 5,650,150.

[0210] In one example, the present disclosure encompasses an anti-CTHRC1 antibody-cytokine fusion protein. In principle, the anti-CTHRC1 antibodies disclosed herein can be 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)).

[0211] In another example, the present disclosure encompasses anti-CTHRC1 antibody-T cell engager fusion proteins. As discussed herein, anti-CTHRC1 antibody-T cell engager fusion proteins comprise a fusion between an anti-CTHRC1 antibody and a ligand for a receptor expressed on a T cell. Examples of such ligands include, but are not limited to, CD40L, OX40L, 4-1BBL, CD80 / 86, ICOSL, and the like. In some embodiments, the ligand is fused to the Fc portion of the anti-CTHRC1 antibody. In some embodiments, the ligand is fused to the C-terminus of the light chain of the anti-CTHRC1 antibody. Such techniques have been described for 4-1BBL (Dafne M. et al., Journal of Immunotherapy 38(8):714-722 (2008)), and similar techniques can be used to generate other antibody-T cell engager fusion proteins.

[0212] B. Certain Methods for Producing Antibodies 1. Screening for anti-CTHRC1 antibodies with desired properties Techniques for generating antibodies that bind to CTHRC1 polypeptides are described above. If desired, antibodies with certain biological properties may be further selected.

[0213] The growth inhibitory effect of the anti-CTHRC1 antibodies of the present invention can be assessed by methods known in the art, for example, using cells that express CTHRC1 polypeptide, either endogenously or after transfection with the CTHRC1 gene. For example, appropriate tumor cell lines and CTHRC1 transfected cells can be treated with various concentrations of the anti-CTHRC1 monoclonal antibodies of the present invention for several days (e.g., 2-7 days) and analyzed by staining with crystal violet or MTT, or by some other colorimetric assay. Another method for measuring proliferation would be by H-thymidine incorporation by cells treated with or without the anti-CTHRC1 antibodies of the present invention. After treatment, cells are harvested, and the amount of radioactivity incorporated into DNA is quantified in a scintillation counter. An appropriate positive control includes treating a selected cell line with a growth inhibitory antibody known to inhibit the growth of that cell line. In vivo tumor cell growth inhibition can be determined in various ways known in the art. The tumor cells can overexpress and / or present CTHRC1 polypeptide. In one embodiment, an anti-CTHRC1 antibody inhibits cell proliferation of CTHRC1-expressing tumor cells in vitro or in vivo by about 25-100%, more preferably about 30-100%, and even more preferably about 50-100% or 70-100% compared to untreated tumor cells at an antibody concentration of about 0.5-30 μg / mL. Growth inhibition can be measured at an antibody concentration of about 0.5-30 μg / mL or about 0.5 nM-200 nM in cell culture, where growth inhibition is determined 1-10 days after exposure of tumor cells to the antibody. An antibody is growth inhibitory in vivo if administration of an anti-CTHRC1 antibody at about 1 μg / kg to about 100 mg / kg body weight results in a reduction in tumor size or tumor cell proliferation within about 5 days to 3 months, preferably about 5-30 days, after the first administration of the antibody.

[0214] To select for anti-CTHRC1 antibodies that induce cell death, loss of membrane integrity, as indicated by, for example, propidium iodide (PI), trypan blue, or 7AAD uptake, can be assessed compared to controls. PI uptake assays can be performed in the absence of complement and immune effector cells. CTHRC1 polypeptide-expressing tumor cells are incubated with medium alone or medium containing the appropriate anti-CTHRC1 antibody (e.g., at about 10 μg / mL). The cells are incubated for a period of 3 days. Following each treatment, the cells are washed and aliquoted into 12 × 75 tubes with 35 mm strainer caps (1 mL per tube, 3 tubes per treatment group) to remove cell clumps. Tubes are then charged with PI (10 μg / mL). Samples can be analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). An anti-CTHRC1 antibody that induces a statistically significant level of cell death as determined by PI uptake can be selected as a cell death-inducing anti-CTHRC1 antibody.

[0215] To screen for antibodies that bind to the epitope on the CTHRC1 polypeptide bound by the antibody of interest, a conventional cross-blocking assay can be performed, such as that described in "Antibodies, A Laboratory Manual," Cold Spring Harbor Laboratory, Ed 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, mutations can be introduced into the antibody sequence, for example, by alanine scanning, to identify contact residues. The mutant antibody is first tested for binding with a polyclonal antibody to ensure proper folding. In a different method, peptides corresponding to different regions of the CTHRC1 polypeptide can be used in a competition assay with the test antibody or with an antibody having a characterized or known epitope.

[0216] In addition, candidate antibodies may also be screened for functionality using 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 in tumor size.

[0217] 2. Specific Library Screening Methods The anti-CTHRC1 antibodies of the present invention can be generated by using combinatorial libraries to screen for antibodies with the desired activity(ies). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are generally described in Hoogenboom et al. (2001) in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ), and in certain embodiments, in Lee et al. (2004) J. Mol. Biol. 340:1073-93.

[0218] In principle, synthetic antibody clones are selected by screening a phage library containing phages displaying various antibody variable region fragments (Fv) fused to phage coat proteins. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. Binding clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles. Any of the anti-CTHRC1 antibodies of the present invention can be obtained by designing a suitable antigen screening procedure to select for the desired phage clone, followed by constructing a full-length anti-CTHRC1 antibody clone using the Fv sequence from the desired phage clone and a suitable constant region (Fc) sequence described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda, MD (1991), vols. 1-3.

[0219] In certain embodiments, the antigen-binding domain of an antibody is formed from two variable (V) regions of approximately 110 amino acids, one from each of the light (VL) and heavy (VH) chains, each displaying three hypervariable loops (HVRs) or complementarity-determining regions (CDRs). The variable domains can be functionally displayed on phage either as single-chain Fv (scFv) fragments in which the VH and VL are covalently linked via a short, flexible peptide, as described in Winter et al., Ann. Rev. Immunol., 12:433-55 (1994), or as Fab fragments in which they are each fused to a constant domain and interact noncovalently. As used herein, scFv-encoding phage clones and Fab-encoding phage clones are collectively referred to as "Fv phage clones" or "Fv clones."

[0220] Repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in phage libraries, which can then be screened for antigen-binding clones, as described by Winter et al., Ann. Rev. Immunol., 12:433-55 (1994). Libraries from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, naive repertoires can be cloned to provide a single source of human antibodies against a wide range of non-self antigens and also self antigens, without any immunization, as described by Griffiths et al., EMBO J. 12:725-34 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and perform in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-8 (1992).

[0221] In certain embodiments, filamentous phage are used to display antibody fragments by fusion to the minor coat protein pill. Antibody fragments can be displayed as single-chain Fv fragments, in which the VH and VL domains are connected on the same polypeptide chain by a flexible polypeptide spacer, as described, for example, by Marks et al., J. Mol. Biol., 222:581-97 (1991), or as Fab fragments, in which one chain is fused to pill and the other is secreted into the bacterial host cell periplasm, and assembly of the Fab coat protein structure displaces a portion of the wild-type coat protein so that it is displayed on the phage surface.

[0222] Generally, nucleic acids encoding antibody gene fragments are obtained from immune cells collected from humans or animals. If a library biased in favor of anti-CTHRC1 clones is desired, a subject is immunized with CTHRC1 to generate an antibody response, and spleen cells and / or circulating B cells, other peripheral blood lymphocytes (PBLs), are collected for library construction. In some embodiments, a human antibody gene fragment library biased in favor of anti-CTHRC1 clones is obtained by generating an anti-CTHRC1 antibody response in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system), such that CTHRC1 immunization generates B cells that produce human antibodies against CTHRC1. The generation of human antibody-producing transgenic mice is described below.

[0223] Further enrichment of the anti-CTHRC1-reactive cell population can be obtained by isolating B cells expressing CTHRC1-specific membrane-bound antibodies using a suitable screening procedure, for example, by cell separation using CTHRC1 affinity chromatography or adsorption of cells to fluorescent dye-labeled CTHRC1, followed by flow-activated cell sorting (FACS).

[0224] Alternatively, the use of spleen cells and / or B cells or other PBLs from unimmunized donors provides a better representation of the possible antibody repertoire and allows for the construction of antibody libraries using any animal species (human or non-human) to which CTHRC1 is not antigenic. For libraries incorporating in vitro antibody gene construction, stem cells are harvested from the subject to provide nucleic acids encoding unrearranged antibody gene segments. Immune cells of interest can be obtained from a variety of animal species, including humans, mice, rats, rabbits, wolves, dogs, cats, pigs, cattle, horses, and avian species.

[0225] Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered and amplified from the cells of interest. In the case of a rearranged VH and VL gene library, the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes, followed by polymerase chain reaction (PCR) using primers matching the 5' and 3' ends of the rearranged VH and VL genes, as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86:3833-7 (1989), thereby generating a diverse V gene repertoire for expression.

[0226] V genes can be amplified from cDNA and genomic DNA using a reverse primer at the 5' end of the exon encoding the mature V domain and a forward primer placed within the J segment, as described by Orlandi et al. (1989) and Ward et al., Nature, 341:544-6 (1989). However, when amplifying from cDNA, the reverse primer can also be placed in the leader exon, as described by Jones et al., Biotechnol., 9:88-9 (1991), and the forward primer can be within the constant region, as described by Sastry et al., Proc. Natl. Acad. Sci. (USA), 86:5728-32 (1989). To maximize complementarity, degeneracy can be incorporated into the primers, as described by 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 configurations present in an immune cell nucleic acid sample, as described, for example, in the method of Marks et al., J. Mol. Biol., 222:581-97 (1991) or in the method of Orum et al., Nucleic Acids Res., 21:4491-98 (1993). When cloning the amplified DNA into an expression vector, rare restriction sites can be introduced into the PCR primers as tags at one end, as described in Orlandi et al. (1989), or further PCR amplification can be performed using tagged primers, as described in Clackson et al., Nature, 352:624-628 (1991).

[0227] Synthetically rearranged V gene repertoires can be generated in vitro from V gene segments. Most human VH gene segments have been cloned, sequenced (as reported in Tomlinson et al., J. Mol. Biol., 227:776-98 (1992)), and mapped (as 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 diverse VH gene repertoires using PCR primers encoding H3 loops of various sequences and lengths, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be generated by concentrating all sequence diversity in a single, long H3 loop, as described by 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 generate synthetic light chain repertoires. Synthetic V gene repertoires based on various VH and VL folds and L3 and H3 lengths will encode antibodies with considerable structural diversity. Following amplification of V gene-encoding DNA, germline V gene segments can be rearranged in vitro according to the method of Hoogenboom and Winter, J. Mol. Biol., 227:381-8 (1992).

[0228] Repertoires of antibody fragments can be constructed by combining VH and VL gene repertoires together in several ways. Each repertoire can be produced in a different vector, and the vectors can be recombined in vitro, as described by Hogrefe et al., Gene, 128:119-26 (1993), or in vivo by combinatorial infection, e.g., the loxP system, as described by Waterhouse et al., Nucl. Acids Res., 21:2265-66 (1993). In vivo recombination techniques take advantage of the double-chain nature of Fab fragments to overcome the limitations on library size imposed by the transformation efficiency of E. coli. Naive VH and VL repertoires are cloned separately, one into a phagemid and the other into a phage vector. These two libraries are then combined by phage infection of phagemid-containing bacteria, such that each cell contains a different combination, with the library size limited only by the number of cells present (approximately 10 clones). Both vectors contain in vivo recombination signals, which allow the VH and VL genes to recombine on a single replicon and be co-packaged into phage virions. These large libraries provide a large number of diverse antibodies with good affinity (Kd of approximately 10 M).

[0229] Alternatively, repertoires may be cloned sequentially into the same vector, as described, for example, in Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or may be assembled together by PCR and then cloned, as described, for example, in Clackson et al., Nature, 352:624-628 (1991). PCR assembly can also be used to link VH and VL DNA with DNA encoding a flexible peptide spacer to form single-chain Fv (scFv) repertoires. In yet another technique, as described in Embleton et al., Nucl. Acids Res., 20:3831-3837 (1992), "in-cell PCR assembly" is used to combine VH and VL genes within lymphocytes by PCR, and the repertoire of linked genes is then cloned.

[0230] Antibodies (either natural or synthetic) produced by naive libraries have moderate affinity (approximately 10 6 ~10 7Although affinity maturation can be mimicked in vitro by constructing a secondary library as described in Winter et al. (1994) (supra) and reselecting from it, affinity maturation can also be mimicked in vitro. For example, mutations can be randomly introduced in vitro by using an error-prone polymerase (as reported in Leung et al., Technique, 1:11-5 (1989)), 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). Additionally, affinity maturation can be performed by randomly mutating one or more CDRs in selected individual Fv clones, for example, using PCR with primers carrying random sequences spanning the CDRs of interest, and screening for higher affinity clones. WO9607754 describes a method for generating a library of light chain genes by inducing mutagenesis in the complementarity-determining regions of immunoglobulin light chains. Another effective approach is to recombine VH or VL domains selected by phage display with a repertoire of naturally occurring V domain variants obtained from unimmunized donors, as described by Marks et al., Biotechnol., 10:779-83 (1992), and screen for higher affinity by several rounds of chain reshuffling. This technique allows the production of antibodies and antibody fragments with affinities of about 10 M or less.

[0231] 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, can be expressed on host cells attached to an adsorption plate, can be used in cell sorting, can be conjugated to biotin for capture on streptavidin-coated beads, or can be used in any other method for panning a phage display library.

[0232] The phage library sample is contacted with the immobilized CTHRC1 under conditions suitable for binding of at least a portion of the phage particles to the adsorbent. Typically, conditions, including pH, ionic strength, temperature, etc., are selected to mimic physiological conditions. The phages bound to the solid phase are washed and then eluted by acid, as described, for example, in Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-82 (1991), or by alkali, as described, for example, in Marks et al., J. Mol. Biol., 222:581-97 (1991), or by CTHRC1 antigen competition, in a procedure similar to the antigen competition method of Clackson et al., Nature, 352:624-8 (1991). Phages can be enriched 20- to 1,000-fold in a single selection round. Furthermore, the enriched phages can be grown in bacterial culture and subjected to further rounds of selection.

[0233] The efficiency of selection depends on many factors, including the dissociation rate during washing and whether multiple antibody fragments on a single phage can simultaneously bind to the antigen. Antibodies with fast dissociation rates (and weak binding affinity) can be retained by using short washes, multivalent phage display, and a high coating density of antigen on the solid phase. High density not only stabilizes phage through multivalent interactions, but also favors the rebinding of dissociated phage. Selection of antibodies with slow dissociation rates (and good binding affinity) can be facilitated by using long washes and monovalent phage display, as described in Bass et al., Proteins, 8:309-314 (1990) and WO92 / 09690, and by using a low coating density of antigen, as described in Marks et al., Biotechnol., 10:779-783 (1992).

[0234] It is possible to select between phage antibodies with different affinities for CTHRC1, even if they have slightly different affinities. However, random mutation of selected antibodies (e.g., as performed in some affinity maturation techniques) can generate many variants, most of which bind to the antigen and a few with higher affinities. Limiting CTHRC1 can result in the competition out of rare high-affinity phages. To retain all higher-affinity variants, phage can be incubated with excess biotinylated CTHRC1, but at a molar concentration lower than the target molar affinity constant of CTHRC1. High-affinity-binding phage can then be captured by streptavidin-coated paramagnetic beads. Such "equilibrium capture" allows antibodies to be selected according to their binding affinity with a sensitivity that allows the isolation of mutant clones with only two-fold higher affinity from a large excess of phage with lower affinity. The conditions used to wash phage bound to a solid phase can also be manipulated to differentiate based on dissociation rates.

[0235] Anti-CTHRC1 clones may 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 (1) isolating anti-CTHRC1 clones from the phage library described above and, optionally, amplifying the population of isolated phage clones by growing the population in a suitable bacterial host, (2) selecting for CTHRC1 and a second protein for which blocking and non-blocking activity are desired, respectively, (3) adsorbing the anti-CTHRC1 phage clones to immobilized CTHRC1, (4) using excess second protein to elute any unwanted clones that recognize CTHRC1 binding determinants that overlap with or are shared with those of the second protein, and (5) eluting the clones that remain adsorbed following step (4). Optionally, clones with the desired blocking / non-blocking properties can be further enriched by repeating the selection procedure described herein one or more times.

[0236] DNA encoding the hybridoma-derived monoclonal antibodies or phage-displayed Fv clones of the present invention can be readily 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 a hybridoma or phage DNA template). Once isolated, the DNA can be placed into expression vectors, which can then be transfected into host cells that do not otherwise produce immunoglobulin proteins, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of the desired monoclonal antibody in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol. 5:256 (1993) and Pluckthun, Immunol. Rev. 130:151 (1992).

[0237] DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding heavy and / or light chain constant regions (e.g., suitable 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 understood that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be derived from any human or animal species. Included in the definition of "chimeric" and "hybrid" antibodies as used herein are Fv clones derived from variable domain DNA of one animal (e.g., human) species and then fused to constant region DNA of another animal species to form the coding sequence(s) for a "hybrid" full-length heavy and / or light chain. In certain embodiments, Fv clones derived from human variable DNA are fused to human constant region DNA to form the coding sequence(s) for a full-length or partial-length human heavy and / or light chain.

[0238] DNA encoding a hybridoma-derived anti-CTHRC1 antibody can also be modified, for example, by substituting coding sequences for human heavy and light chain constant domains for the homologous murine sequences derived from the hybridoma clone (e.g., as described by Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-5 (1984)). DNA encoding a hybridoma- or Fv clone-derived antibody or fragment can be further modified by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, "chimeric" or "hybrid" antibodies having the binding specificity of antibodies derived from the Fv clones or hybridoma clones of the invention can be prepared.

[0239] 3.Generating antibodies using CAR-T cells The anti-CTHRC1 antibodies of the present invention can be generated by screening for antibodies with the desired activity(ies) using a CAR T cell platform. Chimeric antigen receptors (CARs) consist of an extracellular antigen recognition domain (usually a single-chain variable fragment (scFv) antibody) linked to a transmembrane domain and a 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 function, addressing the goal of effector cell activation independent of the major histocompatibility complex. First-generation CARs were typically constructed by fusing an scFv-based TAA-binding domain to a cytoplasmic signaling domain derived from either the zeta chain of the T cell receptor (TCR) / CD3 complex or the gamma chain associated with some Fc receptors (Gross, G. et al., Proc Natl Acad Sci USA 86:10024-8 (1989)). Second-generation CARs (CARv2) have also been developed that contain the signaling region of TCRζ in tandem with signaling domains derived from the T cell costimulatory receptors CD28, 4-1BB (CD137), or OX40 (CD134) (Sanz, L. et al., Trends Immunol 25:85-91 (2004)). Third-generation CARs further combine the signaling potential of two costimulatory domains (e.g., both CD28 and 4-1BB) (Subklewe, M., et al., Transfus Med Hemother 46(1):15-24 (2019)).

[0240] Upon encountering an antigen, the interaction of the transfected CAR can trigger effector functions and mediate tumor cell cytolysis. The practicality and effectiveness of the CAR approach have been demonstrated in various animal models and in ongoing clinical trials using CAR-based engineered T lymphocytes for the treatment of cancer patients. Lipowska-Bhalla, G. et al., Cancer Immunol Immunother 61:953-62 (2012). CARs enable effector cells to target any natural extracellular antigen for which a suitable antibody exists. 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, M. H. et al., Nat Rev Immunol 5:928-40 (2005)).

[0241] Current methods for generating recombinant antibodies are primarily 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 that leverages the functional capabilities of T lymphocytes has recently been described. Alonso-Camino et al., Molecular Therapy Nucleic Acids (2013) 2, e93. Displaying antibodies on the surface of T lymphocytes as part of CAR-mediated signaling would ideally link antigen-antibody interactions to demonstrable changes in cell phenotype due to surface expression of activation markers. Alonso-Camino, V. et al., PLoS ONE 4:e7174 (2009). By using an scFv-based CAR that recognizes a TAA, combining CAR-mediated activation with fluorescence-activated cell sorting (FACS) of CD69+ T cells resulted in at least 10 T cells after two rounds of activation. 3It has been demonstrated that a 2-fold enrichment factor enables the isolation of surface TAA binders, resulting in a homogenous population of T cells expressing a TAA-specific CAR. Alonso-Camino, V, et al., PLoS ONE 4:e7174 (2009).

[0242] C. Preparation of anti-CTHRC1 antibody The following description primarily relates to the production of anti-CTHRC1 antibodies by culturing cells transformed or transfected with a vector containing an anti-CTHRC1 antibody-encoding nucleic acid. It is, of course, contemplated that anti-CTHRC1 antibodies may be prepared using alternative methods well known in the art. For example, the appropriate amino acid sequence, or a portion thereof, may be produced by direct peptide synthesis using solid-phase techniques (e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154 (1963)). In vitro protein synthesis may be performed using manual techniques or automated. Automated synthesis may be accomplished, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using the manufacturer's instructions. Various portions of the anti-CTHRC1 antibody may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-CTHRC1 antibody.

[0243] 1. Isolation of DNA encoding anti-CTHRC1 antibody DNA encoding an anti-CTHRC1 antibody may be obtained from a cDNA library prepared from tissue believed to harbor anti-CTHRC1 antibody mRNA and express it at detectable levels. Thus, human anti-CTHRC1 antibody DNA can be conveniently obtained from a cDNA library prepared from human tissue. Anti-CTHRC1 antibody-encoding genes may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0244] Libraries can be screened using probes (e.g., oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening of cDNA or genomic libraries with selected probes can be carried out 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 means for isolating genes encoding anti-CTHRC1 antibodies is to use PCR techniques (Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[0245] Techniques for screening cDNA libraries are well known in the art. The oligonucleotide sequence selected as a probe should be of sufficient length and sufficiently unambiguous to minimize false positives. The oligonucleotide is preferably labeled so that it can be detected upon hybridization to DNA in the library being screened. Labeling methods are well known in the art and include the use of radioactive labels such as 32P-labeled ATP, biotinylation, or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al. (supra).

[0246] Sequences identified in such library screening methods can be compared and aligned with other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of a molecule or across the full-length sequence can be determined using methods known in the art and as described herein.

[0247] Nucleic acids having protein-coding sequences may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequences disclosed herein for the first time, and, if necessary, conventional primer extension procedures such as those described in Sambrook et al. (supra), to detect mRNA precursors and processing intermediates that may not have been reverse transcribed into cDNA.

[0248] 2. Host Cell Selection and Transformation Host cells are transfected or transformed with the expression or cloning vectors described herein to produce anti-CTHRC1 antibodies and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify genes encoding the desired sequences. Culture conditions, such as media, temperature, pH, etc., can be selected by those skilled in the art without undue experimentation. In general, principles, protocols, and practical techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al. (supra).

[0249] Methods of eukaryotic cell transfection and prokaryotic cell transformation, which refer to the introduction of DNA into a host so that it is replicable, either extrachromosomally or via chromosomal integration, are known to those skilled in the art, such as CaCl2, CaPO4, liposome-mediated, polyethylene glycol (gycol) / DMSO, and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Calcium treatment using calcium chloride, as described by Sambrook et al. (supra), or electroporation is commonly used for prokaryotes. Infection with Agrobacterium tumefaciens is used to transform certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89 / 05859, published June 29, 1989. For mammalian cells lacking 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 are described in U.S. Patent No. 4,399,216. Transformation into yeast is typically performed according to 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 for introducing DNA into cells may also be used, such as intranuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., 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).

[0250] Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells.

[0251] a. Prokaryotic host cells Suitable prokaryotes include, but are not limited to, archaebacteria and eubacteria, e.g., gram-negative or gram-positive organisms, e.g., Enterobacteriaceae, such as E. coli. Various E. 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., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P, disclosed in DD 266,710, published April 12, 1989), Pseudomonas such as P. aeruginosa, Rhizobia, Vitreoscilla, Paracoccus, and Streptomyces. These examples are illustrative and not limiting. E. coli strain W3110 is one particularly preferred host or parent host because it is a common host strain for fermentation of recombinant DNA products. Preferably, the host cells secrete minimal amounts 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 Accession No. 27,325) can be modified to introduce genetic mutations in genes encoding proteins endogenous to the host, examples of which include E. coli W3110 strain 1A2 with the complete tonA genotype; E. coli W3110 strain 9E4 with the complete tonA ptr3 genotype; E. coli W3110 strain 27C7 (ATCC 55,244) with the complete tonA ptr3 phoA E15(argF-lac)169 degP ompT kanr genotype; and E. coli W3110 strain 27C7 with the complete tonA ptr3 phoA E15(argF-lac)169 degP ompT rbs7 ilvG kanr genotype. E. coli W3110 strain 37D6; E. coli W3110 strain 40B4, which is strain 37D6 with a degP deletion mutation that does not result in kanamycin resistance; E. coli W3110 strain 33D3 with the genotype W3110ΔfhuA(ΔtonA)ptr3 lac Iq lacL8ΔompTΔ(nmpc-fepE)degP41 kanR (U.S. Pat. No. 5,639,635); and E. coli strains with mutant periplasmic proteases as disclosed in U.S. Pat. No. 4,946,783, issued August 7, 1990. Other strains and their derivatives, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608), are also suitable. These examples are illustrative rather than limiting. Methods for constructing derivatives of any of the above-mentioned bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). In general, it is necessary to select an appropriate bacterium taking into account the replicability of the replicon in the bacterial cell.For example, E. coli, Serratia, or Salmonella species may be suitably used as hosts when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon. Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may be incorporated into the cell culture if desired. Alternatively, in vitro methods of cloning, such as by PCR or other nucleic acid polymerase reactions, are suitable.

[0252] Full-length antibodies, antibody fragments, and antibody fusion proteins can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. Full-length antibodies have a longer half-life in the blood circulation. Production in E. coli is faster and more cost-effective. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523, which describe translation initiation regions (TIRs) and signal sequences for optimizing expression and secretion, and are incorporated herein by reference. After expression, antibodies can be isolated from E. coli cell paste and purified, for example, by passage through a protein A or G column depending on the isotype. Final purification can be performed similarly to the process for purifying antibodies expressed in, for example, CHO cells.

[0253] b.Eukaryotic host cells In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-CTHRC1 antibody-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Other microorganisms include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 (1981), EP 139,383 published May 2, 1985); e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 (1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al. al., Bio / Technology, 8:135 (1990)), Kluyveromyces hosts such as K. thermotolerans, and K. marxianus (US Pat. No. 4,943,529, Fleer et al., Bio / Technology, 9:968-75 (1991)); yarrowia (EP402,226); Pichia pastoris(EP183,070, Sreekrishna et al.,J.Basic Microbiol.,28:265-278(1988));Candida;Trichoderma reesia(EP244,234);Neurospora crassa(Case et al. al.,Proc.Natl.Acad.Sci.USA,76:5259-5263(1979));Schwanniomyces Schwanniomyces such as A. occidentalis (EP 394,538 published October 31, 1990); as well as, for example, Neurospora, Penicillium, Tolypocladium (WO 91 / 00357 published January 10, 1991), 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, including, but not limited to, yeasts capable of growing on methanol selected from the genera Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species representative of this class of yeast can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

[0254] Suitable host cells for expressing glycosylated anti-CTHRC1 antibodies are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, and plant cells such as cotton, corn, potato, soybean, petunia, tomato, and tobacco cell cultures. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available, and such viruses may be used as the viruses herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

[0255] However, vertebrate cells have received the most attention, and propagation of vertebrate cells in culture (tissue culture) has become routine procedure. Examples of useful mammalian host cell lines are the monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651), the human embryonic kidney cell line (293 cells or 293 cells subcloned to grow in suspension culture, Graham et al., J. Gen. Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL10), 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 CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat liver cells (BRL 3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human liver cells (Hep G2, HB8065), mouse mammary carcinoma (MMT060562, ATCC CCL51), TRI cells (Mather et al., Annals NY Acad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and a human hepatocellular carcinoma cell line (Hep G2).

[0256] Host cells are transformed with the above-described 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.

[0257] 3. Selection and Use of Replicable Vectors For recombinant production of an antibody of the invention, the nucleic acid (e.g., cDNA or genomic DNA) encoding it is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the antibody heavy and light chains). Many vectors are available. The choice of vector will depend, in part, on the host cell to be used. Generally, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin.

[0258] The vector may be in the form of, for example, a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector by a variety of procedures. Generally, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally 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 suitable vectors containing one or more of these components employs standard ligation techniques known to those skilled in the art.

[0259] Anti-CTHRC1 antibodies can be recombinantly produced not only directly but also as fusion polypeptides with heterologous polypeptides, which may be signal sequences or other polypeptides having specific cleavage sites at the N-terminus of the mature protein or polypeptide. Generally, the signal sequence may be a component of the vector, or it may be a part of the anti-CTHRC1 antibody-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence may be, for example, the yeast invertase leader, the alpha factor leader (including the Saccharomyces and Kluyveromyces α-factor leader, the latter described in U.S. Pat. No. 5,010,182), or the acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published April 4, 1990), or a signal described in WO 90 / 13646 published November 15, 1990. In expression in mammalian cells, mammalian signal sequences, such as signal sequences from secreted polypeptides of the same or related species, may also be used to direct protein secretion, as well as viral secretory leaders.

[0260] a. Prokaryotic host cells Polynucleotide sequences encoding polypeptide components of antibodies of the invention can be obtained using standard recombinant techniques. Desired polynucleotide sequences can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the polypeptide-encoding sequence 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. Selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of the heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it will reside.

[0261] Plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are generally used in connection with these hosts. Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells, as well as marking sequences that can provide phenotypic selection in transformed cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322, which contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides a convenient means for identifying transformed cells, is suitable for most Gram-negative bacteria; the 2μ plasmid origin is suitable for yeast; and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by the microorganism for expression of endogenous proteins. Examples of pBR322 derivatives used to express particular antibodies are detailed in Carter et al., US Pat. No. 5,648,237.

[0262] Additionally, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as λGEM™-11 may be utilized to generate recombinant vectors, which can then be used to transform susceptible host cells, such as E. coli LE392.

[0263] The expression vector of the present invention may contain two or more promoter-cistron pairs, one 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 typically divided into two classes: inducible promoters and constitutive promoters. An inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in culture conditions, such as the presence or absence of a nutrient or a change in temperature.

[0264] Numerous promoters recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to the cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they generally allow for greater transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter.

[0265] Promoters recognized by a variety of potential host cells are well known. Suitable promoters for use with prokaryotic hosts include the PhoA promoter, β-galactamase and lactose promoter systems (Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)), alkaline phosphatase, tryptophan (trp) promoter systems (Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36,776), and hybrid promoters such as the tac (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)) or trc promoter. Promoters intended for use in bacterial systems will also contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the anti-CTHRC1 antibody. However, other promoters functional in bacteria (e.g., other known bacterial or phage promoters) are also suitable, whose nucleotide sequences have been published, allowing one of skill in the art to operably link them to cistrons encoding the target light and heavy chains, using linkers or adapters to provide any necessary restriction sites (Siebenlist et al. (1980) Cell 20:269).

[0266] In one aspect of the present invention, each cistron in a recombinant vector contains a secretory signal sequence component that directs translocation of the expressed polypeptide across a membrane. Generally, the signal sequence can be a component of the vector, or it can be part of the target polypeptide DNA inserted into the vector. For purposes of the present invention, the signal sequence selected should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In the case of prokaryotic host cells that do not recognize and process the signal sequence native to the heterologous polypeptide, the signal sequence is substituted with a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA, and MBP. In one embodiment of the present invention, the signal sequence used in both cistrons of the expression system is the STII signal sequence or a variant thereof.

[0267] In another aspect, 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 secretory signal sequence within 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 favor disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0268] 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, properly assembled antibodies of the invention, such adjustment being accomplished, at least in part, by simultaneously adjusting the translational strength for the polypeptide components.

[0269] One technique for adjusting translation strength is disclosed in U.S. Patent No. 5,840,523 to Simmons et al., which 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 can be created with varying translation strengths, thereby providing a convenient means for adjusting this factor to the desired expression level of a particular chain. TIR variants can be generated by conventional mutagenesis techniques, resulting in codon changes that can alter the amino acid sequence; however, silent changes to the nucleotide sequence are preferred. For example, TIR changes can include altering the number or spacing of Shine-Dalgarno sequences in addition to altering the signal sequence. One method for generating variant signal sequences is to generate a "codon bank" at the beginning of the coding sequence that does not alter the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be accomplished by varying 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 can complicate the creation of the bank. This mutagenesis method is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.

[0270] Preferably, a set of vectors is generated with various TIR strengths for each cistron therein. This limited set provides a comparison of the expression levels of each chain and the yield of the desired antibody product under various combinations of TIR strengths. TIR strength can be determined by quantifying the expression level of a reporter gene, as detailed in U.S. Patent No. 5,840,523 to Simmons et al. Based on the comparison of translation strengths, desired individual TIRs are selected and combined in the expression vector construct of the present invention.

[0271] b.Eukaryotic host cells 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.

[0272] (1) Signal sequence components Vectors for use in 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. For expression in mammalian cells, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex gD signal, are available.

[0273] The DNA for such precursor region is ligated in reading frame to DNA encoding the antibody.

[0274] (2) Origin of replication Generally, the origin of replication component is not needed for mammalian expression vectors For example, the SV40 origin may typically be used only because it contains the early promoter.

[0275] (3) Selected gene components Expression and cloning vectors will typically 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 an auxotrophic deficiency, or (c) supply vital nutrients unavailable from complex media, e.g., a gene encoding D-alanine racemase for Bacilli.

[0276] One example of a selection scheme utilizes a drug to arrest growth of the host cell. Cells that are successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0277] Examples of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the anti-CTHRC1 antibody-encoding nucleic acid, 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, an appropriate host cell is a CHO cell line deficient in DHFR activity (e.g., ATCC CRL-9096), prepared and grown as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Alternatively, host cells transformed or co-transformed with a DNA sequence encoding an antibody, a wild-type DHFR protein, and another selectable marker, such as aminoglycoside 3'-phosphotransferase (APH), (particularly wild-type hosts containing endogenous DHFR) can be selected by growing the cells in medium containing a selection agent for the selectable marker, for example, an aminoglycoside antibiotic such as kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

[0278] 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 on tryptophan, e.g., ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)).

[0279] (4) Promoter components Expression and cloning vectors usually contain a promoter operably linked to the anti-CTHRC1 antibody-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known.

[0280] Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream from the site where transcription begins. Another sequence found 70 to 80 bases upstream from the transcription start of many genes is a CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitable for insertion into eukaryotic expression vectors.

[0281] Examples of promoter sequences suitable for use with yeast hosts include promoters 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.

[0282] Other yeast promoters that are inducible promoters with the added 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, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes involved in maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

[0283] Transcription of the anti-CTHRC1 antibody from the vector in mammalian host cells is controlled by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published July 5, 1989), adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and simian virus 40 (SV40), heterologous mammalian promoters such as the actin promoter or immunoglobulin promoter, and heat shock promoters, provided that such promoters are compatible with the host cell system.

[0284] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), concerning the expression of human β-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the long terminal repeat of the Rous sarcoma virus can be used as a promoter.

[0285] (5) Enhancer element components Transcription of DNA encoding an anti-CTHRC1 antibody by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10 to 300 bp, that act on a promoter to increase transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). However, typically, enhancers from eukaryotic viruses will be used. Examples include the SV40 enhancer on the late side of the replication origin (base pairs 100 to 270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) for enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced ​​into the vector at a position 5' or 3' to the anti-CTHRC1 antibody-encoding sequence, but is preferably located at a site 5' from the promoter.

[0286] (6) Transcription termination component Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and stabilization of mRNA. Such sequences are commonly available from the 5' and, occasionally, 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-CTHRC1 antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vector disclosed therein.

[0287] Still other methods, vectors, and host cells suitable for the synthesis of 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.

[0288] 4. Host Cell Culturing The host cells used to produce the anti-CTHRC1 antibody of this invention may be cultured in a variety of media.

[0289] a. Prokaryotic host cells Prokaryotic cells used to produce the polypeptides of the present invention are grown in a medium known in the art and suitable for culturing the selected host cells. An example of a suitable medium is Luria Broth (LB) supplemented with necessary nutritional supplements. In some embodiments, the medium also contains a selection agent selected based on the structure of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing an ampicillin resistance gene.

[0290] In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements may also be included at appropriate concentrations, introduced alone or in mixture with other supplements or media, such as complex nitrogen sources. Optionally, the media may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycollate, dithioerythritol, and dithiothreitol.

[0291] Prokaryotic host cells are cultured at a suitable temperature. For example, for growth of E. coli, preferred temperatures range from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, and even more preferably about 30°C. The pH of the medium may 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.

[0292] When inducible promoter is used in the expression vector of the present invention, protein expression is induced under the conditions suitable for promoter activation.In one aspect of the present invention, PhoA promoter is used to control the transcription of polypeptide.Therefore, transformed host cell is cultured in phosphate-limited medium for induction.In some embodiments, phosphate-limited medium is CRAP medium (see, for example, Simmons et al., J.Immunol.Methods (2002), 263:133-47).As known in the art, various other inducers can be used according to the vector construction employed.

[0293] In one embodiment, the expressed polypeptide of the present invention is secreted into the periplasm of the host cell and recovered therefrom. Protein recovery typically involves disruption of the microorganism, generally by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported into the culture medium and isolated therein. The cells can be removed from the culture, and the culture supernatant filtered and concentrated for further purification of the produced protein. The expressed polypeptide can be further isolated and characterized using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0294] In one aspect of the present invention, antibody production is carried out in large quantities by a fermentation process. A variety of large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentations have a volume of at least 1000 liters, preferably between about 1,000 and 100,000 liters. These fermentors use agitator impellers to distribute oxygen and nutrients, particularly glucose (the preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in fermentors with a volume of approximately 100 liters or less, which can range from about 1 liter to about 100 liters.

[0295] In fermentation processes, induction of protein expression typically begins after cells have been grown under suitable conditions to a desired density, e.g., an OD550 of approximately 180-220, at which point the cells are in early stationary phase. As known in the art and described above, various inducers can be used depending on the vector construct employed. Cells may also be grown for a shorter period before induction. Cells are usually induced for approximately 12-50 hours, although longer or shorter induction times can also be used.

[0296] Various fermentation conditions can be modified to improve the production yield and quality of the polypeptides of the present invention. For example, to improve the proper assembly and folding of secreted antibody polypeptides, prokaryotic host cells can be co-transformed with an additional vector overexpressing a chaperone protein, such as a Dsb protein (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (a peptidyl-prolyl cis-trans isomerase with chaperone activity). Chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274, 19601-5, U.S. Patent No. 6,083,715, U.S. Patent 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.

[0297] To minimize proteolysis of expressed heterologous proteins (especially those sensitive to proteolysis), certain host strains deficient in proteolytic enzymes can be used in the present invention. For example, host cell strains can be modified to introduce genetic mutation(s) in genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several E. coli protease-deficient strains are available and are described, for example, in Joly et al. (1998) (supra), U.S. Patent No. 5,264,365, U.S. Patent No. 5,508,192, and Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0298] In one embodiment, an E. coli strain that is deficient in a proteolytic enzyme and transformed with a plasmid that overexpresses one or more chaperone proteins is used as a host cell in the expression system of the present invention.

[0299] b.Eukaryotic host cells Commercially available media such as Ham's F10 (Sigma), minimal essential medium (MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's modified Eagle's medium (DMEM), Sigma) are suitable for culturing the host cells. In addition, the methods described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al. Any of the media described in 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. Reissue 30,985 may be used as a culture medium for the host cells. Any of these media may optionally contain hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride), or the like. The medium may be supplemented with nutrients (e.g., phosphates, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., the drug GENTAMYCIN™), 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 as would be known to one of skill in the art. Culture conditions, such as temperature, pH, etc., will be those conventionally used with the host cell selected for expression and will be apparent to one of skill in the art.

[0300] 5. Detection of Gene Amplification / Expression Gene amplification and / or expression may be measured directly in a sample, for example, by conventional Southern blotting to quantify mRNA transcription, Northern blotting (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. Alternatively, antibodies capable of recognizing specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes, or DNA-protein duplexes, may be used. The antibody may then be labeled, and the duplex may be bound to a surface, whereby an assay may be performed in which the presence of antibody bound to the duplex can be detected upon formation of the duplex on the surface.

[0301] Alternatively, gene expression may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell cultures or body fluids to directly quantitate expression of the gene product. Antibodies useful for immunohistochemical staining and / or assay of sample fluids may be either monoclonal or polyclonal and may be prepared in any mammal. Advantageously, antibodies may 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.

[0302] 6. Preparation of Anti-CTHRC1 Antibody The anti-CTHRC1 antibody form may be recovered from the culture medium or from host cell lysates. If 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 cycling, sonication, mechanical disruption, or cell lysing agents.

[0303] It may be desirable to purify anti-CTHRC1 antibodies from recombinant cell proteins or polypeptides. The following procedures are representative of suitable purification procedures: fractionation on an ion exchange column; ethanol precipitation; reverse-phase HPLC; chromatography on a cation exchange resin such as silica or DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration, e.g., using Sephadex G-75; a protein A Sepharose column to remove contaminants such as IgG; and a metal chelate column to bind epitope-tagged forms of anti-CTHRC1 antibodies. Various protein purification methods may be used, and such methods are known in the art and are described, for example, in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular anti-CTHRC1 antibody being produced.

[0304] When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate debris (either host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-7 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for approximately 30 minutes. Cell debris can be removed by centrifugation. If the antibody is secreted into the medium, the supernatant of such expression systems is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0305] Antibodies prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species 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 most often agarose, although other matrices are also available. Mechanically stable matrices such as controlled-pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than those achievable with agarose. If the antibody contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Depending on the antibody to be recovered, other techniques for protein purification are also available, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0306] After any preliminary purification step(s), the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5 and generally with a low salt concentration (e.g., about 0-0.25 M salt).

[0307] D. Assay The antibodies of the present invention may be employed 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.).

[0308] Detection labels can be useful for localizing, visualizing, and quantifying binding or recognition events. The labeled antibodies of the present invention can detect cell surface receptors. Another use of detectably labeled antibodies is in bead-based immunocapture methods, which involve conjugating beads with fluorescently labeled antibodies and detecting fluorescent signals upon ligand binding. A similar binding detection technique utilizes the surface plasmon resonance (SPR) effect to measure and detect antibody-antigen interactions.

[0309] Detection labels, such as fluorescent dyes and chemiluminescent dyes (Briggs et al. (1997) J. Chem. Soc., Perkin-Trans. 1:1051-8), provide detectable signals and are commonly available for labeling antibodies, preferably with the following properties: (i) the labeled antibody should produce a very high signal with low background, so that small amounts of antibody can be detected with high sensitivity in both 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 membrane or cell surface binding of labeled antibodies, particularly to live cells, the label preferably (iii) has good aqueous solubility to achieve effective conjugation concentrations and detection sensitivity, and (iv) is non-toxic to live cells so as not to disrupt normal metabolic processes or cause premature cell death.

[0310] Direct quantification of cellular fluorescence intensity and enumeration of fluorescently labeled events, such as cell surface binding of peptide-dye conjugates, can be performed using a system that automates mix-and-read, non-radioactive assays using live cells or beads (FMAT® 8100 HTS 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 in vivo," (1998) Proc. Natl. Acad. Sci. USA 95:618-23; US 6,372,907), apoptosis (Vermes, "A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein-labeled Annexin V," (1995) J. Immunol. Methods 184:39-51), and cytotoxicity assays.Fluorometric microvolume assay technology can be used to identify up- or down-regulation by molecules targeted to the cell surface (Swartzman, "A homogeneous and multiplexed immunoassay for high-throughput screening using fluorometric microvolume assay technology," (1999) Anal. Biochem. 271:143-51).

[0311] The labeled antibodies of the present invention are useful as imaging biomarkers and probes for various biomedical and molecular imaging methods and techniques, 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 a radioactively labeled antibody is administered to an animal or human patient and images of the site in the body where the antibody is localized are obtained (US6528624). Imaging biomarkers can be objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacological responses to therapeutic interventions.

[0312] 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 (T.S. Work and E. Work, Eds.) American Elsevier Publishing Co., New York, Lundblad, R.L. and Noyes, C.M. (1984) Chemical Reagents for Protein Modification, Vols. I and II, CRC Press, New York, Pfleiderer, G. (1985) “Chemical Modification of Proteins”, Modern Methods in Protein Chemistry, H. Tschesche, Ed., Walter DeGryter, Berlin and New York, and Wong (1991) Chemistry of Protein Conjugation and Cross-linking, CRC Press, Boca Raton, Fla.), 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).

[0313] Peptides and proteins labeled with two moieties, a fluorescent reporter and a quencher, in sufficient proximity are subjected to fluorescence resonance energy transfer (FRET). The reporter group is typically a fluorescent dye, which is excited by light of a specific wavelength and transfers energy to an acceptor or quencher group with a Stokes shift appropriate for maximum brightness emission. Fluorescent dyes include highly aromatic molecules such as fluorescein and rhodamine, as well as their derivatives. The fluorescent reporter can be partially or largely quenched by the quencher moiety in the intact peptide. Upon peptide cleavage by peptidases or proteases, a detectable increase in fluorescence can be measured (Knight, C. (1995) "Fluorimetric Assays of Proteolytic Enzymes", Methods in Enzymology, Academic Press, 248:18-34).

[0314] The labeled antibodies of the present invention may also be used as affinity purification agents. In this process, the labeled antibodies are immobilized on a solid phase, such as Sephadex resin or filter paper, using methods well known in the art. The immobilized antibodies are contacted with a sample containing the antigen to be purified, and the support is then washed with a suitable solvent to remove substantially all material in the sample except for the antigen to be purified that is bound to the immobilized polypeptide variant. Finally, the antigen is released from the polypeptide variant by washing the support with another suitable solvent, such as glycine buffer (pH 5.0).

[0315] 1. Activity Assay In one embodiment, assays are provided for identifying anti-CTHRC1 antibodies with 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.

[0316] In certain embodiments, anti-CTHRC1 antibodies are tested for their ability to inhibit cell growth or proliferation in vitro. Assays for inhibiting cell growth or proliferation are well known in the art. Certain assays for cell proliferation, exemplified by the "cell killing" assay described herein, measure cell viability. One such assay is the CellTiter-Glo™ Luminescent Cell Viability Assay, commercially available from Promega (Madison, WI). The assay determines the number of viable cells in culture based on the quantification of ATP present, which is an indicator of metabolically active cells. See Crouch et al. (1993) J. Immunol. Meth. 160:81-8; U.S. Patent No. 6,602,677. This assay may 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 adding a single reagent (CellTiter-Glo® Reagent) directly to cultured cells. This results in cell lysis and the generation of a luminescent signal produced by a 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 as relative light units (RLU).

[0317] Another assay for cell proliferation 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. Like 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).

[0318] In one aspect, anti-CTHRC1 antibodies are tested for their ability to induce cell death in vitro. Assays for cell death induction are well known in the art. In some embodiments, such assays measure loss of membrane integrity, as indicated by, for example, propidium iodide (PI), trypan blue (see Moore et al., Cytotechnology, 17:1-11 (1995)), or 7AAD uptake. 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 2 mM L-glutamine. Thus, the assay is performed in the absence of complement and immune effector cells. Cells are seeded into 100 x 20 mm dishes at a density of 3 x 10 per dish and allowed to adhere overnight. The medium is removed and replaced with fresh medium alone or medium containing various concentrations of antibody. The cells are incubated for a period of 3 days. Following treatment, the monolayer is washed with PBS and detached by trypsinization. The cells are then centrifuged at 1200 rpm at 4°C for 5 minutes, and the pellet is 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 x 75 mm tubes with 35 mm strainer caps (1 mL per tube, 3 tubes per treatment group) to remove cell clumps. Tubes are then spiked with PI (10 μg / mL). Samples are analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickinson). Antibodies that induce statistically significant levels of cell death as determined by PI uptake are thus identified.

[0319] In one embodiment, an anti-CTHRC1 antibody is tested for its ability to induce apoptosis (programmed cell death) in vitro. An exemplary assay for antibodies that induce apoptosis is an annexin binding assay. In an exemplary annexin binding assay, cells are cultured as discussed in the previous paragraph and plated in a dish. The medium is removed and replaced with fresh medium alone or medium containing 0.001-10 μg / mL of antibody. Following a 3-day incubation period, the monolayer is washed with PBS and detached by trypsinization. The cells are then centrifuged as discussed in the previous paragraph, resuspended in Ca2+ binding buffer, and aliquoted into tubes. The tubes then contain labeled annexin (e.g., annexin V-FITC) (1 μg / mL). Samples are analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (BD Biosciences). In this way, antibodies that induce statistically significant annexin binding levels compared to control are identified. Another exemplary assay for antibodies that induce apoptosis is the histone DNA ELISA colorimetric assay for detecting internucleosomal degradation of genomic DNA. Such an assay can be carried out, for example, using a cell death detection ELISA kit (Roche, Palo Alto, CA).

[0320] Cells for use in any of the above in vitro assays include cells or cell lines that naturally express CTHRC1 or that 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 that have been transfected with a nucleic acid encoding CTHRC1.

[0321] In one aspect, the anti-CTHRC1 antibody is tested for its ability to inhibit cell growth or proliferation in vivo. In certain embodiments, the anti-CTHRC1 antibody is tested for its ability to inhibit tumor growth in vivo. An in vivo model system, such as a xenograft model, can be used for such testing. In an exemplary xenograft system, human tumor cells are introduced into a suitable immunodeficient non-human animal, such as a SCID mouse. 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 xenograft system, the human tumor cells are tumor cells derived from a human patient. In certain embodiments, the human tumor cells are introduced into a suitable immunodeficient non-human animal by subcutaneous injection or by implantation into a suitable site, such as the mammary fat pad.

[0322] 2. Binding Assays and Other Assays In one aspect, anti-CTHRC1 antibody is tested for its antigen binding activity.For example, in certain embodiments, anti-CTHRC1 antibody is tested for its ability to bind to CTHRC1 expressed on the surface of cells.FACS assay may be used for this test.

[0323] In one aspect, a competition assay may be used to identify monoclonal antibodies that compete for binding to CTHRC1 with a monoclonal antibody comprising the variable domain of any one of SEQ ID NOS: 1-10, or a chimeric antibody comprising the variable domain of a monoclonal antibody comprising a sequence in Table 3 and Table 4 and a constant domain from IgG1 or IgG4. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or conformational epitope) bound by a monoclonal antibody comprising the variable domain of any one of SEQ ID NOS: 1-10, or a chimeric antibody comprising the variable domain of a monoclonal antibody comprising a sequence in Table 3 and Table 4 and a constant domain from IgG1 or IgG4. Exemplary competition assays include, but are not limited to, routine 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 mapping antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ). Two antibodies are said to bind to the same epitope if each blocks the binding of the other by 50% or more.

[0324] In an exemplary competitive assay, immobilized CTHRC1 is incubated in a solution containing a labeled first antibody that binds to CTHRC1 (e.g., a monoclonal antibody comprising the variable domain of any one of SEQ ID NOS: 1-10, or a chimeric antibody comprising the variable domain of a monoclonal antibody comprising a sequence in Tables 3 and 4 and a constant domain from IgG1 or IgG4) and a second, unlabeled antibody being tested for its ability to compete with the first antibody for binding to CTHRC1. The second antibody may be present in hybridoma supernatant. As a control, immobilized CTHRC1 is incubated in a solution containing the labeled first antibody but not the unlabeled second antibody. After incubation under conditions that allow binding of the first antibody to CTHRC1, excess unbound antibody is removed, and the amount of label associated with immobilized CTHRC1 is measured. If the amount of label associated with immobilized CTHRC1 is substantially reduced in the test sample compared to the control sample, this 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 cells expressing CTHRC1 on their surface or in a membrane preparation obtained from such cells.

[0325] In one aspect, the purified anti-CTHRC1 antibody can be further characterized by a range of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size-exclusion high-pressure liquid chromatography (HPLC), mass spectrometry, ion-exchange chromatography, and papain digestion.

[0326] E.CAR-modified immune cells 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 may be used therapeutically for patients with non-hematological tumors, such as solid tumors arising from breast, CNS, and skin malignancies. In certain embodiments, CAR-NK cells may be used therapeutically for patients with any one of several malignancies. In certain embodiments, CAR-macrophages may be used therapeutically for patients with any one of several malignancies.

[0327] In certain embodiments, the present invention relates to strategies for adoptive cell transfer of T cells or NK cells, or macrophages, transduced to express chimeric antigen receptors (CARs), which are molecules that combine antibody-based specificity for a desired antigen (e.g., a tumor antigen) with, for example, a T cell receptor activating intracellular domain, to generate a chimeric protein that exhibits specific anti-tumor cell immune activity.

[0328] In one aspect, the present invention relates to the use of NK cells genetically modified to stably express a desired CAR. NK cells expressing a CAR are referred to herein as CAR-NK cells or CAR-modified NK cells. Preferably, the cells can be genetically modified to stably express an antibody binding domain on their surface to confer novel antigen specificity. Methods for generating CAR-NK cells are known in the art. See, for example, Glienke et al., Front Pharmacol. 2015;6:21. Assistance for generating CAR-NK cells is commercially available. See, for example, Creative Biolabs Inc., 45-1 Ramsey Road, Shirley, NY 11967, USA.

[0329] In one aspect, the present invention relates to the use of T cells that have been genetically modified to stably express a desired CAR. T cells expressing a CAR are referred to herein as CAR-T cells or CAR-modified T cells. Preferably, the cells can be genetically modified to stably express an antibody binding domain on their surface, conferring novel antigen specificity that is MHC-independent. In some cases, T cells are genetically modified to stably express a CAR that combines the antigen recognition domain of a specific antibody with the intracellular domain of the CD3-zeta chain or FcγRI protein to form a single chimeric protein.

[0330] In one embodiment, the CAR of the present invention comprises an extracellular domain having an antigen recognition domain, a transmembrane domain, and a cytoplasmic domain. The intracellular domain, also known as the cytoplasmic domain, comprises at least one costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to a portion of the CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for efficient lymphocyte responses to antigens. In one embodiment, a transmembrane domain that is 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 domain to the transmembrane domain of the same or different surface membrane protein to minimize interaction with other members of the receptor complex. In one embodiment, the transmembrane domain is a CD8α hinge domain.

[0331] A spacer domain may be incorporated between the extracellular and transmembrane domains of a CAR, or between the cytoplasmic and transmembrane domains of a CAR. As used herein, the term "spacer domain" generally refers to any oligopeptide or polypeptide that functions within a polypeptide chain to link a transmembrane domain to either the extracellular or cytoplasmic domain. A spacer domain may contain up to 300 amino acids, 10-100 amino acids, and often 25-50 amino acids.

[0332] With respect to the cytoplasmic domain, the CAR of the present invention can be designed to include the signaling domain of CD28 and / or 4-1BB, by itself or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR of the present invention. In one embodiment, the cytoplasmic domain of the CAR can be designed to further include the signaling domain of CD3-zeta. For example, the cytoplasmic domain of the CAR can include, but is not limited to, the signaling modules of CD3-zeta, 4-1BB, and CD28, and combinations thereof. Thus, the present invention provides CAR T cells for adoptive therapy and methods of using them.

[0333] In one embodiment, the CAR T cells of the present invention can be generated by introducing into cells a lentiviral vector containing a desired CAR, for example, a CAR containing anti-CTHRC1, the hinge and transmembrane domain of CD8α, and the signaling domains of human 4-1BB and CD3 zeta. The CAR T cells of the present invention can replicate in vivo and provide long-term persistence, which can lead to sustained tumor control.

[0334] In one embodiment, the present invention relates to the administration of genetically modified T cells expressing a CAR for the treatment of patients with or at risk of having cancer using lymphocyte infusion. Preferably, autologous lymphocyte infusion is used for the treatment. Autologous PBMCs are collected from the patient in need of treatment, and the T cells are activated and expanded using methods described herein and known in the art, and then reinfused into the patient. The present invention also includes the treatment of malignancies or autoimmune diseases in which chemotherapy and / or immunotherapy of the patient results in significant immunosuppression in the patient, thereby increasing the patient's risk of developing a malignancy (e.g., CLL).

[0335] The present invention involves the use of T cells expressing an anti-CTHRC1 antibody-derived CAR containing both CD3-zeta and either 4-1BB or CD28 costimulatory domains (also known as CARTPODO T cells). The CARTPODO T cells of the invention can undergo robust T cell proliferation in vivo and establish memory cells specific for cells presenting the CTHRC1 tumor epitope, and such memory cells persist at high levels in the blood and bone marrow for extended periods of time.

[0336] 1. Antigen binding part In one embodiment, the CAR of the present invention comprises a target-specific binding element, also known as an antigen-binding moiety, or targeting arm. The antigen-binding moiety used in the present invention can bind to a CTHRC1 epitope, for example, a CTHRC1 tumor epitope. Thus, the antigen-binding moiety is selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular pathology.

[0337] The CARs of the present invention are engineered to target cells presenting the CTHRC1 epitope by engineering in them an appropriate antigen-binding moiety that specifically binds to the CTHRC1 epitope.

[0338] Preferably, the antigen-binding portion in the CAR of the invention is an scFv or scFab, wherein the nucleic acid sequence of the scFv comprises nucleic acid sequence(s) encoding one or more light chain CDRs and one or more heavy chain CDRs disclosed herein for the anti-CTHRC1 antibodies, and the nucleic acid sequence of the scFab comprises nucleic acid sequence(s) encoding one or more light chain CDRs and one or more heavy chain CDRs disclosed herein for the anti-CTHRC1 antibodies.

[0339] Preferably, the antigen-binding portion of 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: 1-10.

[0340] Preferably, the antigen-binding portion of 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.

[0341] Preferably, the antigen-binding portion of 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.

[0342] Preferably, the antigen-binding portion of 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, and any one of SEQ ID NOs: 2, 4, 6, 8, and 10. More preferably, the antigen-binding portion of 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: 3 and 9, and any one of SEQ ID NOs: 4 and 10.

[0343] In several embodiments, the antigen-binding portion of a CAR of the invention is an scFv or scFab comprising amino acids encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 100 to 109. In several embodiments, the antigen-binding portion of a CAR of the invention is an 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.

[0344] In embodiments, the antigen-binding portion of the CAR of the invention is an scFv or scFab comprising 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.

[0345] In embodiments, the antigen-binding portion of the CAR of the invention is an 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, and a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 101, 103, 105, 107, and 109.

[0346] In embodiments, the antigen-binding portion of the CAR of the invention is an scFv or scFab comprising an amino acid sequence selected from the group consisting of any of the CDR sequences in Table 3 and Table 4.

[0347] Preferably, the antigen-binding portion of 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 the CDR sequences in Table 3 and Table 4, and further comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1 to 10. More preferably, the antigen-binding portion of the CAR of the present invention is an scFv or scFab comprising a heavy chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 150 to 154, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 180 to 184, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 210 to 214, and a light chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 240 to 244, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 270 to 274, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 300 to 304, and further comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 1 to 10.

[0348] In one embodiment, the antigen-binding portion of 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.

[0349] In one embodiment, the antigen-binding portion of 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.

[0350] In one embodiment, the antigen-binding portion of a 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.

[0351] In one embodiment, the antigen-binding portion of a 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.

[0352] In one embodiment, the antigen-binding portion of a CAR of the invention comprises a heavy chain variable region comprising SEQ ID NO:9 and a light chain variable region comprising SEQ ID NO:10.

[0353] In one embodiment, the antigen-binding portion of the CAR of the invention is an scFv or scFab comprising an amino acid sequence having about 80%, 85%, 90%, or 95% identity to the SEQ ID NOs listed above.

[0354] 2. Transmembrane domain Regarding the transmembrane domain, CAR can be designed to include a transmembrane domain that is fused to the extracellular domain of CAR.In one embodiment, the transmembrane domain that is 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 the binding of this domain to the transmembrane domain of the same or different surface membrane protein, in order to minimize the interaction with other members of the receptor complex.

[0355] The transmembrane domain may be derived from either natural or synthetic sources. If the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Particularly useful transmembrane regions in the present invention may be derived from (i.e., comprise at least one transmembrane region or regions) the alpha, beta, or zeta chain of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain may be synthetic, in which case it comprises primarily hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan, and valine are found at each end of the synthetic transmembrane domain. Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, can form the link between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[0356] Preferably, the transmembrane domain in the CAR of the present invention is a CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the 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 the amino acid sequence of SEQ ID NO: 22 of U.S. Patent No. 9,102,760. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22 of U.S. Patent No. 9,102,760.

[0357] In some instances, the transmembrane domain of a CAR of the invention comprises a CD8α 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.

[0358] 3. Cytoplasmic Domain The cytoplasmic domain of the CAR of the present invention, also known as the intracellular signaling domain, is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to the specialized function of a cell. For example, the effector function of a T cell can be cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to a portion of a protein that transmits an effector function signal and guides the cell to perform a specialized function. While the entire intracellular signaling domain can usually be used, it is often not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, it may be used in place of the intact chain, as long as the truncated portion transmits the effector function signal. Thus, the term intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to transmit the effector function signal.

[0359] Preferred examples of intracellular signaling domains for use in the CARs of the invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences that have the same functional capability.

[0360] It is known that signals generated by the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation via the TCR (primary cytoplasmic signaling sequences), and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0361] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0362] Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present invention include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, 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 CD3 zeta.

[0363] In some embodiments, the cytoplasmic domain of a CAR can be designed to include the signaling domain of CD3-zeta, either by itself or in combination with any other desired cytoplasmic domain(s) useful in the context of the CAR of the present invention. For example, the cytoplasmic domain of a CAR can include a CD3 zeta chain portion and at least one costimulatory signaling region. A costimulatory signaling region refers to a portion of a CAR that includes the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for efficient lymphocyte responses 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.

[0364] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention may be linked to each other in random or specified order. Optionally, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[0365] In one embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of CD28. In another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domain of 4-1BB. In yet another embodiment, the cytoplasmic domain is designed to comprise the signaling domain of CD3-zeta and the signaling domains of CD28 and 4-1BB.

[0366] In one embodiment, the cytoplasmic domain in the CAR of the present invention is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the nucleic acid sequence set forth in SEQ ID NO: 17 of U.S. Pat. No. 9,102,760, and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 18 of U.S. Pat. No. 9,102,760.

[0367] In one embodiment, the cytoplasmic domain in the CAR of the present invention is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises 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-zeta comprises a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO: 24 of U.S. Patent No. 9,102,760.

[0368] In one embodiment, the cytoplasmic domain in the CAR of the present invention is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4-1BB comprises the amino acid sequence set forth in SEQ ID NO: 23 of U.S. Pat. No. 9,102,760, and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO: 24 of U.S. Pat. No. 9,102,760.

[0369] 4. Alternative Construction In an alternative embodiment, a CAR may be engineered to contain an antigen-binding domain that does not bind to CTHRC1 but rather binds to an engineered ligand fusion protein (where the engineered ligand fusion protein may comprise a ligand fused to an anti-CTHRC1 antibody of the present disclosure) that contains a ligand that binds to the antigen-binding domain, thus providing orthogonal binding to CTHRC1. By way of non-limiting example, the antigen binding domain can be an engineered receptor, e.g., a modified NKG2D receptor that does not bind the natural ligand, but binds a non-natural ligand, where 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. Appl. Pub. No. 2019 / 0300594, U.S. Pat. Appl. Pub. No. 2020 / 0138866, WO2017 / 222556, and U.S. Pat. Appl. Pub. No. 2016 / 0304578, each of which is incorporated herein by reference.

[0370] 5. Vector The present invention encompasses a DNA construct comprising a CAR sequence, the sequence comprising a nucleic acid sequence of an antigen-binding portion operably linked 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 such as CD3-zeta, CD28, 4-1BB, etc. In some cases, the CAR can comprise any combination of CD3-zeta, CD28, 4-1BB, etc.

[0371] In one embodiment, a CAR of the invention comprises an scFv derived from an anti-CTHRC1 antibody, the hinge and transmembrane domains of human CD8, and the signaling domains of human 4-1BB and CD3 zeta.

[0372] Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, for example, by screening libraries from cells which express the gene, by deriving the gene from a vector known to contain the gene, or by isolating it directly from cells and tissues containing the gene using standard techniques, etc. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0373] The present invention also provides a vector 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, as they allow long-term stable integration of the transgene and its proliferation in daughter cells.Lentivirus vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells such as hepatocytes.They also have the additional advantage of low immunogenicity.

[0374] Briefly summarized, expression of a natural or synthetic nucleic acid encoding a CAR is typically achieved by operably linking a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0375] In addition to the methods described above, the following methods may be used.

[0376] The expression constructs of the present invention may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art (e.g., U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entireties). In another embodiment, the present invention provides a gene therapy vector.

[0377] Nucleic acids can be cloned into several 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.

[0378] Furthermore, the expression vector may be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0379] Several virus-based systems have been developed for gene transfer into mammalian cells.For example, retrovirus provides a convenient platform for gene delivery systems.Selected genes can be inserted into vectors using techniques known in the art and packaged into retroviral particles.Recombinant viruses can then be isolated and delivered to target cells in vivo or ex vivo.Several retroviral systems are known in the art.In some embodiments, adenoviral vectors are used.Several adenoviral vectors are known in the art.In one embodiment, lentiviral vectors are used.

[0380] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although recent studies have shown that some promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, allowing promoter function to be maintained even when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart, after which activity begins to decline. Depending on the promoter, individual elements appear to function either cooperatively or independently to activate transcription.

[0381] One 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 to it. 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, the 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, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0382] To assess the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected via a viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences that allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0383] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that are not present in or expressed by recipient organisms or tissues and encode polypeptides whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at a suitable time after DNA is introduced into recipient cells. Suitable reporter genes may include luciferase, beta-galactosidase, chloramphenicol acetyltransferase, genes encoding secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and may be prepared using known techniques or commercially available. Generally, the construct that exhibits the highest expression level of the reporter gene with the minimum 5' flanking region is identified as the promoter. Such promoter regions may be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0384] Methods for introducing and expressing genes into cells 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 vector can be transferred into the host cell by physical, chemical, or biological means.

[0385] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing 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.

[0386] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0387] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle). When a non-viral delivery system is used, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acid into host cells (in vitro, ex vivo, or in vivo). In another embodiment, nucleic acid may be associated with lipid. Lipid-associated nucleic acids can be encapsulated within the aqueous interior of liposomes, interspersed within the lipid bilayer of liposomes, bound to liposomes via linking molecules associated with both the liposomes and the oligonucleotides, entrapped in liposomes, complexed with liposomes, dispersed in a solution containing lipids, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or in a "collapsed" structure. They can also simply be interspersed in solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include the lipid droplets that occur naturally in the cytoplasm, as well as a class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0388] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid stock solutions in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform evaporates more readily than methanol and is therefore used as the sole solvent. "Liposome" is a generic term encompassing a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions with structures in solution that differ from the typical vesicular structure are also encompassed. For example, lipids may adopt a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0389] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose the cells to the inhibitors of the present invention, various assays may be performed to confirm the presence of the recombinant DNA sequence in the host cells. Such assays include "molecular biological" 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 by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the present invention.

[0390] 6. Source of T cells Prior to the expansion and genetic modification of the T cells of the present invention, a source of T cells is obtained from a subject. T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of T cell lines available in the art may be used. In certain embodiments of the present invention, T cells can be obtained from a blood unit collected from a subject using any number of techniques known to those skilled in the art, such as Ficoll™ separation. In a preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction, and the cells can be placed in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the present invention, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium, and may lack magnesium, and may lack many, if not all, divalent cations. Again, surprisingly, an initial activation step in the absence of calcium results in expanded activation. As one skilled in the art will readily appreciate, the wash step may be accomplished by methods known to those skilled in the art, for example, by using a semi-automated "flow-through" centrifuge (e.g., a Cobe 2991 cell processing machine, a Baxter CytoMate, or a Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells may be washed with, for example, Ca 2+ Contains no Mg 2+ The cells may be resuspended in a variety of biocompatible buffers, such as free PBS, PlasmaLyte A, or other saline solutions with or without buffer. Alternatively, undesirable components of the apheresis sample may be removed and the cells resuspended directly in culture medium.

[0391] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and removing monocytes, for example, by centrifugation through a PERCOLL™ gradient or by elutriation with counterflow centrifugation. Specific subpopulations of T cells, such as CD3 + , CD28 + , CD4 + , CD8 + , CD45RA + , and CD45RO +T cells can be further isolated by positive or negative selection techniques. For example, in one embodiment, T cells are isolated by incubation with anti-CD3 / anti-CD28 (i.e., 3x28)-conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a period sufficient to positively select the desired T cells. In one embodiment, the period is approximately 30 minutes. In a further embodiment, the period ranges from 30 minutes to 36 hours or more, and all integer values ​​therebetween. In a further embodiment, the period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the period is 10 to 24 hours. In a preferred embodiment, the incubation period is 24 hours. In the case of isolation of T cells from patients with leukemia, the use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times can be used to isolate T cells in any situation where there are very few T cells compared to other cell types, such as when isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or immune-compromised individuals. Furthermore, the use of longer incubation times can increase the efficiency of CD8+ T cell capture. Thus, by simply shortening or lengthening the time allowed for T cells to bind to the CD3 / CD28 beads and / or by increasing or decreasing the ratio of beads to T cells (as further described herein), subpopulations of T cells can be preferentially selected for or eliminated at the initiation of culture or at other desired time points. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surfaces, subpopulations of T cells can be preferentially selected for or eliminated at the initiation of culture or at other desired time points. Those skilled in the art 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 the "unselected" cells in the activation and expansion process. The "unselected" cells can also be subjected to additional rounds of selection.

[0392] Enrichment of T cell populations by negative selection can be accomplished using a combination of antibodies directed against surface markers unique to the negatively selected cells. One method is cell sorting and / or selection via negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the negatively selected cells. For example, negative selection can enrich for CD4 + To enrich for cells, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In certain embodiments, CD4 + , CD25 + , CD62L 高 , G.I.T.R. + , and FoxP3 + It may be desirable to enrich for or positively select for regulatory T cells that typically express IL-16. Alternatively, in certain embodiments, regulatory T cells are removed by anti-C25 conjugated beads or other similar selection methods.

[0393] To isolate a desired cell population by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between the 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 a further embodiment, greater than 100 million cells / mL is used. In a further embodiment, a cell concentration of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL is used. In yet another embodiment, a cell concentration from 75, 80, 85, 90, 95, or 100 million cells / mL is used. In further embodiments, a concentration of 125 million or 150 million cells / mL may be used. The use of a higher concentration may result in increased cell yield, cell activation, and cell proliferation. Furthermore, the use of a higher cell concentration allows for more efficient capture of cells that may weakly express a 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 would be desirable to obtain. For example, the use of a higher concentration of cells may result in more efficient capture of CD8 T cells, which normally have weaker CD28 expression. + This allows for more efficient selection of T cells.

[0394] In related embodiments, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surfaces (e.g., particles such as beads), interactions between the particles and the cells are minimized. This selects for cells that express high amounts of the desired antigen that binds to the particles. For example, CD4 + T cells express higher levels of CD28 and, at dilute concentrations, CD8 + In one embodiment, the cell concentration used is 5×10 6In other embodiments, the concentration used is about 1 x 10 5 / mL ~ 1 × 10 6 / mL, and any integer value therebetween.

[0395] In other embodiments, cells may be incubated on a rotator for various lengths of time, at various speeds, at either 2-10°C or room temperature.

[0396] Stimulatory T cells can also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps remove granulocytes and, to some extent, monocytes from the cell population, resulting in a more uniform product. After a washing step to remove plasma and platelets, the cells can be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and may be useful in this regard, one method involves the use of PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% dextran 40 and 5% dextrose, 20% human serum albumin, and 7.5% DMSO, or other suitable cell freezing medium containing, for example, Hespan and PlasmaLyte A. The cells are then frozen to -80°C at a rate of 1°C per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other controlled freezing methods may be used, as may uncontrolled immediate freezing at -20°C or in liquid nitrogen.

[0397] In certain embodiments, cryopreserved cells are thawed and washed as described herein, allowed to stand at room temperature for 1 hour, and then activated using the methods of the invention.

[0398] Also contemplated within the context of the present invention is the collection of a blood sample or apheresis product from a subject at a time prior to when the cells expanded as described herein may be needed. Thus, a source of expanded cells can be collected at any time needed, and desired cells, such as T cells, can be isolated and frozen for later use in T cell therapy for any number of diseases or conditions that would benefit from T cell therapy, such as those described herein. In one embodiment, a blood sample or apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or apheresis is taken from a generally healthy subject who is at risk of developing a disease but has not yet developed the disease, and the desired cells are isolated and frozen for later use. In certain embodiments, T cells can be expanded, frozen, and used at a later time. In certain embodiments, a sample is collected from a patient shortly after diagnosis of a particular disease described herein, but prior to any treatment. In further embodiments, the cells are isolated from a blood sample or apheresis from a subject prior to any number of relevant therapies, including, but not limited to, treatment with drugs such as natalizumab, efalizumab, antivirals, chemotherapy, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolic acid, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and radiation. These drugs either inhibit calcineurin, a calcium-dependent phosphatase (cyclosporine and FK506), or inhibit p70S6 kinase (rapamycin), which is important in growth factor-induced signal transduction (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 a further embodiment, the cells are isolated for a patient and frozen for use after bone marrow or stem cell transplantation, T cell ablative therapy using either a chemotherapy agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide, or an antibody such as OKT3 or CAMPATH. In another embodiment, the cells are isolated before B cell ablative therapy, such as an agent reactive with CD20, e.g., Rituxan, and frozen for later use for treatment following B cell ablative therapy.

[0399] In a further embodiment of the present invention, T cells are obtained from a patient immediately after treatment. In this regard, it has been observed that following certain cancer treatments, particularly treatment with drugs that damage the immune system, the quality of the obtained T cells may be optimal or their ability to expand ex vivo may be improved shortly after treatment, during the period when the patient is typically recovering from the treatment. Similarly, following ex vivo manipulation using the methods described herein, these cells may be in a state favorable for enhanced engraftment and in vivo expansion. Thus, collection of blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery period is contemplated within the context of the present invention. Furthermore, in certain embodiments, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create conditions favorable for the repopulation, recirculation, regeneration, and / or expansion of specific cell types in a subject, particularly during defined periods following therapy. Exemplary cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0400] 7. T cell activation and proliferation Whether before or after genetic modification of the T cells to express a desired CAR, the T cells can be engineered using methods 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, The cells can be activated and expanded generally using methods such as those described in U.S. Patent Application Publication No. 20060121005, U.S. Pat. Nos. 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.

[0401] Generally, T cells of the present invention are expanded by contact with a surface bound with an agent that stimulates CD3 / TCR complex-associated signals and a ligand that stimulates costimulatory molecules on the surface of the T cells. Specifically, T cell populations can be stimulated, for example, by contact with an anti-CD3 antibody or antigen-binding fragment thereof, or an anti-CD2 antibody immobilized on a surface, or by contact with a protein kinase C activator (e.g., bryostatin) in conjunction with a calcium ionophore, as described herein. Costimulation of accessory molecules on the surface of T cells can be achieved using a ligand that binds to the accessory molecule. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions appropriate to stimulate T cell proliferation. CD4 + T cells or CD8 + To stimulate the proliferation of either T cells, anti-CD3 antibodies and anti-CD28 antibodies, such as 9.3, B-T3, and XR-CD28 (Diaclone, Besancon, France), can be used, as well as other methods known in the art (Berg et al., Transplant Proc. 30(8):3975-7, 1998; Haanen et al., J. Exp. Med. 190(9):1319-1328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, (1999)).

[0402] In certain embodiments, the primary stimulatory signal and the costimulatory signal to the T cell can be provided by different protocols. For example, the agents providing each signal can be in solution or bound to a surface. If bound to a surface, the agents can be bound to the same surface (i.e., a "cis" configuration) or to separate surfaces (i.e., a "trans" configuration). Alternatively, one agent can be bound to a surface and the other agent in solution. In one embodiment, the agent providing the costimulatory signal is bound to a cell surface, and the agent providing the primary activation signal is in solution or bound to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents can be in soluble form and then crosslinked to a surface, e.g., a cell expressing an Fc receptor, or an antibody or other binding agent that binds to the agent. In this regard, see, e.g., U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) contemplated for use in the activation and expansion of T cells in the present invention.

[0403] In one embodiment, these two agents are immobilized on beads, either on the same bead (i.e., "cis") or on separate beads (i.e., "trans"). By way of example, the agent providing the primary activation signal is an anti-CD3 antibody or an antigen-binding fragment thereof, and the agent providing the costimulatory signal is an anti-CD28 antibody or an antigen-binding fragment thereof, with both agents being co-immobilized on the same bead at equal molecular weights. In one embodiment, CD4 +A 1:1 ratio of each antibody bound to beads for T cell proliferation and growth is used. In certain aspects of the invention, a ratio of anti-CD3:CD28 antibodies bound to beads is used such that an increase in T cell proliferation is observed compared to the proliferation observed using a 1:1 ratio. In one particular embodiment, an increase of about 1 to about 3-fold is observed compared to the proliferation observed using a 1:1 ratio. In one embodiment, the ratio of CD3:CD28 antibodies bound to beads ranges from 100:1 to 1:100, and all integer values ​​therebetween. In one aspect of the invention, more anti-CD28 antibody than anti-CD3 antibody is bound to the particles, i.e., the CD3:CD28 ratio is less than 1. In certain embodiments of the invention, the ratio of anti-CD28 antibody to anti-CD3 antibody bound to beads is greater than 2:1. In one particular embodiment, a 1:100 ratio of CD3:CD28 antibodies bound to beads is used. In another embodiment, a 1:75 ratio of CD3:CD28 antibodies bound to beads is used. In a further embodiment, a 1:50 ratio of CD3:CD28 antibody bound to beads is used. In another embodiment, a 1:30 ratio of CD3:CD28 antibody bound to beads is used. In a preferred embodiment, a 1:10 ratio of CD3:CD28 antibody bound to beads is used. In another embodiment, a 1:3 ratio of CD3:CD28 antibody bound to beads is used. In yet another embodiment, a 3:1 ratio of CD3:CD28 antibody bound to beads is used.

[0404] Particle-to-cell ratios of 1:500 to 500:1 and any integer value therebetween may be used to stimulate T cells or other target cells. As one of ordinary skill in the art would readily appreciate, the particle-to-cell ratio may depend on the particle size relative to the target cells. For example, small beads may only be able to bind a small number of cells, while larger beads may be able to bind a large number. In certain embodiments, the cell-to-particle ratio is in the range of 1:100 to 100:1 and any integer value therebetween, and in further embodiments, this ratio includes 1:9 to 9:1 and any integer value therebetween, which may also be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28-conjugated particles to T cells that results in T cell stimulation can vary as described above, but 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 one preferred ratio being at least 1:1 particle / T cell. In one embodiment, a particle-to-cell ratio of 1:1 or less is used. In one particular embodiment, a preferred particle:cell ratio is 1:5. In further embodiments, the particle-to-cell ratio can be varied depending on the day of stimulation. For example, in one embodiment, the particle-to-cell ratio is 1:1 to 10:1 on day 1, and then additional particles are added to the cells daily or every other day for up to 10 days, resulting in a final ratio of 1:1 to 1:10 (based on the cell count on the day of addition). In one particular embodiment, the particle-to-cell ratio is 1:1 on day 1 of stimulation and adjusted to 1:5 on days 3 and 5 of stimulation. In another embodiment, particles are added daily or every other day, resulting in a final ratio of 1:1 on day 1 of stimulation and 1:5 on days 3 and 5 of stimulation. In another embodiment, the particle-to-cell ratio is 2:1 on day 1 of stimulation and adjusted to 1:10 on days 3 and 5 of stimulation. In another embodiment, particles are added daily or every other day, resulting in a final ratio of 1:1 on day 1 of stimulation and 1:10 on days 3 and 5 of stimulation. Those skilled in the art will appreciate that a variety of other ratios may be suitable for use in the present invention.Specifically, the ratio will vary depending on particle size and cell size and type.

[0405] In a further embodiment of the invention, cells, such as T cells, are combined with drug-coated beads, after which the beads and cells are separated, and the cells are then cultured. In an alternative embodiment, the drug-coated beads and cells are not separated but are cultured together prior to culture. In a further embodiment, the beads and cells are first concentrated by applying a force, such as a magnetic force, resulting in increased binding of cell surface markers, thereby inducing cell stimulation.

[0406] By way of example, cell surface proteins can be bound by contacting T cells with anti-CD3 and anti-CD28 conjugated paramagnetic beads (3x28 beads). In one embodiment, cells (e.g., 10 4 ~10 9 T cells) and beads (e.g., DYNABEADS® M-450 CD3 / CD28 T paramagnetic beads (1:1 ratio)) are combined in a buffer, preferably PBS (free of divalent cations such as calcium and magnesium). Again, one of skill in the art can readily appreciate that any cell concentration can be used.

[0407] For example, target cells may be very dilute in a sample, constituting only 0.01% of the sample, or the entire sample (i.e., 100%) may contain the desired target cells. Thus, any cell number is within the context of the present invention. In certain embodiments, it may be desirable to significantly reduce the volume in which the particles and cells are mixed together (i.e., increase the cell concentration) to ensure maximum contact between the cells and particles. For example, in one embodiment, a concentration of approximately 2 billion cells / mL is used. In another embodiment, greater than 100 million cells / mL is used. In further embodiments, cell concentrations of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells / mL are used. In yet other embodiments, cell concentrations from 75, 80, 85, 90, 95, or 100 million cells / mL are used. In further embodiments, a concentration of 125 million or 150 million cells / mL may be used. Using a higher concentration may result in increased cell yield, cell activation, and cell proliferation. Furthermore, using a higher cell concentration allows for more efficient capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells. Such cell populations may have therapeutic value and may be desirable to obtain in certain embodiments. For example, using a higher cell concentration allows for more efficient selection of CD8+ T cells, which typically have weaker CD28 expression.

[0408] In one embodiment of the present invention, the mixture may be cultured for a few hours (about 3 hours) to about 14 days, or any integer value of one hour therebetween. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the present invention, the beads and T cells are cultured together for about 8 days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired, so t...

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