WO2023133360A2

WO2023133360A2 - Anti-collagen triple helix repeat containing 1 (cthrc1) antibodies and methods of using the same

Info

Publication number: WO2023133360A2
Application number: PCT/US2023/010527
Authority: WO
Inventors: Sam Cooper; Allison NIXON; Max LONDON; Elizabeth KOCH; Chrispher HARVEY; Michael Briskin
Original assignee: Phenomic Ai
Priority date: 2022-01-10
Filing date: 2023-01-10
Publication date: 2023-07-13
Also published as: AU2023205066A1; CN120202217A; EP4463181A2; JP2025502557A; US20250215070A1; CN119562827A; KR20250112314A; WO2023133361A2; EP4463472A2; AU2023204767A1; KR20250112315A; WO2023133361A3; WO2023133360A3; US20250213710A1

Abstract

The present invention is directed to anti-CTHRCl antibodies, compositions comprising the same, and methods of using such antibodies and compositions for the prevention, diagnosis and treatment of a disease or disorder, such as, e.g., cancer, bone disease, fibrotic disease, arthritis, and osteoporosis.

Description

ANTI-COLLAGEN TRIPLE HELIX REPEAT CONTAINING 1 (CTHRC1)

ANTIBODIES AND METHODS OF USING THE SAME

Cross-Reference to Releated Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63/298,194, filed January 10, 2022, which is incorporated herein by reference in its entirety.

Field of the Invention

[0002] The present invention relates to antibodies and antibody domains that specifically bind to CTHCR1, to compositions thereof, and methods of using the same.

Background of the Invention

[0003] Collagen Triple Helix Repeat Containing 1 (CTHRC1) was identified from a subtractive hybridization cDNA library, seeking genes associated with repair of arterial injury (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 led to disorganization of bone growth plate chondrocytes and incomplete formation of proteoglycan complexes on collagen fibrils, resulting in collagen disorganization and gross deformities. 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, in normal adult homeostatic tissue reports of CTHRC1 expression are limited. For example, low levels of CTHRC1 expression have been seen on smooth muscle cells, but injury is required to see significant CTHRC1 expression (Leclair et al., Arterioscler. Thromb. Vase. Biol., 2008).

[0004] CTHRC1 contains a short motif (12 Gly-X-Y repeat sequences) common in collagen related proteins and is conserved across species (Mei et al., Mediators Inflamm., 2020). At the molecular level a collection of disparate reports suggest CTHRC1 regulates a number of signaling pathways including TGF-P (J. Li et al., EBioMedicine, 2019; Ni et al., Cancer Med., 2018; Zhang et al., Oncogene, 2021), canonical Wnt/p-catenin (Hou et al., Oncotarget., 2015), non-canonical Wnt/PCP pathways (Yamamoto et al., Dev. Cell, 2008), and integrin/FAK (Y.-L. Chen et al., J. Ovarian Res., 2013; Guo et al., PLoS One, 2017). However, no consensus has yet emerged as to its precise molecular mechanism of action. Thus, while early reports indicate CTHRC1 is key for bone growth in development and wound healing in adults, and likely acts downstream of TGF-p/Wnt signaling, it remains a largely uncharacterized protein, with clear gaps in understanding as to its precise function in humans.

[0005] In cancer CTHRC1 overexpression has been reported in colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC), where CTHRC1 correlates with stage and poor survival (W. Liu et al., Oncology Letters, 2016; Ni et al., Cancer Med., 2018; Wang et al., Cancer Set., 2012). Experiments using CRC, PDAC and ovarian cancer lines have suggested that CTHRC1 promotes migratory and invasive behaviors, linked to metastasis (Guo et al., J. Ovarian Res., 2017; Ni et al., Cancer Med., 2018; Park et al., Carcinogenesis, 2013). Finally, in vivo data implicates CTHRC1 in angiogenesis, specifically, xenograft growth was compromised in CTHRC1 knockout mice in tandem with the observation that blood vessel organization was significantly disrupted (Lee et al., Exp. & Mol. Med, 2016). Overall, several reports link CTHRC1 to various pro-tumorigenic roles in cancer, but again mechanistic detail is sparse.

[0006] In addition to promoting cancer, CTHRC1 is also linked to fibrosis. Notably, recent work has linked CTHRC1 expression to a pathological subset of fibroblasts in a mouse model of lung fibrosis, that can also be seen in the lungs of patients with idiopathic pulmonary fibrosis (IPF) (Tsukui et al., Nat. Commun., 2020). Evidence also suggests that CTHRC1 is upregulated in fibrotic liver diseases (J. Li et al., EBioMedicine, 2019). Importantly, depletion of CTHRC1 by genetic knockout suppressed the onset of fibrosis in a rodent model of chemically induced- liver fibrosis (J. Li et al., EBioMedicine, 2019). Taken together this data therefore also implicates CTHRC1 in fibrosis, and fibroblast biology, thus suggesting its pro-tumorigenic role may also be dependent on fibroblastic cells in the tumor microenvironment. Outside of cancer, studies also link CTHRC1 to both a protective, anti-inflammatory activity in Rheumatoid Arthritis (Jin et al., Bone, 2017), where levels of CTHRC1 in the blood can also discriminate healthy from Rheumatoid Arthritis patients (Myngbay et al., Frontiers in Immunology, 2019). CTHRC1 is also implicated as a positive regulator of bone formation and thus protective in Osteoporosis (Chen et al., Bone Research, 2019; Kimura et al., PloS One, 2008).

[0007] Overall, work on CTHRC1 to date has indicated it plays a role in bone development, wound repair, cancer, fibrosis, arthritis, and osteoporosis. However, despite these findings, a lack of any published inhibitors of CTHRC1 has precluded further investigation into the causal role CTHRC1 plays in disease, let alone provided rationale for development of therapeutic inhibitors against CTHRC1 and/or development of mAbs to target payloads to the tumor microenvironment based upon CTHRC1 binding mAbs. Accordingly, there exists a currently unmet need for antibodies and antibody domains that specifically bind to CTHRC1.

Summary of the Invention

[0008] The present invention addresses the foregoing shortcomings in the prior art via the provision and characterization of a range of antibodies and antibody domains to CTHRC1, and methods of use thereof in the prevention, diagnosis, and treatment of cancer, fibrosis and/or fibrotic disease. The anti-CTHRCl antibodies of the present invention are demonstrated to (i) selectively bind to CTHRC1, (ii) block cell adhesion to CTHRC1, (iii) become internalized within cells expressing CTHRC1 upon binding to said cells, and/or iv) recruit CD8+ T cells into the tumor microenvironment. Thus, the specificity and functional effects of the subject anti- CTHRCl antibodies and antibody domains are important in the context of cancer and fibrosis, particularly in disease states where CTHRC1 is upregulated.

[0009] In one aspect, the invention provides anti-CTHRCl antibodies that bind human CTHRC1. In embodiments, the anti-CTHRCl antibody (i) selectively binds CTHRC1, (ii) blocks cell adhesion to CTHRC1, and/or (iii) is internalized upon binding to cells that express CTHRC1.

[0010] In one embodiment, the anti-CTHRCl 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-CTHRCl antibody comprises a heavy chain variable region comprising an amino acid sequence selected from Table 3.

[0011] In one embodiment, the anti-CTHRCl 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-CTHRCl antibody comprises a light chain variable region comprising an amino acid sequence selected from Table 4. [0012] In one embodiment, the anti-CTHRCl antibody comprises a heavy chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 150- 154; a CDR2 sequence selected from the group consisting of SEQ ID NOs: 180-184; and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 210-214.

[0013] In one embodiment, the anti-CTHRCl 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.

[0014] In one embodiment, the anti-CTHRCl 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.

[0015] In one embodiment, the anti-CTHRCl 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.

[0016] In one embodiment, the anti-CTHRCl 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.

[0017] In one embodiment, the anti-CTHRCl 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.

[0018] In one embodiment, the anti-CTHRCl antibody comprises a light chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 240-244; a CDR2 sequence selected from the group consisting of SEQ ID NOs: 270-274; and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 300-304.

[0019] In one embodiment, the anti-CTHRCl 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.

[0020] In one embodiment, the anti-CTHRCl 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. [0021] In one embodiment, the anti-CTHRCl 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.

[0022] In one embodiment, the anti-CTHRCl 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.

[0023] In one embodiment, the anti-CTHRCl 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.

[0024] In one embodiment, the anti-CTHRCl 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.

[0025] In one embodiment, the anti-CTHRCl 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.

[0026] In one embodiment, the anti-CTHRCl 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.

[0027] In one embodiment, the anti-CTHRCl 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.

[0028] In one embodiment, the anti-CTHRCl 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.

[0029] In one embodiment, the anti-CTHRCl antibody has a binding affinity (KD) for CTHRC1 of less than 10 nM, preferably less than 5 nM, more preferably less than 1 nM.

[0030] In one embodiment, the anti-CTHRCl 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-CTHRCl 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-CTHRCl 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-CTHRCl 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- CTHRCl antibody comprises a heavy chain variable region comprising SEQ ID NO: 9 and a light chain variable region comprising SEQ ID NO: 10.

[0031] In one embodiment, the invention provides an anti-CTHRCl antibody that competes 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 for binding to a CTHRC1 epitope.

[0032] Anti-CTHRCl antibodies of the 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 for binding to a CTHRC1 epitope.

[0033] In some embodiments, anti-CTHRCl antibodies of the invention further comprise a human subgroup III heavy chain framework consensus sequence. In one embodiments of these antibodies, these antibodies further comprise a human KI light chain framework consensus sequence.

[0034] In one embodiment, an anti-CTHRCl antibody inhibits or neutralizes one or more human CTHRC1 functions.

[0035] In one embodiment, the anti-CTHRCl antibody is a chimeric, humanized, or human antibody. In one embodiment, the anti-CTHRCl antibody is a monocolonal antibody. In one embodiment, the anti-CTHRCl antibody is an antibody fragment. In one embodiment, the anti- CTHRCl antibody is a single-chain variable fragment. In one embodiment, the anti-CTHRCl antibody is an antibody-drug conjugate (ADC). In one embodiment, the anti-CTHRCl antibody is a radioconjugate.

[0036] In some embodiments, the anti-CTHRCl antibody, or fragment thereof, elicits little to no immunogenic response against the anti-CTHRCl antibody, or fragment thereof, in a subject, for example a human subject. In some embodiments, the invention provides a humanized antibody that elicits and/or is expected to elicit minimal or no human anti-mouse antibody response (HAMA). In one example, an antibody of the invention elicits anti-mouse antibody response that is at or less than a clinically-acceptable level.

[0037] In some aspects, the invention provides nucleic acids comprising DNA encoding any of the herein described anti-CTHRCl antibodies or portions thereof, or CARs or portions thereof. In some embodiments, the nucleic acids comprise any one or more of SEQ ID NOs: 100-109. In embodiments, the invention provides vectors comprising nucleic acids encoding any of the herein described anti-CTHRCl antibodies or portions thereof, or CARs or portions thereof. In some embodiments, the vectors comprise any one or more of SEQ ID NOs: 100-109. In embodiments, the invention provides host cells comprising any such vector. By way of example, the host cells may be CHO cells, E. coli cells, or yeast cells. A process for producing any of the herein described polypeptides is further provided and comprises culturing host cells under conditions suitable for expression of the desired polypeptide and recovering the desired polypeptide from the cell culture.

[0038] In one aspect, the invention provides methods for making an antibody of the invention. In embodiments, the invention provides a method of making a CTHRC1 antibody (which, as defined herein includes full length and fragments thereof), said method comprising expressing in a suitable host cell a recombinant vector of the invention encoding said antibody (or fragment thereof), and recovering said antibody.

[0039] In one aspect, the 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.

[0040] In one aspect, the 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-CTHRCl antibody as herein disclosed, and a light chain variable region of an anti-CTHRCl antibody as herein disclosed.

[0041] In one aspect, the invention provides a CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage, comprising an anti-CTHRCl antibody. In one embodiment, the anti-CTHRCl antibody is an antibody fragment. In one embodiment, the anti- CTHRCl antibody is an scFv.

[0042] In one aspect, the invention provides a method of inhibiting the growth of a cell that displays a CTHRC1 epitope, for example a CTHRC1 tumor epitope, either directly or in a complex, comprising contacting the cell with an anti-CTHRCl antibody or CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage, of the invention. In one embodiment, the anti-CTHRCl antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioconjugate. In embodiments, contacting the cell comprises administering to a patient a therapeutically effective amount of an anti-CTHRCl antibody or CAR modified immune cell of the invention.

[0043] In one aspect, the invention provides a method of inhibiting metastasis of a tumor that displays a CTHRC1 epitope, for example a CTHRC1 tumor epitope, either directly or in a complex, comprising contacting a cell of the tumor with an anti-CTHRCl antibody or CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage, of the invention. In one embodiment, the anti-CTHRCl antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioconjugate. In embodiments, contacting the cell comprises administering to a patient a therapeutically effective amount of an anti-CTHRCl antibody or CAR modified immune cell of the invention.

[0044] In one aspect, the invention provides a method of inducing death of a cell that displays a CTHRC1 epitope, for example a CTHRC1 tumor epitope, comprising contacting the cell with an anti-CTHRCl antibody or CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage, of the invention. In one embodiment, the anti-CTHRCl antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioconjugate. In embodiments, contacting the cell comprises administering to a patient a therapeutically effective amount of an anti-CTHRCl antibody or CAR modified immune cell of the invention.

[0045] In one aspect, the invention provides a method of decreasing size of a tumor comprised of a cell that displays a CTHRC1 epitope, for example a CTHRC1 tumor epitope, comprising contacting the cell with an anti-CTHRCl antibody or CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage, of the invention. In one embodiment, the anti- CTHRCl antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioconjugate. In embodiments, contacting the cell comprises administering to a patient a therapeutically effective amount of an anti-CTHRCl antibody or CAR modified immune cell of the invention.

[0046] In one aspect, the invention provides a method of inhibiting vascularization of a tumor comprising a cell that displays a CTHRC1 tumor epitope, comprising contacting the cell with an anti-CTHRCl antibody or CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage, of the invention. In one embodiment, the anti-CTHRCl antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioconjugate. In embodiments, contacting the cell comprises administering to a patient a therapeutically effective amount of an anti-CTHRCl antibody or CAR modified immune cell of the invention.

[0047] In one aspect, the invention provides a method of exhibiting cytostatic activity on a tumor cell or cancer associated fibroblasts displaying CTHRC1, comprising contacting the cell/cancer associated fibroblasts with an anti-CTHRCl antibody or CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage, of the invention. In one embodiment, the anti-CTHRCl antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioconjugate. In embodiments, contacting the cell comprises administering to a patient a therapeutically effective amount of an anti-CTHRCl antibody or CAR modified immune cell of the invention.

[0048] In one aspect, the invention provides a method of preventing suppression of immune cells in a tumor microenvironment, comprising contacting at least one cell of the tumor microenvironment with an anti-CTHRCl antibody or CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage, of the invention. In one embodiment, the anti- CTHRCl antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioconjugate. In embodiments, contacting the cell comprises administering to a patient a therapeutically effective amount of an anti-CTHRCl antibody or CAR modified immune cell of the invention.

[0049] In one aspect, the invention provides a method of enhancing infiltration of anti-tumor immune cells in the tumor microenvironment in vivo, comprising contacting at least one cell of the tumor microenvironment with an anti-CTHRCl antibody of the invention, preferably in combination with a cellular immunotherapy, e.g., an allogeneic or autologous T or NK cell therapy. In embodiments, contacting the cell comprises administering to a patient a therapeutically effective amount of an anti-CTHRCl antibody in conjunction with administering a cellular immunotherapy, e.g., a CAR-T or CAR-NK cell therapy.

[0050] In embodiments of the invention, a cell displaying a CTHRC1 tumor epitope is a cancer cell.

[0051] In one aspect, the invention provides for a method for treating or preventing a cell proliferative disorder associated with increased expression and/or display of CTHRC1, said method comprising administering to the subject an effective amount of an anti-CTHRCl antibody or CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR- macrophage, of the invention. In one embodiment, the anti-CTHRCl antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioconjugate. In one embodiment, said cell proliferative disorder is cancer. [0052] In one aspect, the invention provides a method of inhibiting tumor metastasis in a subject having cancer, comprising administering to the subject an effective amount of an anti- CTHRC1 antibody or CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR- macrophage, of the invention. In one embodiment, the anti-CTHRCl antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioconjugate.

[0053] In one aspect, the invention provides a method of decreasing tumor size in a subject having cancer, comprising administering to the subject an effective amount of an anti-CTHRCl antibody or CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR- macrophage, of the invention. In one embodiment, the anti-CTHRCl 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 cancer, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colon adenocarcinoma, B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck cancer, kidney clear cell cancer, kidney papillary cell cancer, myeloid leukemia, liver hepatocellular cancer, lung adenocarcinoma, lung squamous cancer, ovarian cancer, pancreatic adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, melanoma, stomach adenocarcinoma, testicular germ cell cancer, 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, comprising administering to the subject a therapeutically effective amount of an anti-CTHRCl antibody or CAR modified immune cell, such as a CAR-T or CAR- NK cell, or a CAR-macrophage, of the invention. In embodiments, the anti-CTHRCl antibody is administered in conjunction with radiation therapy.

[0056] In one embodiment, the invention provides a method of treating a subject afflicted with a fibrotic disease, comprising administering to the subject an effective amount of an anti- CTHRCl antibody or CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR- macrophage, of the invention. In one embodiment, the anti-CTHRCl antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioconjugate. In embodiments, the subject is a human subject. [0057] In embodiments, the fibrotic disease can be selected from the group consisting of idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, pulmonary arterial hypertension, kidney fibrosis, keratinosis, non-alcoholic fatty liver disease (NASH), scleroderma, rheumatoid arthritis, Crohn’s disease, ulcerative colitis, myelofibrosis and systemic lupus erythematosus.

[0058] In one aspect, the invention provides a pharmaceutical composition, comprising an anti-CTHRCl antibody and a pharmaceutically acceptable carrier. In one aspect, the invention provides a pharmaceutical composition, comprising a CAR modified immune cell, such as a CAR-T or CAR-NK cell, or a CAR-macrophage, of the invention and a pharmaceutically acceptable carrier. In one embodiment, the anti-CTHRCl antibody is used in the form of an ADC. In one embodiment, the ADC comprises a radioconjugate.

[0059] In one aspect, the invention provides methods for making an anti-CTHRCl antibody. In one aspect, the invention provides methods for making a CAR modified immune cell disclosed herein. In one embodiment, the invention provides methods for making an ADC comprising an anti-CTHRCl antibody.

[0060] In one aspect, the invention provides a method of determining the presence of CTHRC1, for example a CTHRC1 epitope, for example a CTHRC1 tumor epitope, in a subject or in a biological sample from a subject. In one embodiment, the method comprises contacting a sample with an anti-CTHRCl antibody and determining binding of the anti-CTHRCl antibody to the sample, wherein binding of the anti-CTHRCl antibody to the sample is indicative of the presence of the CTHRC1 epitope in the sample.

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

[0062] In one aspect, the invention provides a method for determining the prognosis for a subject diagnosed with cancer, comprising detecting the presence of a CTHRC1 epitope, for example a CTHRC1 tumor epitope, in the subject or in a biological sample from the subject. In one embodiment, the method involves detecting the presence of the CTHRC1 epitope in the 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 CAR modified immune cells, preferably a CAR-T or CAR-NK cells, or CAR macrophages, 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] Also provided herein are kits and methods of using the same. Brief Description of the Drawings

[0070] FIG. 1 is a heat map illustrating enzyme-linked immunosorbent assay (ELISA) data that shows identification of 12 clones with selective binding to human and/or rat CTHRC1.

[0071] FIG. 2 is a bar graph illustrating assessment of cell adhesion to CTHRC1, Periostin, and ECM protein fibronectin in a panel of fibroblast cell lines (BJ, skin; CCD-8Lu, lung; CCDI8C0, colon) and cancer cell lines (SKOV3, ovarian; Mia PaCa2, pancreatic; HCT116, colorectal).

[0072] FIG. 3 is a bar graph illustrating cell adhesion of ovarian cancer cells to CTHRC1 and the ECM proteins vitronectin and fibronectin following treatment with various integrin blocking antibodies.

[0073] FIG. 4 is a bar graph illustrating selective blocking of cell adhesion by CTHRC 1 S-M5 (AB987) and CTHRC 1S-M23 (AB988) in ovarian cancer cells.

[0074] FIGS. 5A-5B illustrate that CTHRC 1 mRNA is a top ranked marker of cancer- accociated fibroblasts (CAFs) in cancer-rich, immune-cold, tumor samples. Aggregated 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) (FIG. 5A). Genes expressed by CAFs were compared between the two groups, to determine which associate most with cancer rich samples (Wilcoxon Rank). These genes were then filtered down to those specifically expressed by CAFs in all samples (top 500; Wilcoxon rank). CTHRC 1 was the 11^th highest ranked gene in the analysis (FIG. 5B).

[0075] FIG. 6 illustrates that CTHRC 1 is highly upregulated in cancer vs. normal adjacent tissues, as determined in bulk RNA measurements analyzed from The Cancer Genome Atlas. Across the set of indications profiled, the highest levels of CTHRC 1 expression was seen in solid cancers, notably breast, lung, ovarian, pancreatic, sarcoma, melanoma, and uterine carcinosarcomas (P < 0.001 in all cases tested). Collectively this indicates that CTHRC 1 expression is highest in more desmoplastic, stromal rich cancers, in line with the notion that it is principally a CAF secreted target.

[0076] FIG. 7 depicts a series of survival plots showing CTHRC 1 survival curves for a number of solid tumors where CTHRC1 is associated with poor survival. Values were derived from The Cancer Genome Atlas. An online tool, GEPIA, was used to calculate and plot the survival curves.

[0077] FIG. 8 illustrates that CTHRC1 levels increase with stage in liver cancer (left) and colorectal cancer (right) alongside other indications (not shown). In both cases, p < 0.05; Student’s T test between Stage I and Stage IV. Stage II and Stage III display intermediate CTHRC1 expression levels.

[0078] FIGS. 9A-9C are histograms showing bulk-RNA expression level of known stromal targets FAP (FIG. 9A) and LRRC15 (FIG. 9B) alongside CTHRC1 (FIG. 9C) in pancreatic cancer samples (The Cancer Genome Atlas) vs. all normal tissue samples (GTEX). The histograms highlight that a significant therapeutic window exists for targeting CTHRC1 in pancreatic cancer, based on bulk-RNA measurements. This window is similar, if not larger, than that of the known/developed stromal targets FAP and LRRC15.

[0079] FIG. 10 is a dataset illustrating that very high levels of CTHRC1 expression is seen in CAFs across many solid cancers, as well as cancer epithelial expression in breast, pancreatic, lung, ovarian, and skin cancers. In contrast, minimal CTHRC1 expression is seen in normal tissue. The data was obtained based on previously generated large integrated single-cell RNA- sequence Atlas to enable probing of gene-expression at the single cell level across numberous cancer and normal tissue samples (Swechha, 2021). The data highlights a potentially large therapeutic window for blocking CTHRC1 and value in using mAbs against CTHRC1 for targeting payloads, e.g., ADCs, to the tumor microenvironment.

[0080] FIG. 11 is a dataset using the same atlas discussed with regard to FIG. 10, performed for LRRC15, a known non-toxic stromal target, where antibodies ADCs have been engineered and shown to be safe in the clinic. Low levels of LRRC15 were seen in normal tissue as compared to CTHRC1. In cancerous single-cell RNA (scRNA) datasets it was observed that LRRC15 expression, unlike CTHRC1, is more selectively localized to specific CAFs in specific breast cancers, and low levels of expression are also seen on sarcoma cancer cells.

[0081] FIG. 12 is a graph illustrating quantitative ELISA of CTHRC1 in mono-culture and co-culture. Supernatants from a set of different mono-cultures (fibroblast or cancer cells) and co-cultures (fibroblasts and cancer cells) were profiled for CTHRC1 levels. CTHRC1 is expressed at low levels in fibroblast mono-cultures (BJ, CCDI8-C0) and upregulated in co- culture, indicating that interactions between fibroblasts and cancer cells drives CTHRC1 expression.

[0082] FIG. 13 shows a series of images of tissue from three mouse models probed with CTHRC1 specific mAbs. Broad staining was observed within tumor regions, indicating CTHRC1 protein localizes to cancer regions in vivo.

[0083] FIG. 14 shows a series of images of tissue from three human cancers, showing variation in CTHRC1 expression patterns at the protein level, indicative of different expression dynamics. In Head and Neck and Melanoma cancer samples, CTHRC1 localizes to interfaces between cancer and stromal tissue. In pancreatic cancer, CTHRC1 is seen broadly expressed on regions dense in CAFs/stromal tissue.

[0084] FIGS. 15A-15B illustrate that CTHRC1 expression is seen in human cancer cell lines (FIG. 15A), and that CTHRC1 mAbs M14 and M23 bind to the surface of a range of human cancer cell lines that express CTHRC1 (FIG. 15B). Binding of the same mAbs was seen to EMT6 cancer cells, which express CTHRC1 in vivo, as shown in FIG. 13. As illustrated, the higher affinity mAb M14 shows a greater level of cell-surface binding than the lower affinity mAb M23.

[0085] FIGS. 16A-16D are graphs showing that CTHRC1 mAbs are rapidly internalized by cancer cells with or without the addition of 50 nM exogenous CTHRC1. Internalization was much faster in SKOV3 ovarian cells that express high levels of CTHRC1, and exogenous CTHRC1 also had less of an effect on the internalization rate (FIGS. 16A-16B) compared to KP4 pancreatic cells (FIGS. 16C-16D). In line with higher levels of surface binding, CTHRC1S-M14 (FIG. 16A and FIG. 16C) internalized faster than CTHRC1S-M23 (FIG. 16B and FIG. 16D)

[0086] FIG. 17 shows graphs illustrating that CTHRC1 mAbs are internalized by mouse EMT6 and 4T1 cancer cells without the addition of exogenous CTHRC1. Internalization was observed to occur muct faster in EMT6 cancer cells vs. 4T1 cells. This is in line with cancer cells in this model having a more mesenchymal appearance. Similar to human cell lines, a greater rate of internalization was observed for CTHRC1S-M14 (left) versus CTHRC1S-M23 (right). [0087] FIG. 18 shows graphs illustrating that CTHRC 1 mAbs conjugated to MMAE (Vedotin) results in selective killing of SKOV3 cells.

[0088] FIG. 19A-19C illustrate efficacy of CTHRC 1 tested in the syngeneic mouse breast tumor model, EMT6. Results are shown for three different anti-CTHRCl antibodies, specifically M5 (FIG. 19A), M14 (FIG. 19B), and M14 (FIG. 19C).

[0089] FIG. 20A-20B illustrate efficacy of anti-CTHRCl (clone M5) in the PD-1 resistant Pan02 pancreatic cancer model.

[0090] FIG. 21 illustrates the effect of preconditioning of cells with anti-CTHRCl antibody on infiltration of CD8 T cells.

Detailed Description of the Invention

General Techniques

[0091] 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 the art. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second 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. Ausubel et al., eds., 1987, and periodic updates); “PCR: The Polymerase Chain Reaction”, (Mullis et al., ed., 1994); “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988); “Phage Display: A Laboratory Manual” (Barbas et al., 2001).

[0092] 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.

II. Definitions

[0093] For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will 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.

[0094] The term “Collagen Triple Helix Repeat Containing 1 (CTHRC1)”, as used herein, refers to any native CTHRC1 from any vertebrate source, including mammals such as primates (e.g., humans, primates, and rodents (e.g., mice and rats), unless otherwise indicated. 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.

[0095] The term “Collagen Triple Helix Repeat Containing 1” encompasses “full-length,” unprocessed CTHRC1 as well as any form of CTHRC1 that results from processing in the cell. The term encompasses naturally occurring variants of CTHRC1, e.g., splice variants, allelic variants and isoforms. The CTHRC1 polypeptides described herein may be isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods. A “native sequence CTHRC1 polypeptide” comprises a polypeptide having the same amino acid sequence as the corresponding CTHRC1 polypeptide derived from nature. Such native sequence CTHRC1 polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term “native sequence CTHRC1 polypeptide” specifically encompasses naturally-occurring truncated or secreted forms of the specific CTHRC1 polypeptide (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide. 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 shown in the accompanying disclosure.

[0096] A “modification” of an amino acid residue/position, as used herein, refers to a change of a primary amino acid sequence as compared to a starting amino acid sequence, wherein the change results from a sequence alteration involving said amino acid residue/positions. For example, typical modifications include substitution of the residue (or at said position) with another amino acid (e.g., a conservative or non-conservative substitution), insertion of one or more (generally fewer than 5 or 3) amino acids adjacent to said residue/position, and deletion of said residue/position. An “amino acid substitution”, or variation 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 alteration in at least one physicobiochemical activity of the variant polypeptide compared to a polypeptide comprising the starting (or “wild type”) amino acid sequence. For example, in the case of an antibody, a physicobiochemical activity that is altered can be binding affinity, binding capability and/or binding effect upon a target molecule.

[0097] The term “antibody” is used in the broadest sense and specifically covers, for example, single anti-CTHRCl monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full length or intact monoclonal antibodies), anti-CTHRCl antibody compositions with polyepitopic specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desired biological activity), formed from at least two intact antibodies, single chain anti-CTHRCl antibodies, and fragments of anti-CTHRCl antibodies (see below), including Fab, Fab’, F(ab’)2 and Fv fragments, diabodies, single domain antibodies (sdAbs), as long as they exhibit the desired biological or immunological activity. Also included among anti-CTHRCl antibodies, and among fragments in particular, are portions of anti-CTHRCl antibodies (and combinations of portions of anti- CTHRCl antibodies, for example, scFv) that may be used as targeting arms, directed to e.g., a CTHRC1 tumor epitope, in chimeric antigenic receptors of CAR-T cells, CAR-NK cells, or CAR-macrophages. Such fragments are not necessarily proteolytic fragments but rather portions of polypeptide sequences that can confer affinity for target. The term “immunoglobulin” (Ig) is used interchangeably with antibody herein. An antibody can be, for example, human, humanized and/or affinity matured.

[0098] The terms “anti-CTHRCl antibody”, “CTHRC1 antibody”, and “an antibody that binds to CTHRC1” are used interchangeably. Anti-CTHRCl antibodies are preferably capable of binding with sufficient affinity such that the antibody is useful as a diagnostic and/or therapeutic agent, whether in isolation or as part of fusion protein, cell, or cell composition.

[0099] In one embodiment, CTHRC1 antibody is used herein to specifically refer to an anti- CTHRCl monoclonal antibody that (i) comprises the heavy chain variable domain of any one of SEQ ID nOs: 1, 3, 5, 7, and 9; and/or the light chain variable domain of any one of SEQ ID nOs: 2, 4, 6, 8, and 10, or (ii) comprises one, two, three, four, five, or six of the CDRs shown in Table 3 or Table 4. [00100] An “isolated antibody” is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials which would interfere with therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

[00101] The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to a 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 intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and y chains and four CH domains for p and a isotypes. Each L chain has at the N-terminus, a variable domain (VL) 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 of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994, at page 71 and Chapter 6.

[00102] The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated a, 6, a, y, and p, respectively. The y and a classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[00103] The “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as “VH” or “VH” The variable domain of the light chain may be referred to as “VL” or “VL”. These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.

[00104] The term “variable” refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a P-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the P-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, 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)).

[00105] An “intact” antibody is one which comprises an antigen-binding site as well as a CL and at least heavy chain constant domains, CHI, CH2 and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

[00106] “Antibody fragments” comprise a portion of an intact antibody, preferably the antigen binding 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. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-62 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, an antibody fragment comprises an antigen binding site of the intact antibody and thus retains the ability to bind antigen. Also included among anti-CTHRCl antibody fragments are portions of anti-CTHRCl antibodies (and combinations of portions of anti-CTHRCl antibodies, for example, scFv) that may be used as targeting arms, directed to e.g., a CTHRC1 tumor epitope, in chimeric antigenic receptors of CAR-T cells or CAR-NK cells, or CAR macrophages. Such fragments are not necessarily proeteolytic fragments but rather portions of polypeptide sequences that can confer affinity for target.

[00107] 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 along with the variable region domain of the H chain (VH), and the first constant domain of one heavy chain (CHI). Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment which roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Fab’ fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation 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.

[00108] The Fc fragment comprises the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of antibodies are determined by sequences in the Fc region, which region is also the part recognized by Fc receptors (FcR) found on certain types of cells.

[00109] “Fv” is the minimum antibody fragment which contains a complete antigen-recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. In a single-chain Fv (scFv) species, one heavy - and one light-chain variable domain can be covalently linked by a flexible peptide linker such that the light and heavy chains can associate in a “dimeric” structure analogous to that in a two- chain Fv species. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute 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 comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. [00110] “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise 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 sFv to form a desired structure for antigen binding. For a review of sFv, see, e.g., Pluckthun 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 anti-CTHRCl antibody derived scFv is used as the targeting arm of a CAR- T cell, a CAR-NK cell, or a CAR-macrophage disclosed herein.

[00111] The term “monoclonal antibody” as used herein 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 naturally occurring mutations that may be present in minor amounts. 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, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The modifier “monoclonal” is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature, 256: 495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (e.g., U.S. Patent No. 4,816,567). The “monoclonal antibodies” may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352: 624-8 (1991) and Marks et al., J. Mol. Biol., 222: 581-97 (1991), for example.

[00112] The term “hypervariable region”, “HVR”, or “HV”, when used herein refers to the regions of an antibody variable domain which are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six hypervariable regions; three in the VH (Hl, H2, H3), and three in the VL (LI, L2, L3). A number of hypervariable region delineations are in use and are encompassed herein. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The end 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 the insertions at H35A and H35B; if neither 35 A nor 35B is present, the loop ends at 32; if only 35 A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software. The “contact” hypervariable regions are based on an analysis of the available complex crystal structures. The residues from each of these hypervariable regions are noted below.

Loop Kabat AbM Chothia Contact

LI L24-L34 L24-L34 L24-L34 L30-L36

L2 L50-L56 L50-L56 L50-L56 L46-L55

L3 L89-L97 L89-L97 L89-L97 L89-L96

Hl H31-H35B H26-H35B H26-H32..34 H30-H35B

(Kabat Numbering)

Hl H31-H35 H26-H35 H26-H32 H30-H35

(Chothia Numbering)

H2 H50-H65 H50-H58 H52-H56 H47-H58

H3 H95-H102 H95-H102 H95-H102 H93-H101

[00113] Hypervariable regions may comprise “extended hypervariable regions” as follows: 24- 36 or 24-34 (LI), 46-56 or 50-56 (L2) and 89-97 (L3) in the VL and 26-35B (Hl), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102 or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[00114] “Framework” or “FR” residues are those variable domain residues other than the hypervariable region residues herein defined.

[00115] The term “variable domain residue numbering as in Kabat” or “amino acid position numbering as in Kabat”, and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies 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 include a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc according to Kabat) after heavy chain FR residue 82. The Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.

[00116] The Kabat numbering system is generally used when referring to a residue 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 a residue in an immunoglobulin heavy chain constant region (e.g., the EU index reported in Kabat et al., supra). The “EU index as in Kabat” refers to the residue numbering of the human IgGl EU antibody. Unless stated otherwise herein, references to residue numbers in the variable domain of antibodies means residue numbering by the Kabat numbering system.

[00117] A “blocking” antibody or an “antagonist” antibody is one which inhibits or reduces biological activity of the antigen it binds. Preferred blocking antibodies or antagonist antibodies substantially or completely inhibit the biological activity of the antigen. In one embodiment, an anti-CTHRCl antibody is provided, which is an antagonist antibody.

[00118] An antibody that “binds” an antigen or epitope of interest is one that binds the antigen or epitope with sufficient affinity that is measurably different from a non-specific interaction. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity.

[00119] An antibody that inhibits the growth of tumor cells is one that results in measurable growth inhibition of cancer cells. In one embodiment, an anti-CTHRCl antibody is capable of inhibiting the growth of cancer cells displaying a CTHRC1 tumor epitope. As referred to herein, a CTHRC1 tumor epitope comprises a CTHRC1 epitope capable of being bound by an anti- CTHRCl antibody, or fragment thereof, as herein disclosed, or capable of being at least partially bound by an antibody or other molecule that competes with an anti-CTHRCl antibody as herein disclosed for binding to said epitope. Preferred growth inhibitory anti-CTHRCl antibodies inhibit growth of CTHRC1 -expressing tumor cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control, the control typically being tumor cells not treated with the antibody being tested.

[00120] Anti-CTHRCl antibodies may (i) inhibit tumor metastasis in vivo; (ii) inhibit tumor growth in vivo; (iii) decrease tumor size in vivo; (iv) inhibit tumor vascularization in vivo; (v) exhibit cycotoxic activity activity on tumor cells and cancer associated fibroblasts expressing CTHRC1 in vivo; (vi) exhibit cytostatic activity on tumor cells or cancer associated fibroblasts expressing CTHRC1 in vivo; or (vii) prevent suppression of immune-cells in the tumor microenvironment in vivo.

[00121] The term “antagonist” is used in the broadest sense, and includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of 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, etc. Methods for identifying antagonists of a CTHRC1 polypeptide, may comprise contacting a CTHRC1 polypeptide, with a candidate antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the CTHRC1 polypeptide.

[00122] The terms “cancer” and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A “tumor” comprises one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), skin cancer, melanoma, lung cancer including small-cell lung cancer, non-small cell lung cancer (“NSCLC”), adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), glioblastoma, cervical cancer, ovarian cancer (e.g., high grade serous ovarian carcinoma), liver cancer (e.g., hepatocellular carcinoma (HCC)), bladder cancer (e.g., urothelial bladder cancer), testicular (germ cell tumor) cancer, hepatoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer (e.g., renal cell carcinoma, nephroblastoma or Wilms’ tumor), prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer. Additional examples of cancer include, without limitation, adrenocortical cancer, cholangiocarcinoma, colon adenocarcinoma, B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, kidney clear cell cancer, kidney papillary cell cancer, myeloid leukemia, lung adenocarcinoma, lung squamous cancer, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, stomach adenocarcinoma, thymoma, uterine corpus, and uterine carcinosarcoma.

[00123] The term “metastatic cancer” means the state of cancer where the cancer cells of a tissue of origin are transmitted from the original site to one or more sites elsewhere in the body, by the blood vessels or lymphatics, to form one or more secondary tumors in one or more organs besides the tissue of origin. A prominent example is metastatic breast cancer.

[00124] As used herein, an “CTHRC1 -associated cancer” is a cancer that is associated with over-expression of a CTHRC1 gene or gene product and/or is associated with display of a CTHRC1 tumor epitope. Suitable control cells can be, for example, cells from an individual who is not affected with cancer or non-cancerous cells from the subject who has cancer.

[00125] The present methods include methods of treating a subject having cancer. Particularly cancer that is associated with expression of a CTHRC1 tumor epitope. The present methods also include methods for modulating certain cell behaviours, particularly cancer cell behaviours, particularly cancer cells displaying a CTHRC1 tumor epitope.

[00126] The terms “cell proliferative disorder” and “proliferative disorder” refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[00127] “ Tumor”, as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.

[00128] The term “fibrotic disease” as used herein broadly refers to a number of different diseases that are characterized by the development of organ fibrosis, examples which include but are not limited to idiopathic pulmonary fibrosis (TPF) and scleroderma. The term “fibrosis” refers to the development of fibrous connective tissue as a representative response to injury or damage. Fibrosis may occur as part of normal healing or responsive to excess tissue deoposition that occurs as part of a pathological process.

[00129] The terms “predictive” and “prognostic” as used herein are also interchangeable. In one sense, the methods for prediction or prognostication are to allow the person practicing a predictive/prognostic method of the invention to select patients that are deemed (usually in advance of treatment, but not necessarily) more likely to respond to treatment with an anti-cancer agent, preferably an anti-CTHRCl antibody or a CAR engineered cell of the invention.

III. Compositions and Methods of the Invention

A. Anti-CTHRCl Antibodies

[00130] In one embodiment, the present invention provides anti-CTHRCl antibodies which may find use herein as therapeutic agents. Exemplary antibodies include polyclonal, monoclonal, chimeric, humanized, and human antibodies.

1. Polyclonal Antibodies

[00131] Polyclonal antibodies may be raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized. For example, the antigen can be conjugated to keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOC12, or R'N=C=NR, where R and R1 are different alkyl groups.

[00132] Animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 pg or 5 pg of the 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 Vs to 1/10 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 also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably used to enhance the immune response.

2. Monoclonal Antibodies

[00133] A monoclonal antibody (mAb) to an antigen-of-interest can be prepared by using any technique known in the art. These include, but are 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). The Selected Lymphocyte Antibody Method (SLAM) (Babcook, J.S., et al., A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc Natl Acad Sci U S A, 1996. 93 (15): p. 7843-8. ) and (McLean G et al., 2005, J Immunol. 174(8): 4768-78. Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, and IgD and any subclass thereof. The hybridoma producing the mAbs of use in this invention may be cultivated in vitro or in vivo.

[00134] 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).

[00135] In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may 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 a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[00136] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium which may contain one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. [00137] 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 a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Va., USA. Human myeloma and mouse-human heteromyeloma cell lines also have 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)).

[00138] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[00139] 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).

[00140] Once hybridoma cells that produce 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 culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal, e.g., by intraperitoneal injection of the cells into mice.

[00141] 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 ionexchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc.

[00142] DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol. 5: 256-62 (1993) and Pliickthun, Immunol. Rev. 130: 151-88 (1992).

[00143] 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- 83 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21 : 2265-6 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies.

[00144] The DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (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 the immunoglobulin coding sequence with all or part of the coding sequence for a nonimmunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

3. Chimeric, Humanized, and Human Antibodies

[00145] In some embodiments, the anti-CTHRCl antibody is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 : 6851-5 (1984)). In one example, a chimeric antibody comprises a non- human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or nonhuman primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[00146] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a nonhuman antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise 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.

[00147] The anti-CTHRCl antibodies of the invention may comprise humanized antibodies or human antibodies. Humanized forms of non-human (e.g., murine or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from a complementary 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 instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the 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)).

[00148] 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 domain. 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 the 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.

[00149] As is known in the art, and as described in greater detail herein, the amino acid position/boundary delineating a hypervariable region of an antibody can vary, depending on the context and the various definitions known in the art (as described below). Some positions within a variable domain may be viewed as hybrid hypervariable positions in that these positions can be deemed to be within a hypervariable region under one set of criteria while being deemed to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in extended hypervariable regions (as further defined below). The invention provides antibodies comprising modifications in these hybrid hypervariable positions. In one embodiment, these hypervariable positions include one or more positions 26-30, 33-35B, 47-49, 57-65, 93, 94 and 101-102 in a 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 a light chain variable domain. In one embodiment, an antibody of the invention comprises a human variant human subgroup consensus framework sequence modified at one or more hybrid hypervariable positions.

[00150] An antibody of the invention can comprise any suitable human or human consensus light chain framework sequences, provided the antibody exhibits the desired biological characteristics (e.g., a desired binding affinity). In one embodiment, an antibody of the invention comprises at least a portion (or all) of the framework sequence of human K light chain. In one embodiment, an antibody of the invention comprises at least a portion (or all) of human K subgroup I framework consensus sequence. [00151] 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 source which is non-human. These non-human amino acid residues are often referred to as “import” residues, which are typically taken from an “import” variable domain. Humanization can be essentially performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534- 1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially 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.

[00152] The choice of human variable domains, both light and heavy, to be used in making the humanized antibodies is very important to reduce antigenicity and HAMA response (human antimouse antibody) when the antibody is intended for human therapeutic use. Reduction or elimination of a HAMA response is a significant aspect of 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 invention provides antibodies that are humanized such that HAMA response is reduced or eliminated. Variants of these antibodies can further be obtained using routine 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 which is closest to that of the rodent is identified and the human framework region (FR) within it 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 a particular framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same framework may 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)).

[00153] For example, an amino acid sequence from an antibody as described herein can serve as a starting (parent) sequence for diversification of the framework and/or hypervariable sequence(s). A selected framework sequence to which a starting hypervariable sequence is linked is referred to herein as an acceptor human framework. While the acceptor human frameworks may be from, or derived from, a human immunoglobulin (the VL and/or VH regions thereof), preferably the acceptor human frameworks are from, or derived from, a human consensus framework sequence as such frameworks that have been demonstrated to have minimal, or no, immunogenicity in human patients.

[00154] Where the acceptor is derived from a human immunoglobulin, one may optionally select a human framework sequence that is selected based on its homology to the donor framework sequence by aligning the donor framework sequence with various human framework sequences in a collection of human framework sequences, and select the most homologous framework sequence as the acceptor.

[00155] In one embodiment, human consensus frameworks herein are from, or derived from, VH subgroup III and/or VL kappa subgroup I consensus framework sequences.

[00156] While the acceptor may be identical in sequence to the human framework sequence selected, whether that be from a human immunoglobulin or a human consensus framework, the present invention contemplates that the acceptor sequence may comprise pre-existing amino acid substitutions relative to the human immunoglobulin sequence or human consensus framework sequence. These pre-existing substitutions are preferably minimal; usually four, three, two or one amino acid differences only relative to the human immunoglobulin sequence or consensus framework sequence.

[00157] Hypervariable region residues of the non-human antibody are incorporated into the VL and/or VH acceptor human frameworks. For example, one may incorporate residues corresponding to the Kabat CDR residues, the Chothia hypervariable loop residues, the Abm residues, and/or contact residues. Optionally, the extended hypervariable region residues as follows are incorporated: 24-34 (LI), 50-56 (L2) and 89-97 (L3), 26-35B (Hl), 50-65, 47-65 or 49-65 (H2) and 93-102, 94-102, or 95-102 (H3). [00158] While “incorporation” of hypervariable region residues is discussed herein, it will be appreciated that this can be achieved in various ways, for example, nucleic acid encoding the desired amino acid sequence can be generated by mutating nucleic acid encoding the mouse variable domain sequence so that the framework residues thereof are changed to acceptor human framework residues, or by mutating nucleic acid encoding the human variable domain sequence so that the hypervariable domain residues are changed to non-human residues, or by synthesizing nucleic acid encoding the desired sequence, etc.

[00159] As described herein, hypervariable region-grafted variants may be generated by Kunkel mutagenesis of nucleic acid encoding the human acceptor sequences, using a separate oligonucleotide for each hypervariable region. Kunkel et al., Methods Enzymol. 154:367-382 (1987). Appropriate changes can be introduced within the framework and/or hypervariable region, using routine techniques, to correct and re-establish proper hypervariable region-antigen interactions.

[00160] Phage(mid) display (also referred to herein as phage display in some contexts) can be used as a convenient and fast method for generating and screening many different potential variant antibodies in a library generated by sequence randomization. However, other methods for making and screening altered antibodies are available to the skilled person.

[00161] Phage(mid) display technology has provided a powerful tool for generating and selecting novel proteins which bind to a ligand, such as an antigen. Using the techniques of phage(mid) display allows the generation of large libraries of protein variants which can be rapidly sorted for those sequences that bind to a target molecule with high affinity. Nucleic acids encoding variant polypeptides are generally fused to a nucleic acid sequence encoding a viral coat protein, such as the gene III protein or the gene VIII protein. Monovalent phagemid display systems where the nucleic acid sequence encoding the protein or polypeptide is fused to a nucleic acid sequence encoding a portion of the gene III protein have been developed. (Bass, S., Proteins, 8:309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3:205 (1991)). In a monovalent phagemid display system, the gene fusion is expressed at low levels and wild type gene III proteins are also expressed so that infectivity of the particles is retained. Methods of generating peptide libraries and screening those libraries have been disclosed in many patents (e.g., U.S. Pat. No. 5,723,286, U.S. Pat. No. 5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,427,908 and U.S. Pat. No. 5,498,530).

[00162] Libraries of antibodies or antigen binding polypeptides have been prepared in a number of ways including by altering a single gene by inserting random DNA sequences or by cloning a family of related genes. Methods for displaying antibodies or antigen binding fragments using phage(mid) display have been described in U.S. Pat. Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727. The library is then screened for expression of antibodies or antigen binding proteins with the desired characteristics.

[00163] Methods of substituting an amino acid of choice into a template nucleic acid are well established in the art, some of which are described herein. For example, hypervariable region residues can be substituted using the Kunkel method (e.g., Kunkel et al., Methods Enzymol. 154:367-382 (1987)).

[00164] The sequence of oligonucleotides includes one or more of the designed codon sets for the hypervariable region residues to be altered. A codon set is a set of different nucleotide triplet sequences used to encode desired variant amino acids. Codon sets can be represented using symbols to designate particular nucleotides or equimolar mixtures of nucleotides as shown in below according to the IUB code.

IUB Codes

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) [00165] For example, in the codon set DVK, D can be nucleotides A or G or T; V can be A or G or C; and K can be G or T. This codon set can present 18 different codons and can encode amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

[00166] Oligonucleotide or primer sets can be synthesized using standard methods. A set of oligonucleotides can be synthesized, for example, by solid phase synthesis, containing sequences that represent all possible combinations of nucleotide triplets provided by the codon set and that will encode the desired group of amino acids. Synthesis of oligonucleotides with selected nucleotide “degeneracy” at certain positions is well known in that art. Such sets of nucleotides having certain codon sets can be synthesized using commercial nucleic acid synthesizers (available from, for example, Applied Biosystems, Foster City, Calif.), or can be obtained commercially (for example, from Life Technologies, Rockville, Md.). Therefore, a set of oligonucleotides synthesized having a particular codon set will typically include a plurality of oligonucleotides with different sequences, the differences established by the codon set within the overall sequence. Oligonucleotides, as used according to the invention, have sequences that allow for hybridization to a variable domain nucleic acid template and also can include restriction enzyme sites for cloning purposes.

[00167] In one method, nucleic acid sequences encoding variant amino acids can be created 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). Briefly, nucleic acid sequences encoding variant amino acids are created by hybridizing an oligonucleotide set encoding the desired codon sets to a DNA template, where the template is the single-stranded form of the plasmid containing a variable region nucleic acid template sequence. After hybridization, DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus incorporate the oligonucleotide primer and will contain the codon sets as provided by the oligonucleotide set.

[00168] Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation(s). This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule. The oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al., Proc. Nat'l. Acad. Sci. USA, 75:5765 (1978).

[00169] The DNA template is generated by those vectors that are either derived from bacteriophage M13 vectors (the commercially available M13 mp 18 and M13 mp 19 vectors are suitable), or those vectors that contain a single-stranded phage origin of replication as described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus, the DNA that is to be mutated can be inserted into one of these vectors in order to generate single-stranded template. Production of the single-stranded template is described in sections 4.21-4.41 of Sambrook et al., above.

[00170] To alter the native DNA sequence, the oligonucleotide is hybridized to the 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. A heteroduplex molecule is thus formed such that one strand of DNA encodes the mutated form of gene 1, and the other strand (the original template) encodes the native, unaltered sequence of gene 1. This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. coll JM101. After growing the cells, they are plated onto agarose plates and screened using the oligonucleotide primer radiolabelled with a 32-Phosphate to identify the bacterial colonies that contain the mutated DNA.

[00171] The method described immediately above may be modified such that a homoduplex molecule is created wherein both strands of the plasmid contain the mutation(s). The modifications are as follows: The single stranded oligonucleotide is annealed to the singlestranded template as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTT), is combined with a modified thiodeoxyribocytosine called dCTP-(aS) (which can be obtained from Amersham). This mixture is added to the template-oligonucleotide complex. Upon addition of DNA polymerase to this mixture, a strand of DNA identical to the template except for the mutated bases is generated. In addition, this new strand of DNA will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion. After the template strand of the double-stranded heteroduplex is nicked with an appropriate restriction enzyme, the template strand can be digested with ExoIII nuclease or another appropriate nuclease past the region that contains the site(s) to be mutagenized. The reaction is then stopped to leave 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.

[00172] As indicated previously the sequence of the oligonucleotide set is of sufficient length to hybridize to the template nucleic acid and may also, but does not necessarily, contain restriction sites. The DNA template can be generated by those vectors that are either derived from bacteriophage Ml 3 vectors or vectors that contain a single-stranded phage origin of replication as described by Viera et al. Meth. Enzymol., 153:3 (1987). Thus, the DNA that is to be mutated must be inserted into one of these vectors in order to generate single-stranded template. Production of the single-stranded template is described in sections 4.21-4.41 of Sambrook et al., supra.

[00173] According to another method, antigen binding may be restored during humanization of antibodies through the selection of repaired hypervariable regions (see, e.g., US application Ser. No. 11/061,841, filed Feb. 18, 2005). The method includes 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 may be accompanied by modifications in the acceptor framework sequence.

[00174] 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 established by the codon sets provided within the sequence of the oligonucleotides. The upstream and downstream oligonucleotide sets, along with a variable domain template nucleic acid sequence, can be used in a polymerase chain reaction to generate a “library” of PCR products. The PCR products can be referred to as “nucleic acid cassettes”, as they can be fused with other related or unrelated nucleic acid sequences, for example, viral coat proteins and dimerization domains, using established molecular biology techniques. [00175] The sequence of the PCR primers includes one or more of the designed codon sets for the solvent accessible and highly diverse positions in a hypervariable region. As described above, a codon set is a set of different nucleotide triplet sequences used to encode desired variant amino acids.

[00176] Antibody selectants that meet the desired criteria, as selected through appropriate screening/selection steps can be isolated and cloned using standard recombinant techniques.

[00177] 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 which illustrate and display probable three-dimensional conformational structures 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., the analysis of residues that influence the ability of the candidate immunoglobulin to bind 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, the hypervariable region residues are directly and most substantially involved in influencing antigen binding.

[00178] Various forms of a humanized anti-CTHRCl antibody 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 IgGl antibody.

[00179] 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, it has been described that the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ -line mutant mice results in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90: 2551 (1993); Jakobovits et al., Nature, 362: 255-8 (1993); Bruggemann et al., Year in Immuno. 7: 33 (1993); U.S. Pat. Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97/17852).

[00180] 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 a 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 the B-cell. Phage display can be performed in a variety of formats, reviewed in, e.g., 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 diverse array of anti -oxazol one antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from unimmunized human donors can be constructed and antibodies to a diverse array of antigens (including self-antigens) can be isolated 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).

[00181] As discussed above, human antibodies may also be generated by in vitro activated B cells (see, e.g., U.S. Pat. Nos. 5,567,610 and 5,229,275).

[00182] In another embodiment, the antibodies of this 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 mice referred to herein as the HuMAb Mouse™ and KM Mouse™, respectively, and are collectively referred to herein as “human Ig mice.”

[00183] The HuMAb Mouse™ (Medarex, Inc.) contains human immunoglobulin gene miniloci that encode unrearranged human heavy (p and y) and K light chain immunoglobulin sequences, together with targeted mutations that inactivate the endogenous p. and K chain loci (see e.g., Lonberg, et al. (1994) Nature 368(6474): 856-9). Accordingly, the mice exhibit reduced expression of mouse IgM or K, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to generate high affinity human IgGK 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. ( \ 99S) Intern. Rev. Immunol. 13: 65-93, and Harding, F. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci. 764: 536-46). Preparation and use of the HuMAb Mouse™, and the genomic modifications carried by such mice, is further described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5: 647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3720-4; Choi et al. (1993) Nature Genetics 4: 117-23; Chen, J. et al. (1993) EMBO J. 12: 21-830; Tuaillon et al., (1994) J. Immunol. 152: 2912-20; Taylor, L. et al. (1994) International Immunology 6: 579-91; and Fishwild, D. et al. (1996) Nature Biotechnology 14: 845-51, the contents of all of which are hereby specifically incorporated by reference in their entirety. See further, U.S. Pat. Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016; 5,814,318; 5,874,299; and 5,770,429; U.S. Pat. No. 5,545,807; PCT 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.

[00184] In another embodiment, human antibodies of this disclosure can be raised using a mouse that carries human immunoglobulin sequences on transgenes and transchomosomes, such as a mouse that carries a human heavy chain transgene and a human light chain transchromosome. This mouse is referred to herein as a “KM Mouse™” and is described in detail in PCT Publication WO 02/43478.

[00185] Still further, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-CTHRCl antibodies of this disclosure. For example, an alternative transgenic system referred to as the Xenomouse (Abgenix, Inc.) can be used; such mice are described in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181; 6,114,598; 6,150,584 and 6,162,963.

[00186] Moreover, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to raise anti-CTHRCl antibodies of this disclosure. For example, mice carrying both a human heavy chain transchromosome and a human light chain tranchromosome, referred to as “TC mice” can be used; such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97: 722-7. As another example, cows carrying human heavy and light chain transchromosomes have been described in the art (e.g., Kuroiwa et al. (2002) Nature Biotechnology 20: 889-94 and PCT application No. WO 2002/092812) and can be used to raise anti-CTHRCl antibodies of this disclosure. Additional examples of transgenic animals that can be used to produce anti-CTHCRl 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. Pat. No. 8,907,157; European Pat. No. 2152880B1; European Pat. No. 2336329B1). Yet another example includes the use of VELOCIMMUNE® Technology (see, for example, U.S. Pat. No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®. Briefly, the VELOCIMMUNE® technology involves generation of a transgenic mouse having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mouse produces an antigen-binding protein, e.g., antibody, comprising a human variable region and a mouse constant region in response to antigenic stimulation. The DNA encoding the variable regions of the heavy and light chains of the antibody are isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in a cell capable of expressing the fully human antibody.

4. Antibody Fragments

[00187] In certain circumstances there are advantages of 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.

[00188] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were derived 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. coH, thus allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio/Technology 10:163-7 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Pat. No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv) (see WO 93/16185; U.S. Pat. No. 5,571,894; and U.S. Pat. No. 5,587,458). Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv (see Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a “linear antibody”, e.g., as described in U.S. Pat. No. 5,641,870 for example.

[00189] In one embodiment, an anti-CTHRCl antibody derived scFv is used in a CAR modified immune cell, such as a CAR-T or CAR-NK cell, or CAR-macrophage. Included among anti- CTHRCl antibody fragments are portions of anti-CTHRCl antibodies (and combinations of portions of anti-CTHRCl antibodies, for example, scFv) that may be used as targeting arms, directed to CTHRC1 tumor epitope, in chimeric antigenic receptors of CAR-T or CAR-NK cells, or CAR-macrophages. Such fragments are not necessarily proeteolytic fragments but rather portions of polypeptide sequences that can confer affinity for target.

5. Bispecific Antibodies

[00190] Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies may bind to two different epitopes of an CTHRC1 protein as described herein. Other such antibodies may combine a CTHRC1 binding site with a binding site for another protein. Alternatively, an anti-CTHRCl arm may be combined with an arm which binds to a triggering molecule on a leukocyte such as a T-cell receptor molecule (e.g., CD3), or Fc receptors for IgG (FcyR), such as FcyRI (CD64), FcyRII (CD32) and FcyRIII (CD16), so as to focus and localize cellular defense mechanisms to the CTHRC1- expressing cell. Bispecific antibodies may also be used to localize cytotoxic agents to cells which express CTHRC1. These antibodies possess a CTHRC1 -binding arm and an arm which binds the cytotoxic agent (e.g., saporin, anti-interferon-a, 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).

[00191] WO 96/16673 describes a bispecific anti-ErbB2/anti-FcyRIII antibody and U.S. Patent No. 5,837,234 discloses a bispecific anti-ErbB2/anti-FcyRI antibody. A bispecific anti- ErbB2/Fca antibody is shown in WO98/02463. U.S. Patent No. 5,821,337 teaches a bispecific anti-ErbB2/anti-CD3 antibody.

[00192] Methods for making bispecific antibodies are known in the art. Traditional production of full length bispecific antibodies is based on the co-expression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al., Nature 305: 537-9 (1983)). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually done by affinity chromatography steps, is rather cumbersome, and the product yields are low. Similar procedures are disclosed in WO 93/08829, and in Traunecker et al., EMBO J. 10:3655-3659 (1991).

6, Antibody Variants and Modifications a) Substitution, Insertion, and Deletion variants

[00193] In addition to the anti-CTHRCl antibodies described herein, it is contemplated that anti-CTHRCl antibody variants can be prepared. Anti-CTHRCl antibody variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired antibody or polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-translational processes of the anti-CTHRCl antibody, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics.

[00194] Variations in the anti-CTHRCl antibodies described herein, can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a substitution, deletion or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native sequence antibody or polypeptide. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the anti-CTHRCl antibody. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the anti-CTHRCl antibody with that of homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed 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.

[00195] Anti-CTHRCl antibody fragments are provided herein. Such fragments may be truncated at the N-terminus or C-terminus, or may lack internal residues, for example, when compared with a full-length native antibody or protein. Certain fragments lack amino acid residues that are not essential for a desired biological activity of the anti-CTHRCl antibody.

[00196] Anti-CTHRCl antibody fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative approach involves generating antibody or polypeptide fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired antibody or polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, anti-CTHRC 1 antibody fragments share at least one biological and/or immunological activity with the native anti-CTHRCl antibody disclosed herein.

[00197] In particular embodiments, conservative substitutions of interest are shown in Table 1 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 1, or as further described below in reference to amino acid classes, are introduced and the products screened.

Table 1

Original Exemplary Preferred

Residue Substitutions Substitutions

Ala (A) val; leu; ile val

Arg (R) lys; gin; asn lys

Asn (N) gin; his; lys; arg gin

Asp (D) glu glu

Cys (C) ser ser

Gin (Q) asn asn

Glu (E) asp asp

Gly (G) pro; ala ala

His (H) asn; gin; lys; arg arg lie (I) leu; val; met; ala; phe; norleucine leu

Leu (L) norleucine; ile; val; met; ala; phe ile

Lys (K) arg; gin; asn arg

Met (M) leu; phe; ile leu

Phe (F) leu; val; ile; ala; tyr leu

Pro (P) ala ala

Ser (S) thr thr

Thr (T) ser ser

Trp (W) tyr; phe tyr

Tyr (Y) trp; phe; thr; ser phe

Val (V) ile; leu; met; phe; ala; norleucine leu [00198] Substantial modifications in function or immunological identity of the anti-CTHRCl antibody are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

(1) hydrophobic: norleucine, met, ala, val, leu, ile;

(2) neutral hydrophilic: cys, ser, thr;

(3) acidic: asp, glu; (4) basic: asn, gin, his, lys, arg;

(5) residues that influence chain orientation: gly, pro; and

(6) aromatic: trp, tyr, phe.

[00199] Non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

[00200] The variations 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 selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317: 415 (1986)) or other known techniques can be performed on the cloned DNA to produce the anti-CTHRCl antibody variant DNA.

[00201] Scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely 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. Further, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (W.H. Freeman & Co., N. Y.); Chothia, J. Mol. Biol., 150: 1 (1976)). If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.

[00202] Any cysteine residue not involved in maintaining the proper conformation of the anti- CTHRCl antibody also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to the anti-CTHRCl antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[00203] 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 way 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 substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene III product of Ml 3 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigenantibody complex to identify contact points between the antibody and CTHRC1 polypeptide. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

[00204] Nucleic acid molecules encoding amino acid sequence variants of the anti-CTHRCl antibody are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (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 an earlier prepared variant or a non-variant version of the anti-CTHRCl antibody. b) Modifications

[00205] Covalent modifications of anti-CTHRCl antibodies are included within the scope of this invention. One type of covalent modification includes reacting targeted amino acid residues of an anti-CTHRCl antibody with an organic derivatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues of the anti-CTHRCl antibody. Derivatization with bifunctional agents is useful, for instance, for crosslinking anti-CTHRCl antibody to a water-insoluble support matrix or surface for use in the method for purifying anti- CTHRCl antibodies, and vice-versa. Commonly used crosslinking agents include, e.g., 1,1- bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N- maleimido-l,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[00206] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the a-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, 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.

[00207] Another type of covalent modification of the anti-CTHRCl antibody included within the scope of this invention comprises altering the native glycosylation pattern of the antibody or polypeptide. “Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moi eties found in native sequence anti-CTHRCl antibody (either by removing the underlying glycosylation site or by deleting the glycosylation by chemical and/or enzymatic means), and/or adding one or more glycosylation sites that are not present in the native sequence anti-CTHRCl antibody. In addition, the phrase includes qualitative changes in the glycosylation of the native proteins, involving a change in the nature and proportions of the various carbohydrate moieties present.

[00208] 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-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[00209] Addition of glycosylation sites to the anti-CTHRCl antibody is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the abovedescribed tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original anti-CTHRCl antibody (for O-linked glycosylation sites). The anti- CTHRC1 antibody amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the anti-CTHRCl antibody at preselected bases such that codons are generated that will translate into the desired amino acids.

[00210] Another means of increasing the number of carbohydrate moi eties on the anti-CTHRC 1 antibody is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[00211] Removal of carbohydrate moi eties present on the anti-CTHRCl antibody may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem., 118: 131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exoglycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987). c) Fc Region Variants

[00212] It may be desirable to modify the antibody of the invention with respect to effector function, e.g., so as to enhance antigen-dependent cell-mediated cyotoxicity (ADCC) and/or complement dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions in an 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, B. J. Immunol. 148: 2918-22 (1992). Homodimeric antibodies with enhanced anti-tumor activity may also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53: 2560-5 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-30 (1989). To increase the serum half life of the antibody, one may incorporate a salvage receptor binding epitope into the antibody (especially an antibody fragment) as described in U.S. Patent 5,739,277, for example. As used herein, the term “salvage receptor binding epitope” refers to an epitope of the Fc region of an IgG molecule (e.g., IgGl, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule. d) Cysteine Engineered Antibody Variants

[00213] 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 particular 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 and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate, as described further herein. Cysteine engineered antibodies can be generated as described, e.g, in U.S. Patent No. 7,521,541. e) Immunoconjugates

[00214] The presently disclosed subject matter also provides immunoconjugates, which include an antibody, disclosed herein, conjugated to one or more cytotoxic agents, such as chemotherapeutic agents or drugs, growth inhibitory agents, proteins, peptides, toxins ( e.g ., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioactive isotopes. For example, an antibody of the disclosed subject matter can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other binding molecules, such as another antibody, antibody fragment, peptide or binding mimetic.

[00215] In certain embodiments, an immunoconjugate is an antibody-drug conjugate (ADC) in which an antibody of the present disclosure is conjugated to one or more drugs, including but not limited to, a maytansinoid (see U.S. Patent Nos. 5,208,020, 5,416,064 and European Patent EP 0425235 Bl); an auristatin such as monomethyl auri statin drug moieties DE and DF (MMAE and MMAF) (see U.S. Patent Nos. 5,635,483 and 5,780,588, and 7,498,298); a dolastatin; a calicheamicin or derivative 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)); an anthracycline such as daunomycin or doxorubicin (see Kratz et al., Current Med. Chem. 13 :477-523 (2006); Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006); Torgov et al., Bioconj . Chem. 16:717- 721 (2005); Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000); Dubowchik et al., Bioorg. & Med. Chem. Letters 12: 1529-1532 (2002); King et al., J. Med. Chem. 45:4336-4343 (2002); and U.S. Patent No. 6,630,579); methotrexate; vindesine; a taxane such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel; a trichothecene; and CC1065. In certain embodiments, an immunoconjugate includes an antibody as described herein conjugated to an enzymatically active toxin or fragment thereof, including but not limited to diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.

[00216] In certain embodiments, an immunoconjugate includes an antibody, as described herein, conjugated to a radioactive atom to form a radioconjugate. A variety of radioactive isotopes are available for the production of radioconjugates. Non-limiting examples include At²¹¹, Ac²²⁵, 1¹³¹, I ¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu. When a radioconjugate is used for detection, it can include a radioactive atom for scintigraphic studies, for example tc99m or I¹²³, or a spin label for nuclear magnetic resonance (NMR) imaging (also known as magnetic resonance imaging, mri), such as iodine-123, iodine-131, indium-1 1 1, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese or iron.

[00217] Conjugates of an antibody fragment and cytotoxic agent can be made using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2 -pyridyldithio) propionate (SPDP), succinimidyl-4-(N-maleimidom ethyl) cyclohexane- 1 -carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoro-2, 4-dinitrobenzene). For example, a ricin immunotoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon- 14-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX- DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See WO 94/1 1026. The linker can be a“ cleavable linker” facilitating release of a cytotoxic drug in the cell. For example, an acid-labile linker, peptidase-sensitive linker, photolabile linker, dimethyl linker or 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 immunuoconjugates 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., U.S.A). f) Antibody fusions

[00218] The presently disclosed subject matter also encompasses antibody fusions. For example, proteins can be linked together either through chemical or genetic manipulation using methods known in the art. See, for example, Gillies et al., Proc. Nat’l Acad. Sci. USA 89: 1428- 1432 (1992) and US Patent No. 5,650,150.

[00219] In one example, the present disclosure encompasses an anti-CTHRCl antibodycytokine fusion protein. In principle, an anti-CTHRCl antibody as herein disclosed can be fused to any cytokine via the use of recombinant molecular biological techniques. In a preferred embodiment, the anti-CTHRCl antibody is fused to IL-2 (Gillies, S., Protein Engineering, Design and Selection 26(10): 561-569 (2013); Klein, C. et al., Oncolmmunology 6:3 (2017).

[00220] In another example, the present disclosure encompasses an anti-CTHRCl antibody-T- cell engager fusion protein. Discussed herein, anti-CTHRCl antibody-T-cell engager fusion proteins comprise fusions between an anti-CTHRCl 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 embodiments, the ligand is fused to an Fc portion of an anti-CTHRCl antibody. In embodiments, the ligand is fused to a C-terminus of a light chain of an anti-CTHRCl antibody. Such an approach is described with regard to 4-1BBL (Dafne M. et al., Journal of Immunotherapy 38(8): 714-722 (2008), and a similar approach can be used for generation of other antibody-T-cell engager fusion proteins.

B. Certain Methods of Making Antibodies

1. Screening for Anti-CTHRCl Antibodies with the Desired Properties [00221] Techniques for generating antibodies that bind to CTHRC1 polypeptides have been described above. One may further select antibodies with certain biological characteristics, as desired.

[00222] The growth inhibitory effects of an anti-CTHRCl antibody of the invention may be assessed by methods known in the art, e.g., using cells which express a CTHRC1 polypeptide either endogenously or following transfection with the CTHRC1 gene. For example, appropriate tumor cell lines and CTHRC1 -transfected cells may be treated with an anti- CTHRC1 monoclonal antibody of the invention at various concentrations for a few days (e.g., 2-7) days and stained with crystal violet or MTT or analyzed by some other colorimetric assay. Another method of measuring proliferation would be by comparing 3H-thymidine uptake by the cells treated in the presence or absence an anti-CTHRCl antibody of the invention. After treatment, the cells are harvested and the amount of radioactivity incorporated into the DNA quantitated in a scintillation counter. Appropriate positive controls include treatment of a selected cell line with a growth inhibitory antibody known to inhibit growth of that cell line. Growth inhibition of tumor cells in vivo can be determined in various ways known in the art. The tumor cell may be one that overexpresses and/or displays a CTHRC1 polypeptide. The anti-CTHRCl antibody will inhibit cell proliferation of a CTHRC1 -expressing tumor cell in vitro or in vivo by about 25-100% compared to the untreated tumor cell, more preferably, by about 30-100%, and even more preferably by about 50-100% or 70-100%, in one embodiment, at an antibody concentration of about 0.5 to 30 pg/mL. Growth inhibition can be measured at an antibody concentration of about 0.5 to 30 pg/mL or about 0.5 nM to 200 nM in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antibody. The antibody is growth inhibitory in vivo if administration of the anti-CTHRCl antibody at about 1 pg/kg to about 100 mg/kg body weight results in reduction in tumor size or reduction of tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days. [00223] To select for an anti-CTHRCl antibody which induces cell death, loss of membrane integrity as indicated by, e.g., propidium iodide (PI), trypan blue or 7AAD uptake may be assessed relative to control. A PI uptake assay can be performed in the absence of complement and immune effector cells. CTHRC1 polypepti de-expressing tumor cells are incubated with medium alone or medium containing the appropriate anti-CTHRCl antibody (e.g, at about 10 pg/mL). The cells are incubated for a 3 day time period. Following each treatment, cells are washed and aliquoted into 35 mm strainer-capped 12 x 75 tubes (1 mL per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive PI (10 pg/mL). Samples may be analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). Those anti-CTHRCl antibodies that induce statistically significant levels of cell death as determined by PI uptake may be selected as cell death-inducing anti-CTHRCl antibodies.

[00224] To screen for antibodies which bind to an epitope on a CTHRC1 polypeptide bound by an antibody of interest, a routine cross-blocking assay such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine if a test antibody binds the same site or epitope as a known anti-CTHRCl antibody. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, the antibody sequence can be mutagenized such as by alanine scanning, to identify contact residues. The mutant antibody is initially tested for binding with polyclonal antibody to ensure proper folding. In a different method, peptides corresponding to different regions of a CTHRC1 polypeptide can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.

[00225] In addition, candidate antibodies may also be screened for function using one or more of the following: in vivo screening for inhibition of metastasis, inhibition of chemotaxis by an in vitro method (e.g., U.S. 2010/0061978, incorporated herein by reference in its entirety), inhibition of vascularization, inhibition of tumor growth, and decrease in tumor size.

2. Certain Library Screening Methods

[00226] Anti-CTHRCl antibodies of the invention can be made by using combinatorial libraries to screen for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are described generally 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.

[00227] In principle, synthetic antibody clones are selected by screening phage libraries containing phage that display various fragments of antibody variable region (Fv) fused to phage coat protein. 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 the non-binding clones in the library. The binding clones are then eluted from the antigen, and can be further enriched by additional cycles of antigen adsorption/elution. Any of the anti-CTHRCl antibodies of the invention can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest followed by construction of a full length anti-CTHRCl antibody clone using the Fv sequences from the phage clone of interest and suitable constant region (Fc) sequences described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vol s. 1-3.

[00228] In certain embodiments, the antigen-binding domain of an antibody is formed from two variable (V) regions of about 110 amino acids, one each from the light (VL) and heavy (VH) chains, that both present three hypervariable loops (HVRs) or complementarity-determining regions (CDRs). Variable domains can be displayed functionally on phage, either as single-chain Fv (scFv) fragments, in which VH and VL are covalently linked through a short, flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non- covalently, as described in Winter et al., Ann. Rev. Immunol., 12: 433-55 (1994). As used herein, scFv encoding phage clones and Fab encoding phage clones are collectively referred to as “Fv phage clones” or “Fv clones.”

[00229] Repertoires of VH and VL genes can be separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be searched for antigenbinding clones as described in Winter et al., Ann. Rev. Immunol., 12: 433-55 (1994). Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned to provide a single source of human antibodies to a wide range of non-self 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 made synthetically by cloning the unrearranged V-gene segments from stem cells and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-8 (1992).

[00230] In certain embodiments, filamentous phage is used to display antibody fragments by fusion to the minor coat protein pill. The antibody fragments can be displayed as single chain Fv fragments, in which VH and VL domains are connected on the same polypeptide chain by a flexible polypeptide spacer, e.g., as described 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 where assembly of a Fab-coat protein structure which becomes displayed on the phage surface by displacing some of the wild type coat proteins, e.g., as described in Hoogenboom et al., Nucl. Acids Res., 19: 4133-7 (1991).

[00231] In general, nucleic acids encoding antibody gene fragments are obtained from immune cells harvested from humans or animals. If a library biased in favor of anti-CTHRCl clones is desired, the subject is immunized with CTHRC1 to generate an antibody response, and spleen cells and/or circulating B cells other peripheral blood lymphocytes (PBLs) are recovered for library construction. In some embodiments, a human antibody gene fragment library biased in favor of anti-CTHRCl clones is obtained by generating an anti-CTHRCl antibody response in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) such that CTHRC1 immunization gives rise to B cells producing human antibodies against CTHRC1. The generation of human antibodyproducing transgenic mice is described below.

[00232] Additional enrichment for anti-CTHRCl reactive cell populations can be obtained by using a suitable screening procedure to isolate B cells expressing CTHRC1 -specific membrane bound antibody, e.g., by cell separation using CTHRC1 affinity chromatography or adsorption of cells to fluorochrome-labeled CTHRC1 followed by flow-activated cell sorting (FACS). [00233] Alternatively, the use of spleen cells and/or B cells or other PBLs from an unimmunized donor provides a better representation of the possible antibody repertoire, and also permits the construction of an antibody library using any animal (human or non-human) species in 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. The immune cells of interest can be obtained from a variety of animal species, such as human, mouse, rat, lagomorpha, luprine, canine, feline, porcine, bovine, equine, and avian species, etc.

[00234] Nucleic acid encoding antibody variable gene segments (including VH and VL segments) are recovered from the cells of interest and amplified. In the case of rearranged VH and VL gene libraries, the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes followed by polymerase chain reaction (PCR) with primers matching the 5' and 3' ends of rearranged VH and VL genes as described in Orlandi et al., Proc. Natl. Acad. Set. (USA), 86: 3833-7 (1989), thereby making diverse V gene repertoires for expression.

[00235] The V genes can be amplified from cDNA and genomic DNA, with back primers at the 5' end of the exon encoding the mature V-domain and forward primers based within the J- segment as described in Orlandi et al. (1989) and in Ward et al., Nature, 341 : 544-6 (1989). However, for amplifying from cDNA, back primers can also be based in the leader exon as described in Jones et al., Biotechnol ., 9: 88-9 (1991), and forward primers within the constant region as described in Sastry et al., Proc. Natl. Acad. Set. (USA), 86: 5728-32 (1989). To maximize complementarity, degeneracy can be incorporated in the primers as described in Orlandi et al. (1989) or Sastry et al. (1989). In certain embodiments, library diversity is maximized by using PCR primers targeted to each V-gene family in order to amplify all available VH and VL arrangements present in the immune cell nucleic acid sample, e.g., as described in the method of Marks et al., J. Mol. Biol., 222: 581-97 (1991) or as described in the method of Orum et al., Nucleic Acids Res., 21 : 4491-98 (1993). For cloning of the amplified DNA into expression vectors, rare restriction sites can be introduced within the PCR primer as a tag at one end as described in Orlandi et al. (1989), or by further PCR amplification with a tagged primer as described in Clackson et al., Nature, 352: 624-628 (1991). [00236] Repertoires of synthetically rearranged V genes can be derived in vitro from V gene segments. Most of the human VH-gene segments have been cloned and sequenced (reported in Tomlinson et al., J. Mol. Biol., 227: 776-98 (1992)), and mapped (reported in Matsuda et al., Nature Genet., 3: 88-94 (1993); these cloned segments (including all the major conformations of the Hl and H2 loop) can be used to generate diverse VH gene repertoires with PCR primers encoding H3 loops of diverse sequence and length as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). VH repertoires can also be made with all the sequence diversity focused in a long H3 loop of a single length as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89: 4457-61 (1992). Human VK and VX segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23: 1456-61 (1993)) and can be used to make synthetic light chain repertoires. Synthetic V gene repertoires, based on a range of VH and VL folds, and L3 and H3 lengths, will encode antibodies of considerable structural diversity. Following amplification of V-gene encoding DNAs, germline V-gene segments can be rearranged in vitro according to the methods of Hoogenboom and Winter, J. Mol. Biol., 227: 381-8 (1992).

[00237] Repertoires of antibody fragments can be constructed by combining VH and VL gene repertoires together in several ways. Each repertoire can be created in different vectors, and the vectors recombined in vitro, e.g., as described in Hogrefe et al., Gene, 128: 119-26 (1993), or in vivo by combinatorial infection, e.g., the loxP system described in Waterhouse et al., Nucl. Acids Res., 21 : 2265-66 (1993). The in vivo recombination approach exploits the two-chain nature of Fab fragments to overcome the limit on library size imposed by E. coli transformation efficiency. Naive VH and VL repertoires are cloned separately, one into a phagemid and the other into a phage vector. The two libraries are then combined by phage infection of phagemid-containing bacteria so that each cell contains a different combination and the library size is limited only by the number of cells present (about 1012 clones). Both vectors contain in vivo recombination signals so that the VH and VL genes are recombined onto a single replicon and are co-packaged into phage virions. These huge libraries provide large numbers of diverse antibodies of good affinity (Kd-1 of about 10-8 M).

[00238] Alternatively, the repertoires may be cloned sequentially into the same vector, e.g., as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88: 7978-7982 (1991), or assembled together by PCR and then cloned, e.g., as described in Clackson et al., Nature, 352: 624-628 (1991). PCR assembly can also be used to join VH and VL DNAs with DNA encoding a flexible peptide spacer to form single chain Fv (scFv) repertoires. In yet another technique, “in cell PCR assembly” is used to combine VH and VL genes within lymphocytes by PCR and then clone repertoires of linked genes as described in Embleton et al., Nucl. Acids Res., 20: 3831-3837 (1992).

[00239] The antibodies produced by naive libraries (either natural or synthetic) can be of moderate affinity (Kd-1 of about 10⁶ to 10⁷ M-l), but affinity maturation can also be mimicked in vitro by constructing and reselecting from secondary libraries as described in Winter et al. (1994), supra. For example, mutation can be introduced at random in vitro by using error-prone polymerase (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, e.g., using PCR with primers carrying random sequence spanning the CDR of interest, in selected individual Fv clones and screening for higher affinity clones. WO 9607754 described a method for inducing mutagenesis in a complementarity determining region of an immunoglobulin light chain to create a library of light chain genes. Another effective approach is to recombine the VH or VL domains selected by phage display with repertoires of naturally occurring V domain variants obtained from unimmunized donors and screen for higher affinity in several rounds of chain reshuffling as described in Marks et al., Biotechnol., 10: 779-83 (1992). This technique allows the production of antibodies and antibody fragments with affinities of about 10-9 M or less.

[00240] Screening of the libraries can be accomplished by various techniques known in the art. For example, CTHRC1 can be used to coat the wells of adsorption plates, expressed on host cells affixed to adsorption plates or used in cell sorting, or conjugated to biotin for capture with streptavidin-coated beads, or used in any other method for panning phage display libraries.

[00241] The phage library samples are contacted with immobilized CTHRC1 under conditions suitable for binding at least a portion of the phage particles with the adsorbent. Normally, the conditions, including pH, ionic strength, temperature and the like are selected to mimic physiological conditions. The phages bound to the solid phase are washed and then eluted by acid, e.g., as described in Barbas et al., Proc. Natl. Acad. Sci USA, 88: 7978-82 (1991), or by alkali, e.g., as described in Marks et al., J. Mol. Biol., 222: 581-97 (1991), or by CTHRC1 antigen competition, e.g., 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 round of selection. Moreover, the enriched phages can be grown in bacterial culture and subjected to further rounds of selection.

[00242] The efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and whether multiple antibody fragments on a single phage can simultaneously engage with antigen. Antibodies with fast dissociation kinetics (and weak binding affinities) can be retained by use of short washes, multivalent phage display and high coating density of antigen in solid phase. The high density not only stabilizes the phage through multivalent interactions but favors rebinding of phage that has dissociated. The selection of antibodies with slow dissociation kinetics (and good binding affinities) can be promoted by use of long washes and monovalent phage display as described in Bass et al., Proteins, 8: 309-314 (1990) and in WO 92/09690, and a low coating density of antigen as described in Marks et al., Biotechnol., 10: 779-783 (1992).

[00243] It is possible to select between phage antibodies of different affinities, even with affinities that differ slightly, for CTHRC1. However, random mutation of a selected antibody (e.g., as performed in some affinity maturation techniques) is likely to give rise to many mutants, most binding to antigen, and a few with higher affinity. With limiting CTHRC 1 , rare high affinity phage could be competed out. To retain all higher affinity mutants, phages can be incubated with excess biotinylated CTHRC 1, but with the biotinylated CTHRC 1 at a concentration of lower molarity than the target molar affinity constant for CTHRC 1. The high affinity -binding phages can then be captured by streptavidin-coated paramagnetic beads. Such “equilibrium capture” allows the antibodies to be selected according to their affinities of binding, with sensitivity that permits isolation of mutant clones with as little as two-fold higher affinity from a great excess of phages with lower affinity. Conditions used in washing phages bound to a solid phase can also be manipulated to discriminate on the basis of dissociation kinetics.

[00244] Anti-CTHRCl clones may be selected based on activity. In certain embodiments, the invention provides anti -CTHRC 1 antibodies that bind to living cells that naturally express CTHRC 1. In one embodiment, the invention provides anti-CTHRCl 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-CTHRCl antibodies can be selected by (1) isolating anti-CTHRCl clones from a phage library as described above, and optionally amplifying the isolated population of phage clones by growing up the population in a suitable bacterial host; (2) selecting CTHRC1 and a second protein against which blocking and non-blocking activity, respectively, is desired; (3) adsorbing the anti-CTHRCl phage clones to immobilized CTHRC1; (4) using an excess of the second protein to elute any undesired clones that recognize CTHRC1 -binding determinants which overlap or are shared with the binding determinants of the second protein; and (5) eluting the clones which remain adsorbed following step (4). Optionally, clones with the desired blocking/non-blocking properties can be further enriched by repeating the selection procedures described herein one or more times.

[00245] DNA encoding hybridoma-derived monoclonal antibodies or phage display Fv clones of the invention is 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 hybridoma or phage DNA template). Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of the desired monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of antibodyencoding DNA include Skerra et al., Curr. Opinion in Immunol. 5: 256 (1993) and Pluckthun, Immunol. Rev. 130: 151 (1992).

[00246] DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding heavy chain and/or light chain constant regions (e.g., the appropriate DNA sequences can be obtained from Kabat et al., supra) to form clones encoding full or partial length heavy and/or light chains. It will be appreciated that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and that such constant regions can be obtained from any human or animal species. An Fv clone derived from the variable domain DNA of one animal (such as human) species and then fused to constant region DNA of another animal species to form coding sequence(s) for “hybrid,” full length heavy chain and/or light chain is included in the definition of “chimeric” and “hybrid” antibody as used herein. In certain embodiments, an Fv clone derived from human variable DNA is fused to human constant region DNA to form coding sequence(s) for full- or partial-length human heavy and/or light chains.

[00247] DNA encoding anti-CTHRCl antibody derived from a hybridoma can also be modified, for example, by substituting the coding sequence for human heavy- and light-chain constant domains in place of homologous murine sequences derived from the hybridoma clone (e.g., as in the method of 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 joining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide. In this manner, “chimeric” or “hybrid” antibodies are prepared that have the binding specificity of the Fv clone or hybridoma clone-derived antibodies of the invention.

3. Generation of antibodies using CAR T-cells

[00248] Anti-CTHRCl antibodies of the invention can be made by using CAR T-cell platforms to screen for antibodies with the desired activity or activities. Chimeric antigen receptors (CARs) are composed of an extracellular antigen recognition domain (usually a single-chain variable fragment (scFv) antibody) attached to transmembrane and cytoplasmic signaling domains. 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 of effector functions, addressing the goal of major histocompatibility complex-independent activation of effector cells. First-generation CARs were constructed through the fusion of a scFv-based TAA- binding domain to a cytoplasmic signaling domain typically derived either from the C, chain of the T cell receptor (TCR)/CD3 complex or from the y chain associated with some Fc receptors (Gross, G. et al., Proc Natl Acad Sci USA 86: 10024-8 (1989)). Second-generation CARs (CARv2) comprising the signaling region of the TCR C, in series with the signaling domain derived from the T-cell co-stimulatory receptors CD28, 4-1BB (CD137) or 0X40 (CD134) have also been developed (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).

[00249] Upon encountering antigen, the interaction of a genetically transferred CAR triggers effector functions and can mediate cytolysis of tumor cells. The utility and effectiveness of the CAR approach have been demonstrated in a variety of animal models, and ongoing clinical trials using CAR-based genetically engineered T lymphocytes for the treatment of cancer patients. Lipowska-Bhalla, G. et al., Cancer Immunol Immunother 61 : 953-62 (2012). CARs enable targeting of effector cells toward any native extracellular antigen for which a suitable antibody exists. Engineered cells can be targeted not only to proteins but also to structures such as carbohydrate and glycolipid tumor antigens (Mezzanzanica, D. et al., Cancer Gene Ther 5: 401- 7 (1998); Kershaw, MH. et al., Nat Rev Immunol 5: 928-40 (2005)).

[00250] Current methods for the generation of recombinant antibodies are mainly based on the use of purified proteins. Hoogenboom, H.R. et al., Nat Biotechnol 23: 1105-1116 (2005). However, a mammalian cell-based antibody display platform has recently been described, which takes advantage of the functional capabilities of T lymphocytes. Alonso-Camino et al, Molecular Therapy Nucleic Acids (2013) 2, e93. The display of antibodies on the surface of T lymphocytes, as a part of a CAR-mediating signaling, may ideally link the antigen-antibody interaction to a demonstrable change in cell phenotype, due to the surface expression of activation markers. Alonso-Camino, V. et al., PLoS ONE 4: e7174 (2009). By using a scFv-based CAR that recognizes a TAA, it has been demonstrated that combining CAR-mediated activation with fluorescence-activated cell sorting (FACS) of CD69+ T cells makes it possible to isolate binders to surface TAA, with an enrichment factor of at least 10³-fold after two rounds, resulting in a homogeneous population of T cells expressing TAA-specific CAR. Alonso-Camino, V, et al., PLoS ONE -. e7174 (2009).

C. Preparation of Anti-CTHRCl Antibodies

[00251] The description below relates primarily to production of anti-CTHRCl antibodies by culturing cells transformed or transfected with a vector containing anti-CTHRCl antibodyencoding nucleic acid. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare anti-CTHRCl antibodies. For instance, the appropriate amino acid sequence, or portions 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 by automation. Automated synthesis may be accomplished, for instance, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using 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-CTHRCl antibody.

1. Isolation of DNA Encoding Anti-CTHRCl Antibody

[00252] DNA encoding anti-CTHRCl antibody may be obtained from a cDNA library prepared from tissue believed to possess the anti-CTHRCl antibody mRNA and to express it at a detectable level. Accordingly, human anti-CTHRCl antibody DNA can be conveniently obtained from a cDNA library prepared from human tissue. The anti-CTHRCl antibodyencoding gene may also be obtained from a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[00253] Libraries can be screened with probes (such as oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cDNA or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means to isolate the gene encoding anti- CTHRCl antibody is to use PCR methodology (Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[00254] Techniques for screening a cDNA library are well known in the art. The oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. The oligonucleotide is preferably labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32P-labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.

[00255] Sequences identified in such library screening methods can be compared and aligned to 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 the molecule or across the full-length sequence can be determined using methods known in the art and as described herein.

[00256] Nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al., supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

[00257] Host cells are transfected or transformed with expression or cloning vectors described herein for anti-CTHRCl antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

[00258] Methods of eukaryotic cell transfection and prokaryotic cell transformation, which means introduction of DNA into the host so that the DNA is replicable, either as an extrachromosomal or by chromosomal integrant, are known to the ordinarily skilled artisan, for example, CaCh, CaPC , liposome-mediated, polyethylene-gycol/DMSO and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al., Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in U.S. Patent No. 4,399,216. Transformations into yeast are typically carried out according to the method 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, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, may also be used. 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). [00259] Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. a. Prokaryotic Host Cells

[00260] Suitable prokaryotes include but are not limited to archaebacteria and eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as K12 strain 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 lyphimiiriiim, 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 12 April 1989), Pseudomonas such as P. aeruginosa, Rhizobia, Vitreoscilla, Paracoccus and Streptomyces. These examples are illustrative rather than limiting. E. coli strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, D.C.: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Deposit No. 27,325) may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. co/z'W3110 strain 1A2, which has the complete genotype tonA ; E. co/z'W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3 phoA El 5 (argF-lac)169 degP ompT kanr; E. coli W3110 strain 37D6, which has the complete genotype tonA ptr3 pho A E 15 (argF -lac) 169 degP ompT rbs7 ilvG kanr; E. coli W3110 strain 40B4, which is strain 37D6 with a non- kanamycin resistant degP deletion mutation; E. coli W3110 strain 33D3 having genotype W3110 AfhuA (AtonA) ptr3 lac Iq lacL8 AompTA(nmpc-fepE) degP41 kanR (U.S. Pat. No. 5,639,635) and an E. coli strain having mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990. Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli 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 having defined genotypes are known in the art and described in, for example, Bass et al., Proteins, 8:309-314 (1990). It is generally necessary to select the appropriate bacteria taking into consideration replicability of the replicon in the cells of a bacterium. For example, E. coli, Serratia, or Salmonella species can be suitably used as the host 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 desirably be incorporated in the cell culture. Alternatively, in vitro methods of cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.

[00261] Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. Full length antibodies have greater half life in circulation. Production in E. coll is faster and more cost efficient. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. 5,648,237; U.S. 5,789,199 and U.S. 5,840,523, which describe translation initiation region (TIR) and signal sequences for optimizing expression and secretion, these patents incorporated herein by reference. After expression, the antibody is isolated from the E. coll cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed e.g., in CHO cells. b. Eukaryotic Host Cells

[00262] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for anti-CTHRCl antibody-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290: 140 (1981); EP 139,383 published 2 May 1985); Kluyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al., Bio/Technology, 9: 968-75 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacterio!., 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., Bio/Technology, 8: 135 (1990)), K. thermotolerans, and K. marxianus,' yarrowia (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol., 28:265-278 (1988)); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 (1979)); Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulcms (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)). Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torul opsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982).

[00263] Suitable host cells for the expression of glycosylated anti-CTHRCl antibody are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells, such as cell cultures of cotton, com, potato, soybean, petunia, tomato, and tobacco. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-l variant of Autographa califomica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present invention, particularly for transfection of Spodoptera frugiperda cells.

[00264] However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BEK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).

[00265] Host cells are transformed with the above-described expression or cloning vectors for anti-CTHRCl antibody production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

3. Selection and Use of a Replicable Vector

[00266] 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 that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends in part on the host cell to be used. Generally, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin.

[00267] The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety of procedures. In general, 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 which are known to the skilled artisan.

[00268] The anti-CTHRCl antibody may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. In general, the signal sequence may be a component of the vector, or it may be a part of the anti-CTHRCl 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 the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders. For yeast secretion the signal sequence may be, e.g., the yeast invertase leader, alpha factor leader (including Saccharomyces and Kluyveromyces a-factor leaders, the latter described in U.S. Patent No. 5,010,182), or acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published 4 April 1990), or the signal described in WO 90/13646 published 15 November 1990. In mammalian cell expression, mammalian signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders. a. Prokaryotic Host Cells

[00269] Polynucleotide sequences encoding polypeptide components of the antibody of the invention can be obtained using standard recombinant techniques. Desired polynucleotide sequences may be isolated and sequenced from antibody producing cells such as hybridoma cells. Alternatively, polynucleotides can be synthesized using nucleotide synthesizer or PCR techniques. Once obtained, sequences encoding the polypeptides are inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in prokaryotic hosts. Many vectors that are available and known in the art can be used for the purpose of the present invention. Selection of an appropriate vector will depend mainly on the size of the nucleic acids 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 heterologous polynucleotide, or both) and its compatibility with the particular host cell in which it resides.

[00270] In general, plasmid vectors containing replicon and control sequences which are derived from species compatible with the host cell are used in connection with these hosts. Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells, as well as marking sequences which are capable of providing 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 easy means for identifying transformed cells, is suitable for most Gram-negative bacteria, the 2p 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 bacteriophage may also contain, or be modified to contain, promoters which can be used by the microbial organism for expression of endogenous proteins. Examples of pBR322 derivatives used for expression of particular antibodies are described in detail in Carter et al., U.S. Patent No. 5,648,237.

[00271] In addition, phage vectors containing replicon and control sequences that are compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as GEM™-11 may be utilized in making a recombinant vector which can be used to transform susceptible host cells such as E. coli LE392.

[00272] The expression vector of the invention may comprise two or more promoter-ci stron pairs, encoding each of the polypeptide components. A promoter is an untranslated regulatory sequence located upstream (5') to a cistron that modulates its expression. Prokaryotic promoters typically fall into two classes, inducible and constitutive. Inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to changes in the culture condition, e.g.,the presence or absence of a nutrient or a change in temperature.

[00273] A large number of promoters recognized by a variety of potential host cells are well known. The selected promoter can be operably linked to 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 invention. Both the native promoter sequence and many heterologous promoters may be used to direct amplification and/or expression of the target genes. In some embodiments, heterologous promoters are utilized, as they generally permit greater transcription and higher yields of expressed target gene as compared to the native target polypeptide promoter.

[00274] Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the PhoA promoter, the P-galactamase and lactose promoter systems (Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281 :544 (1979)), alkaline phosphatase, a tryptophan (trp) promoter system (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 the trc promoter. Promoters for use in bacterial systems also will contain a Shine-Dalgamo (S.D.) sequence operably linked to the DNA encoding anti-CTHRCl antibody. However, other promoters that are functional in bacteria (such as other known bacterial or phage promoters) are suitable as well. Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to cistrons encoding the target light and heavy chains (Siebenlist et al. (1980) Cell 20: 269) using linkers or adaptors to supply any required restriction sites.

[00275] In one aspect of the invention, each cistron within the recombinant vector comprises a secretion signal sequence component that directs translocation of the expressed polypeptides across a membrane. In general, the signal sequence may be a component of the vector, or it may be a part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purpose of this invention should be one that is recognized and processed (i.e. cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the signal sequences native to the heterologous polypeptides, the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leaders, LamB, PhoE, PelB, OmpA and MBP. In one embodiment of the invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[00276] In another aspect, the production of the immunoglobulins according to the invention can occur in the cytoplasm of the host cell, and therefore does not require the presence of secretion signal sequences within each cistron. In that regard, immunoglobulin light and heavy chains are expressed, folded and assembled to form functional immunoglobulins within the cytoplasm. Certain host strains (e.g., the A. coll trxB- strains) provide cytoplasm conditions that are favorable for disulfide bond formation, thereby permitting proper folding and assembly of expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[00277] The present invention provides an expression system in which the quantitative ratio of expressed polypeptide components can be modulated in order to maximize the yield of secreted and properly assembled antibodies of the invention. Such modulation is accomplished at least in part by simultaneously modulating translational strengths for the polypeptide components.

[00278] One technique for modulating translational strength is disclosed in Simmons et al., U.S. Pat. No. 5,840,523. It utilizes variants of the translational initiation region (TIR) within a cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be created with a range of translational strengths, thereby providing a convenient means by which to adjust this factor for the desired expression level of the specific chain. TIR variants can be generated by conventional mutagenesis techniques that result in codon changes which can alter the amino acid sequence, although silent changes in the nucleotide sequence are preferred. Alterations in the TIR can include, for example, alterations in the number or spacing of Shine-Dalgamo sequences, along with alterations in the signal sequence. One method for generating mutant signal sequences is the generation of a “codon bank” at the beginning of a coding sequence that does not change the amino acid sequence of the signal sequence (i.e., the changes are silent). This can be accomplished by changing the third nucleotide position of each codon; additionally, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions that can add complexity in making the bank. This method of mutagenesis is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4: 151-158.

[00279] Preferably, a set of vectors is generated with a range of TIR strengths for each cistron therein. This limited set provides a comparison of expression levels of each chain as well as the yield of the desired antibody products under various TIR strength combinations. TIR strengths can be determined by quantifying the expression level of a reporter gene as described in detail in Simmons et al. U.S. Pat. No. 5, 840,523. Based on the translational strength comparison, the desired individual TIRs are selected to be combined in the expression vector constructs of the invention. b. Eukaryotic Host Cells

[00280] The 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.

(1) Signal sequence component

[00281] A vector for use in a eukaryotic host cell may also contain a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex gD signal, are available.

[00282] The DNA for such precursor region is ligated in reading frame to DNA encoding the antibody. (2) Origin of replication

[00283] Generally, an 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.

(3) Selection gene component

[00284] Expression and cloning vectors will typically contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

[00285] One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

[00286] An example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the anti-CTHRCl antibody-encoding nucleic acid, such as DHFR or thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc. An appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity (e.g., ATCC CRL-9096), prepared and propagated 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 a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'- phosphotransferase (APH) can be selected by cell growth in medium containing a selection agent for the selectable marker such as an aminoglycosidic antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

[00287] A suitable selection gene for use in yeast is the trpl 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 trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4- 1 (Jones, Genetics, 85: 12 (1977)).

(4) Promoter Component

[00288] 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.

[00289] Virtually alleukaryotic genes have an AT -rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may 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 suitably inserted into eukaryotic expression vectors.

[00290] Examples of suitable promoting sequences for use with yeast hosts include the 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.

[00291] Other yeast promoters, which are inducible promoters having the additional advantage of transcription 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 responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

[00292] Anti-CTHRCl antibody transcription from vectors in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems.

[00293] 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 Hindlll E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982) on expression of human P-interferon cDNA in mouse cells under the control of a thymidine kinase promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(5) Enhancer Element Component

[00294] Transcription of a DNA encoding the anti-CTHRCl antibody by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), 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) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5' or 3' to the anti-CTHRCl antibody coding sequence, but is preferably located at a site 5' from the promoter.

(6) Transcription Termination Component

[00295] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding anti-CTHRCl antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector disclosed therein.

[00296] Still other methods, vectors, and host cells suitable for adaptation to the synthesis of anti-CTHRCl antibody 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.

4. Culturing the Host Cells

[00297] The host cells used to produce the anti-CTHRCl antibody of this invention may be cultured in a variety of media. a. Prokaryotic Host Cells

[00298] Prokaryotic cells used to produce the polypeptides of the invention are grown in media known in the art and suitable for culture of the selected host cells. Examples of suitable media include luria broth (LB) plus necessary nutrient supplements. In some embodiments, the media also contains a selection agent, chosen based on the construction of the expression vector, to selectively permit growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to media for growth of cells expressing ampicillin resistant gene.

[00299] Any necessary supplements besides carbon, nitrogen, and inorganic phosphate sources may also be included at appropriate concentrations introduced alone or as a mixture with another supplement or medium such as a complex nitrogen source. Optionally the culture medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thiogly collate, di thioerythritol and dithiothreitol.

[00300] The prokaryotic host cells are cultured at suitable temperatures. For A. coll growth, for example, the preferred temperature ranges from about 20°C to about 39°C, more preferably from about 25°C to about 37°C, even more preferably at about 30°C. The pH of the medium may be any pH ranging from about 5 to about 9, depending mainly on the host organism. For A. coli, the pH is preferably from about 6.8 to about 7.4, and more preferably about 7.0.

[00301] If an inducible promoter is used in the expression vector of the invention, protein expression is induced under conditions suitable for the activation of the promoter. In one aspect of the invention, PhoA promoters are used for controlling transcription of the polypeptides. Accordingly, the transformed host cells are cultured in a phosphate-limiting medium for induction. In some embodiments, the phosphate-limiting medium is the C.R.A.P medium (see, e.g., Simmons et al., J. Immunol. Methods (2002), 263 : 133-47). A variety of other inducers may be used, according to the vector construct employed, as is known in the art.

[00302] In one embodiment, the expressed polypeptides of the present invention are secreted into and recovered from the periplasm of the host cells. Protein recovery typically involves disrupting the microorganism, generally by such means as osmotic shock, sonication or lysis. Once cells are disrupted, cell debris or whole cells may be removed by centrifugation or filtration. The proteins may be further purified, for example, by affinity resin chromatography. Alternatively, proteins can be transported into the culture media and isolated therein. Cells may be removed from the culture and the culture supernatant being filtered and concentrated for further purification of the proteins produced. The expressed polypeptides can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assay.

[00303] In one aspect of the invention, antibody production is conducted in large quantity by a fermentation process. Various large-scale fed-batch fermentation procedures are available for production of recombinant proteins. Large-scale fermentations have at least 1000 liters of capacity, preferably about 1,000 to 100,000 liters of capacity. These fermentors use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon/energy source). Small scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.

[00304] In a fermentation process, induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in the early stationary phase. A variety of inducers may be used, according to the vector construct employed, as is known in the art and described above. Cells may be grown for shorter periods prior to induction. Cells are usually induced for about 12-50 hours, although longer or shorter induction time may be used. [00305] To improve the production yield and quality of the polypeptides of the invention, various fermentation conditions can be modified. For example, to improve the proper assembly and folding of the secreted antibody polypeptides, additional vectors overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG) or FkpA (a peptidylprolyl cis, trans-isomerase with chaperone activity) can be used to co-transform the host prokaryotic cells. The 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.

[00306] To minimize proteolysis of expressed heterologous proteins (especially those that are proteolytically sensitive), certain host strains deficient for proteolytic enzymes can be used for the present invention. For example, host cell strains may be modified to effect genetic mutation(s) in the genes encoding known bacterial proteases such as Protease III, OmpT, DegP, Tsp, Protease I, Protease Mi, Protease V, Protease VI and combinations thereof. Some E. coli protease-deficient strains are available and described in, for example, Joly et al. (1998), supra; U.S. Patent No. 5,264,365; U.S. Patent No. 5,508,192; Hara et al., Microbial Drug Resistance, 2 :63-72 (1996).

[00307] In one embodiment, E. coli strains deficient for proteolytic enzymes and transformed with plasmids overexpressing one or more chaperone proteins are used as host cells in the expression system of the invention. b. Eukaryotic Host Cells

[00308] Commercially available media such as Ham’s F 10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco’s Modified Eagle Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58: 44 (1979), Barnes et al., Anal. BiochemACl'. 255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Patent Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), 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 that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordinarily skilled artisan.

5. Detecting Gene Amplification/Expression

[00309] Gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA (Thomas, Proc. Natl. Acad. Set. USA, 77: 5201-5 (1980)), dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

[00310] Gene expression, alternatively, may be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of 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. Conveniently, the antibodies may be prepared against a native sequence CTHRC1 polypeptide or against a synthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to CTHRC1 DNA and encoding a specific antibody epitope.

6. Purification of Anti-CTHRCl Antibody

[00311] Forms of anti-CTHRCl antibody may be recovered from 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 employed in expression of anti- CTHRC1 antibody can be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.

[00312] It may be desired to purify anti-CTHRCl antibody from recombinant cell proteins or polypeptides. The following procedures are exemplary of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS- PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Sepharose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the anti-CTHRCl antibody. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymology, 182 (1990); Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular anti-CTHRCl antibody produced.

[00313] When using recombinant techniques, the antibody can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10: 163- 7 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, 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.

[00314] The antibody composition prepared from the 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 that is present in the antibody. Protein A can be used to purify antibodies that are based on human yl, y2 or y4 heavy chains (Lindmark et al., J. Immunol. Meth. 62: 1-13 (1983)). Protein G is recommended for all mouse isotypes and for human y3 (Guss et al., EMBO J. 5: 15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CH3 domain, the Bakerbond ABX™resin (J. T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™ chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[00315] Following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, and generally at low salt concentrations (e.g., from about 0-0.25M salt).

D. Assays

[00316] The antibody 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.).

[00317] A detection label may be useful for localizing, visualizing, and quantitating a binding or recognition event. The labelled antibodies of the invention can detect cell-surface receptors. Another use for detectably labelled antibodies is a method of bead-based immunocapture comprising conjugating a bead with a fluorescent labelled antibody and detecting a fluorescence signal upon binding of a ligand. Similar binding detection methodologies utilize the surface plasmon resonance (SPR) effect to measure and detect antibody-antigen interactions.

[00318] Detection labels such as fluorescent dyes and chemiluminescent dyes (Briggs et al (1997) J. Chem. Soc., Perkin-Trans. 1 : 1051-8) provide a detectable signal and are generally applicable for labelling antibodies, preferably with the following properties: (i) the labelled antibody should produce a very high signal with low background so that small quantities of antibodies can be sensitively detected in both cell-free and cell-based assays; and (ii) the labelled antibody should be photostable so that the fluorescent signal may be observed, monitored and recorded without significant photo bleaching. For applications involving cell surface binding of labelled antibody to membranes or cell surfaces, especially live cells, the labels preferably (iii) have good water-solubility to achieve effective conjugate concentration and detection sensitivity and (iv) are non-toxic to living cells so as not to disrupt the normal metabolic processes of the cells or cause premature cell death.

[00319] Direct quantification of cellular fluorescence intensity and enumeration of fluorescently labelled events, e.g., cell surface binding of peptide-dye conjugates may be conducted on a system (FMAT® 8100 HTS System, Applied Biosystems, Foster City, Calif.) that automates mix-and-read, non-radioactive assays with live cells or beads (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 labelled antibodies also include cell surface receptor binding assays, inmmunocapture 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 6372907), apoptosis (Vermes, “A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V” (1995) J. Immunol. Methods 184:39-51) and cytotoxicity assays. Fluorometric microvolume assay technology can be used to identify the up or down regulation by a molecule that is 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).

[00320] Labelled antibodies of the invention are useful as imaging biomarkers and probes by the various methods and techniques of biomedical and molecular imaging such as: (i) MRI (magnetic resonance imaging); (ii) MicroCT (computerized tomography); (iii) SPECT (single photon emission computed tomography); (iv) PET (positron emission tomography) Chen et al Bioconjugate Chem. 15: 41-9 (2004); (v) bioluminescence; (vi) fluorescence; and (vii) ultrasound. Immunoscintigraphy is an imaging procedure in which antibodies labeled with radioactive substances are administered to an animal or human patient and a picture is taken of sites in the body where the antibody localizes (US 6528624). Imaging biomarkers may be objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention.

[00321] Peptide labelling 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).

[00322] Peptides and proteins labelled with two moieties, a fluorescent reporter and quencher in sufficient proximity undergo fluorescence resonance energy transfer (FRET). Reporter groups are typically fluorescent dyes that are excited by light at a certain wavelength and transfer energy to an acceptor, or quencher, group, with the appropriate Stokes shift for emission at maximal brightness. Fluorescent dyes include molecules with extended aromaticity, such as fluorescein and rhodamine, and their derivatives. The fluorescent reporter may be partially or significantly quenched by the quencher moiety in an intact peptide. Upon cleavage of the peptide by a peptidase or protease, a detectable increase in fluorescence may be measured (Knight, C. (1995) “Fluorimetric Assays of Proteolytic Enzymes”, Methods in Enzymology, Academic Press, 248:18-34).

[00323] The labelled antibodies of the invention may also be used as an affinity purification agent. In this process, the labelled antibody is immobilized on a solid phase such a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody is contacted with a sample containing the antigen to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the antigen to be purified, which is bound to the immobilized polypeptide variant. Finally, the support is washed with another suitable solvent, such as glycine buffer, pH 5.0, that will release the antigen from the polypeptide variant.

1. Activity assays

[00324] In one aspect, assays are provided for identifying anti-CTHRCl antibodies thereof having biological activity. Biological activity may include, e.g., 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 having such biological activity in vivo and/or in vitro are also provided.

[00325] In certain embodiments, an anti-CTHRCl antibody is tested for its ability to inhibit cell growth or proliferation in vitro. Assays for inhibition of cell growth or proliferation are well known in the art. Certain assays for cell proliferation, exemplified by the “cell killing” assays described herein, measure cell viability. One such assay is the CellTiter-GloTM Luminescent Cell Viability Assay, which is commercially available from Promega (Madison, WI). That assay determines the number of viable cells in culture based on quantitation of ATP present, which is an indication of metabolically active cells. See Crouch et al (1993) J. Immunol. Meth. 160: 81- 8, US Pat. No. 6602677. The assay may be conducted in 96- or 384-well format, making it amenable to 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 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 culture. Data can be recorded by luminometer or CCD camera imaging device. The luminescence output is expressed as relative light units (RLU).

[00326] 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-GloTM 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).

[00327] In one aspect, an anti-CTHRCl antibody is tested for its ability to induce cell death in vitro. Assays for induction of cell death are well known in the art. In some embodiments, such assays measure, e.g., loss of membrane integrity as indicated by uptake of propidium iodide (PI), trypan blue (see Moore et al. Cytotechnology, 17: 1-11 (1995)), or 7AAD. In an exemplary PI uptake assay, cells are cultured in Dulbecco’s Modified Eagle 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 at a density of 3 x 106 per dish in 100 x 20 mm dishes and allowed to attach overnight. The medium is removed and replaced with fresh medium alone or medium containing various concentrations of the antibody. The cells are incubated for a 3-day time period. Following treatment, monolayers are washed with PBS and detached by trypsinization. Cells are then centrifuged at 1200 rpm for 5 minutes at 4 °C, the pellet resuspended in 3 mL cold Ca2+ binding buffer (10 mM Hepes, pH 7.4, 140 mM NaCl, 2.5 mM CaC12) and aliquoted into 35 mm strainercapped 12 x 75 mm tubes (1 mL per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive PI (10 pg/mL). Samples are analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickinson). Antibodies which induce statistically significant levels of cell death as determined by PI uptake are thus identified.

[00328] In one aspect, an anti-CTHRCl 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 and seeded in dishes as discussed in the preceding paragraph. The medium is removed and replaced with fresh medium alone or medium containing 0.001 to 10 pg/mL of the antibody. Following a three-day incubation period, monolayers are washed with PBS and detached by trypsinization. Cells are then centrifuged, resuspended in Ca2+ binding buffer, and aliquoted into tubes as discussed in the preceding paragraph. Tubes then receive labeled annexin (e.g., annexin V-FITC) (1 pg/mL). Samples are analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (BD Biosciences). Antibodies that induce statistically significant levels of annexin binding relative to control are thus identified. Another exemplary assay for antibodies that induce apoptosis is a histone DNA ELISA colorimetric assay for detecting intemucleosomal degradation of genomic DNA. Such an assay can be performed using, e.g., the Cell Death Detection ELISA kit (Roche, Palo Alto, CA).

[00329] 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 have been transfected with nucleic acid encoding CTHRC1.

[00330] In one aspect, an anti- CTHRC1 antibody thereof is tested for its ability to inhibit cell growth or proliferation in vivo. In certain embodiments, an anti- CTHRC1 antibody thereof is tested for its ability to inhibit tumor growth in vivo. In vivo model systems, such as xenograft models, can be used for such testing. In an exemplary xenograft system, human tumor cells are introduced into a suitably immunocompromised non-human animal, e.g., a SCID mouse. An antibody of the invention is administered to the animal. The ability of the antibody to inhibit or decrease tumor growth is measured. In certain embodiments of the above xenograft system, the human tumor cells are tumor cells from a human patient. In certain embodiments, the human tumor cells are introduced into a suitably immunocompromised non-human animal by subcutaneous injection or by transplantation into a suitable site, such as a mammary fat pad.

2. Binding assays and other assays

[00331] In one aspect, an anti-CTHRCl antibody is tested for its antigen binding activity. For example, in certain embodiments, an anti- CTHRC1 antibody is tested for its ability to bind to CTHRC1 expressed on the surface of a cell. A FACS assay may be used for such testing.

[00332] In one aspect, competition assays may be used to identify a monoclonal antibody that competes with a monoclonal antibody comprising the variable domains of any one of SeQ ID NOs: 1-10 or a chimeric antibody comprising the variable domain of the monoclonal antibody comprising the sequences of Table 3 and Table 4, and constant domains from IgGl or IgG4 for binding to CTHRC1. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or a conformational epitope) that is bound by a monoclonal antibody comprising the variable domains of any one of SeQ ID NOs: 1-10 or a chimeric antibody comprising the variable domain of the monoclonal antibody comprising the sequences of Table 3 and Table 4, and constant domains from IgGl 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 an epitope to which an antibody binds 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 binding of the other by 50% or more.

[00333] In an exemplary competition assay, immobilized CTHRC1 is incubated in a solution comprising a first labeled antibody that binds to CTHRC1 (e.g., a monoclonal antibody comprising the variable domains of any one of SeQ ID NOs: 1-10 or a chimeric antibody comprising the variable domain of the monoclonal antibody comprising the sequences of Table 3 and Table 4, and constant domains from IgGl or IgG4) and a second unlabeled antibody that is being tested for its ability to compete with the first antibody for binding to CTHRC1. The second antibody may be present in a hybridoma supernatant. As a control, immobilized CTHRC 1 is incubated in a solution comprising the first labeled antibody but not the second unlabeled antibody. After incubation under conditions permissive for binding of the first antibody to CTHRC 1, excess unbound antibody is removed, and the amount of label associated with immobilized CTHRC 1 is measured. If the amount of label associated with immobilized CTHRC 1 is substantially reduced in the test sample relative to the control sample, then that indicates that the second antibody is competing with the first antibody for binding to CTHRC 1. In certain embodiments, immobilized CTHRC 1 is present on the surface of a cell or in a membrane preparation obtained from a cell expressing CTHRC 1 on its surface.

[00334] In one aspect, purified anti-CTHRCl antibodies can be further characterized by a series of assays including, but not limited to, N-terminal sequencing, amino acid analysis, nondenaturing size exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography and papain digestion.

E. CAR Modified Immune Cells

[00335] In certain embodiments, the invention relates to compositions and methods for treating cancer including but not limited to hematologic malignancies and solid tumors. In certain embodiments, CAR modified immune cells are used. CAR-T cells can be used therapeutically for patients suffering from non-hematological tumors such as solid tumors arising from, for example, breast, CNS, and skin malignancies. In certain embodiments, CAR-NK cells can be used therapeutically for patients suffering from any one of a number of malignancies. In certain embodiments, CAR-macrophages can be used therapeutically for patients suffering from any one of a number of malignancies.

[00336] In certain embodiments, the present invention relates to a strategy of adoptive cell transfer of T cells or NK cells, or macrophages transduced to express a chimeric antigen receptor (CAR). CARs are molecules that combine antibody -based specificity for a desired antigen (e.g., tumor antigen) with, for example, a T cell receptor-activating intracellular domain to generate a chimeric protein that exhibits a specific anti-tumor cellular immune activity.

[00337] 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 cell can be genetically modified to stably express an antibody binding domain on its surface, conferring novel antigen specificity. Methods for generating CAR-NK cells are known in the art. For example, see Glienke et al., Front Pharmacol. 2015; 6: 21. Services for generating CAR-NK cells are commercially available. See for example Creative Biolabs Inc., 45-1 Ramsey Road, Shirley, NY 11967, USA.

[00338] In one aspect, the present invention relates to the use of T cells 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 cell can be genetically modified to stably express an antibody binding domain on its surface, conferring novel antigen specificity that is MHC independent. In some instances, the T cell is genetically modified to stably express a CAR that combines an antigen recognition domain of a specific antibody with an intracellular domain of the CD3-zeta chain or FcyRI protein into a single chimeric protein.

[00339] In one embodiment, the CAR of the invention comprises an extracellular domain having an antigen recognition domain, a transmembrane domain, and a cytoplasmic domain. The intracellular domain or otherwise 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 comprising the intracellular domain of a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigens receptors or their ligands that are required for an efficient response of lymphocytes to antigen. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In another embodiment, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In one embodiment, the transmembrane domain is the CD8a hinge domain.

[00340] Between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR, there may be incorporated a spacer domain. As used herein, the term “spacer domain” generally means any oligo- or polypeptide that functions to link the transmembrane domain to, either the extracellular domain or, the cytoplasmic domain in the polypeptide chain. A spacer domain may comprise up to 300 amino acids, 10 to 100 amino acids and often 25 to 50 amino acids.

[00341] With respect to the cytoplasmic domain, the CAR of the invention can be designed to comprise the CD28 and/or 4- IBB signaling domain by itself or be combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. In one embodiment, the cytoplasmic domain of the CAR can be designed to further comprise the signaling domain of CD3-zeta. For example, the cytoplasmic domain of the CAR can include but is not limited to CD3-zeta, 4-1BB and CD28 signaling modules and combinations thereof. Accordingly, the invention provides CAR T cells and methods of their use for adoptive therapy.

[00342] In one embodiment, the CAR T cells of the invention can be generated by introducing a lentiviral vector comprising a desired CAR, for example a CAR comprising anti-CTHRCl, CD8a hinge and transmembrane domain, and human 4-1BB and CD3zeta signaling domains, into the cells. The CAR T cells of the invention are able to replicate in vivo resulting in longterm persistence that can lead to sustained tumor control.

[00343] In one embodiment the invention relates to administering a genetically modified T cell expressing a CAR for the treatment of a patient having cancer or at risk of having cancer using lymphocyte infusion. Preferably, autologous lymphocyte infusion is used in the treatment. Autologous PBMCs are collected from a patient in need of treatment and T cells are activated and expanded using the methods described herein and known in the art and then infused back into the patient. The invention also includes treating a malignancy or an autoimmune disease in which chemotherapy and/or immunotherapy in a patient results in significant immunosuppression in the patient, thereby increasing the risk of the patient of developing a malignancy (e.g., CLL).

[00344] The invention includes using T cells expressing an anti-CTHRCl antibody derived CAR including both CD3-zeta and either the 4-1BB or CD28 costimulatory domain (also referred to as CARTPODO T cells). The CARTPODO T cells of the invention can undergo robust in vivo T cell expansion and can establish memory cells specific for cells displaying CTHRC1 tumor epitope, which memory cells persist at high levels for an extended amount of time in blood and bone marrow.

1. Antigen Binding Moiety

[00345] In one embodiment, the CAR of the invention comprises a target-specific binding element otherwise referred to as an antigen binding moiety, or targeting arm. Antigen binding moi eties used in the present invention are capable of binding a CTHRC1 epitope, for example a CTHRC1 tumor epitope. As such, the antigen binding moiety is chosen to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.

[00346] A CAR of the invention is engineered to target a cell displaying the CTHRC1 epitope by way of engineering an appropriate antigen binding moiety that specifically binds to the CTHRC1 epitope.

[00347] Preferably, the antigen binding moiety portion in the CAR of the invention is scFv, or scFab wherein the nucleic acid sequence of the scFv comprises the nucleic acid sequence(s) that encode for one or more light chain CDRs and one or more heavy chain CDRs disclosed herein for anti-CTHRCl antibodies, and wherein the nucleic acid sequence of the scFab comprises the nucleic acid sequence(s) that encode for one or more light chain CDRs and one or more heavy chain CDRs disclosed herein for anti-CTHRCl antibodies.

[00348] Preferably, the antigen binding moiety portion in the CAR of the 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.

[00349] Preferably, the antigen binding moiety portion in the CAR of the 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.

[00350] Preferably, the antigen binding moiety portion in the CAR of the 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.

[00351] Preferably, the antigen binding moiety portion in the CAR of the 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 moiety portion in the CAR of the 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.

[00352] In embodiments, the antigen binding moiety portion in the CAR of the invention is an scFv, or scFab comprising an amino acid that is encoded by a nucleotide sequence selected from the group consisting of SeQ ID NOs: 100-109. In embodiments, the antigen binding moiety portion in the CAR of the invention is an scFv, or scFab comprising an amino acid that is encoded by a nucleotide sequence selected from the group consisting of any one of SeQ ID NOs: 100, 102, 104, 106, and 108.

[00353] In embodiments, the antigen binding moiety portion in the CAR of the invention is an scFv, or scFab comprising an amino acid that is encoded by a nucleotide sequence selected from the group consisting of any one of SeQ ID NOs: 101, 103, 105, 107, and 109.

[00354] In embodiments, the antigen binding moiety portion in the CAR of the invention is an scFv, or scFab comprising an amino acid that is encoded by a nucleotide sequence selected from the group consisting of any one of SeQ ID NOs: 100, 102, 104, 106, and 108; and from the group consisting of any one of SeQ ID NOs: 101, 103, 105, 107, and 109.

[00355] In embodiments, the antigen binding moiety portion in the CAR of the invention is an scFv, or scFab comprising an amino acid sequence selected from the group consisting of any CDR sequence in Table 3 and Table 4. [00356] Preferably, the antigen binding moiety portion in the CAR of the invention is an scFv, or scFab comprising an amino acid sequence selected from the group consisting of any CDR sequence in Table 3 and Table 4; and further comprises an amino acid sequence selected from the group consisting of any one of SeQ ID NOs: 1-10. More preferably, the antigen binding moiety portion of the CAR of the 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: 150-154; a CDR2 sequence selected from the group consisting of SeQ ID NOs: 180-184; and a CDR3 sequence selected from the group consisting of SeQ ID NOs: 210-214, and a light chain variable region comprising a CDR1 sequence selected from the group consisting of SeQ ID NOs: 240-244; a CDR2 sequence selected from the group consisting of SeQ ID NOs: 270-274; and a CDR3 sequence selected from the group consisting of SeQ ID NOs: 300-304; and further comprises an amino acid sequence selected from the group consisting of any one of SeQ ID NOs: 1-10.

[00357] In one embodiment, the antigen binding moiety portion in the CAR of the invention comprises a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO:2.

[00358] In one embodiment, the antigen binding moiety portion in the CAR of the invention comprises a heavy chain variable region comprising SEQ ID NO:3 and a light chain variable region comprising SEQ ID NO:4.

[00359] In one embodiment, the antigen binding moiety portion in the CAR of the invention comprises a heavy chain variable region comprising SEQ ID NO:5 and a light chain variable region comprising SEQ ID NO: 6.

[00360] In one embodiment, the antigen binding moiety portion in the CAR of the invention comprises a heavy chain variable region comprising SEQ ID NO:7 and a light chain variable region comprising SEQ ID NO: 8.

[00361] In one embodiment, the antigen binding moiety portion in the 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. [00362] In one embodiment, the antigen binding moiety portion in 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 recited above.

2. Transmembrane Domain

[00363] With respect to the transmembrane domain, the CAR can be designed to comprise a transmembrane domain that is fused to the extracellular domain of the CAR. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CAR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[00364] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Transmembrane regions of particular use in this invention may be derived from (i.e. comprise at least the transmembrane region(s) of) 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, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. Preferably a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the cytoplasmic signaling domain of the CAR. A glycine-serine doublet provides a particularly suitable linker.

[00365] Preferably, the transmembrane domain in the CAR of the invention is the CD8 transmembrane domain. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence of SEQ ID NO: 16 of US Patent No. 9,102,760. In one embodiment, the CD8 transmembrane domain comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 22 of US Patent No. 9,102,760. In another embodiment, the CD8 transmembrane domain comprises the amino acid sequence of SEQ ID NO: 22 of US Patent No. 9,102,760. [00366] In some instances, the transmembrane domain of the CAR of the invention comprises the CD8a hinge domain. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence of SEQ ID NO: 15 of US Patent No. 9,102,760. In one embodiment, the CD8 hinge domain comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 21 of US Patent No. 9,102,760. In another embodiment, the CD8 hinge domain comprises the amino acid sequence of SEQ ID NO: 21 of US Patent No. 9,102,760.

3. Cytoplasmic Domain

[00367] The cytoplasmic domain or otherwise the intracellular signaling domain of the CAR of the invention is responsible for activation of at least one of the normal effector functions of the immune cell in which the CAR has been placed in. The term “effector function” refers to a specialized function of a cell. Effector function of a T cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term “intracellular signaling domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire intracellular signaling domain can be employed, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular signaling domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular signaling domain is thus meant to include any truncated portion of the intracellular signaling domain sufficient to transduce the effector function signal.

[00368] Preferred examples of intracellular signaling domains for use in the CAR 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 engagement, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functional capability.

[00369] It is known that signals generated through the TCR alone are insufficient for full activation of the T cell and that a secondary or co-stimulatory signal is also required. Thus, T cell activation can be said to be mediated by two distinct classes of cytoplasmic signaling sequence: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal (secondary cytoplasmic signaling sequences). [00370] Primary cytoplasmic signaling sequences regulate primary activation of the TCR complex either in a stimulatory way, or in an inhibitory way. Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs or IT AMs.

[00371] Examples of IT AM containing primary cytoplasmic signaling sequences that are of particular use in the 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 cytoplasmic signaling molecule in the CAR of the invention comprises a cytoplasmic signaling sequence derived from CD3 zeta.

[00372] In some embodiments, the cytoplasmic domain of the CAR can be designed to comprise the CD3-zeta signaling domain by itself or combined with any other desired cytoplasmic domain(s) useful in the context of the CAR of the invention. For example, the cytoplasmic domain of the CAR can comprise a CD3 zeta chain portion and at least one costimulatory signaling region. The costimulatory signaling region refers to a portion of the CAR comprising the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or their ligands that is required for an efficient response of lymphocytes to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, CD40, PD-1, ICOS, lymphocyte function- associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, and a ligand that specifically binds with CD83, and the like.

[00373] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR of the invention may be linked to each other in a random or specified order. Optionally, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage. A glycine-serine doublet provides a particularly suitable linker.

[00374] 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 domain of CD28 and 4-1BB. [00375] In one embodiment, the cytoplasmic domain in the CAR of the 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 US Patent No. 9,102,760 and the signaling domain of CD3-zeta comprises the nucleic acid sequence set forth in SEQ ID NO: 18 of US Patent No. 9,102,760.

[00376] In one embodiment, the cytoplasmic domain in the CAR of the invention is designed to comprise the signaling domain of 4-1BB and the signaling domain of CD3-zeta, wherein the signaling domain of 4- IBB comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 23 of US Patent No. 9,102,760 and the signaling domain of CD3-zeta comprises the nucleic acid sequence that encodes the amino acid sequence of SEQ ID NO: 24 of US Patent No. 9,102,760.

[00377] In one embodiment, the cytoplasmic domain in the CAR of the 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 US Patent No. 9,102,760 and the signaling domain of CD3-zeta comprises the amino acid sequence set forth in SEQ ID NO: 24 of US Patent No. 9,102,760.

4. Alternative Construction

[00378] In alternative embodiments, the CAR can be engineered to include an antigen binding domain that can bind to an engineered ligand fusion protein that includes a ligand that binds to the antigen binding domain rather than to CTHRC1, where the engineered ligand fusion protein can include the ligand fused to an anti-CTHRCl antibody of the present disclosure, thus providing orthogonal binding to CTHRC1. By way of example, but not limitation, the antigen binding domain can be an engineered receptor, such as a modified NKG2D receptor that does not bind its natural ligand but which binds a non-natural ligand, where the non-natural ligand is the ligand portion of the engineered ligand fusion protein, such as those described in U.S. Patent No. 10,259,858, U.S. Patent No. 10,259,858, U.S. Patent Application Publication No. 2019/0300594, U.S. Patent Application Publication No. 2020/0138866, WO 2017/222556, and U.S. Patent Application Publication No. 2016/0304578, each of which is incorporated herein by reference.

5. Vectors [00379] The present invention encompasses a DNA construct comprising sequences of a CAR, wherein the sequence comprises the nucleic acid sequence of an antigen binding moiety operably linked to the nucleic acid sequence of an intracellular domain. An exemplary intracellular domain that can be used in the CAR of the invention includes but is not limited to the intracellular domain of CD3-zeta, CD28, 4-1BB, and the like. In some instances, the CAR can comprise any combination of CD3-zeta, CD28, 4-1BB, and the like.

[00380] In one embodiment, the CAR of the invention comprises an anti-CTHRCl antibody derived scFv, human CD8 hinge and transmembrane domain, and human 4- IBB and CD3zeta signaling domains.

[00381] The nucleic acid sequences coding for the desired molecules can be obtained using recombinant methods known in the art, such as, for example by screening libraries from cells expressing the gene, by deriving the gene from a vector known to include the same, or by isolating directly from cells and tissues containing the same, using standard techniques. Alternatively, the gene of interest can be produced synthetically, rather than cloned.

[00382] The present invention also provides vectors in which a DNA of the present invention is inserted. Vectors derived from retroviruses such as the lentivirus are suitable tools to achieve long-term gene transfer since they allow long-term, stable integration of a transgene and its propagation in daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses such as murine leukemia viruses in that they can transduce nonproliferating cells, such as hepatocytes. They also have the added advantage of low immunogenicity.

[00383] In brief summary, the expression of natural or synthetic nucleic acids encoding CARs is typically achieved by operably linking a nucleic acid encoding the CAR polypeptide or portions thereof to a promoter and incorporating the construct into an expression vector. The vectors can be suitable for replication and integration eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.

[00384] In addition to the methods described above, the following methods may be used. [00385] 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. Pat. Nos. 5,399,346, 5,580,859, 5,589,466, incorporated by reference herein in their entireties). In another embodiment, the invention provides a gene therapy vector.

[00386] The nucleic acid can be cloned into a number of types of vectors. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[00387] Further, the expression vector may be provided to a cell 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 in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers, (e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).

[00388] A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. A number of retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. A number of adenovirus vectors are known in the art. In one embodiment, lentivirus vectors are used.

[00389] Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved 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 before activity begins to decline. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[00390] 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 levels of expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is Elongation Growth Factor-la (EF-la). 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, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Further, the invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the invention. The use of an inducible promoter provides a molecular switch capable of turning on expression of the polynucleotide sequence which it is operatively linked when such expression is desired or turning off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to a metallothionine promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[00391] In order to assess the expression of a CAR polypeptide or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes, such as neo and the like.

[00392] Reporter genes are used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, betagalactosidase, chloramphenicol acetyl transferase, 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 obtained commercially. In general, the construct with the minimal 5' flanking region showing the highest level of expression of reporter gene is identified as the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription.

[00393] Methods of introducing and expressing genes into a cell are known in the art. In the context of an expression vector, the vector can be readily introduced into a host cell, e.g., mammalian, bacterial, yeast, or insect cell by any method in the art. For example, the expression vector can be transferred into a host cell by physical, chemical, or biological means.

[00394] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising 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 the introduction of a polynucleotide into a host cell is calcium phosphate transfection.

[00395] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially 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 lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

[00396] Chemical means for introducing a polynucleotide into a host cell 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., an artificial membrane vesicle).

In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[00397] 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, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20. degree. C. Chloroform is used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner 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 the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5: 505-10). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.

[00398] Regardless of the method used to introduce exogenous nucleic acids into a host cell or otherwise expose a cell to the inhibitor of the present invention, in order to confirm the presence of the recombinant DNA sequence in the host cell, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the invention.

6. Sources of T Cells

[00399] Prior to expansion and genetic modification of the T cells of the invention, a source of T cells is obtained from a subject. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, 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 unit of blood collected from a subject using any number of techniques known to the skilled artisan, such as Ficoll™ separation. In one preferred embodiment, cells from the circulating blood of an individual 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, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing steps. In one embodiment of the invention, the cells are washed with phosphate buffered saline (PBS). In an alternative embodiment, the wash solution lacks calcium and may lack magnesium or may lack many if not all divalent cations. Again, surprisingly, initial activation steps in the absence of calcium lead to magnified activation. As those of ordinary skill in the art would readily appreciate a washing step may be accomplished by methods known to those in the art, such as by using a semi-automated “flow-through” centrifuge (for example, the Cobe 2991 cell processor, the Baxter CytoMate, or the Haemonetics Cell Saver 5) according to the manufacturer’s instructions. After washing, the cells may be resuspended in a variety of biocompatible buffers, such as, for example, Ca²⁺-free, Mg²⁺-free PBS, PlasmaLyte A, or other saline solution with or without buffer. Alternatively, the undesirable components of the apheresis sample may be removed and the cells directly resuspended in culture media.

[00400] In another embodiment, T cells are isolated from peripheral blood lymphocytes by lysing the red blood cells and depleting the monocytes, for example, by centrifugation through a PERCOLL™ gradient or by counterflow centrifugal elutriation. A specific subpopulation 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 time period sufficient for positive selection of the desired T cells. In one embodiment, the time period is about 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one preferred embodiment, the incubation time period is 24 hours. For isolation of T cells from patients with leukemia, use of longer incubation times, such as 24 hours, can increase cell yield. Longer incubation times may be used to isolate T cells in any situation where there are few T cells as compared to other cell types, such in isolating tumor infiltrating lymphocytes (TIL) from tumor tissue or from immune-compromised individuals. Further, use of longer incubation times can increase the efficiency of capture of CD8+ T cells. Thus, by simply shortening or lengthening the time T cells are allowed to bind to the CD3/CD28 beads and/or by increasing or decreasing the ratio of beads to T cells (as described further herein), subpopulations of T cells can be preferentially selected for or against at culture initiation or at other time points during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and/or anti-CD28 antibodies on the beads or other surface, subpopulations of T cells can be preferentially selected for or against at culture initiation or at other desired time points. The skilled artisan would recognize that multiple rounds of selection can also be used in the context of this invention. In certain embodiments, it may be desirable to perform the selection procedure and use the “unselected” cells in the activation and expansion process. “Unselected” cells can also be subjected to further rounds of selection. [00401] Enrichment of a T cell population by negative selection can be accomplished with a combination of antibodies directed to surface markers unique to the negatively selected cells. One method is cell sorting and/or selection via negative magnetic immunoadherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the cells negatively selected. For example, to enrich for CD4⁺ cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDl lb, CD16, HLA-DR, and CD8. In certain embodiments, it may be desirable to enrich for or positively select for regulatory T cells which typically express CD4⁺, CD25⁺, CD62L^hi, GITR⁺, and FoxP3⁺. Alternatively, in certain embodiments, T regulatory cells are depleted by anti-C25 conjugated beads or other similar method of selection.

[00402] For isolation of a desired population of cells by positive or negative selection, the concentration of cells and surface (e.g., particles such as beads) can be varied. In certain embodiments, it may be desirable to significantly decrease the volume in which beads and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and beads. For example, in one embodiment, a concentration of 2 billion cells/mL is used. In one embodiment, a concentration of 1 billion cells/mL is used. In a further embodiment, greater than 100 million cells/mL is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further embodiments, concentrations of 125 or 150 million cells/mL can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells, or from samples where there are many tumor cells present (i.e., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and would be desirable to obtain. For example, using high concentration of cells allows more efficient selection of CD8⁺ T cells that normally have weaker CD28 expression.

[00403] In a related embodiment, it may be desirable to use lower concentrations of cells. By significantly diluting the mixture of T cells and surface (e.g., particles such as beads), interactions between the particles and cells is minimized. This selects for cells that express high amounts of desired antigens to be bound to the particles. For example, CD4⁺ T cells express higher levels of CD28 and are more efficiently captured than CD8⁺ T cells in dilute concentrations. In one embodiment, the concentration of cells used is 5xlO⁶/mL. In other embodiments, the concentration used can be from about lxl0⁵/mL to lxlO⁶/mL, and any integer value in between.

[00404] In other embodiments, the cells may be incubated on a rotator for varying lengths of time at varying speeds at either 2-10°C or at room temperature.

[00405] T cells for stimulation can also be frozen after a washing step. Wishing not to be bound by theory, the freeze and subsequent thaw step provides a more uniform product by removing granulocytes and to some extent monocytes in the cell population. After the washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and will be useful in this context, one method involves using PBS containing 20% DMSO and 8% human serum albumin, or culture media 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 media containing for example, Hespan and PlasmaLyte A, the cells then are frozen to -80° C. at a rate of 1 ° per minute and stored in the vapor phase of a liquid nitrogen storage tank. Other methods of controlled freezing may be used as well as uncontrolled freezing immediately at -20° C. or in liquid nitrogen.

[00406] In certain embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest for one hour at room temperature prior to activation using the methods of the present invention.

[00407] Also contemplated in the context of the invention is the collection of blood samples or apheresis product from a subject at a time period prior to when the expanded cells as described herein might be needed. As such, the source of the cells to be expanded can be collected at any time point necessary, and desired cells, such as T cells, 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 an apheresis is taken from a generally healthy subject. In certain embodiments, a blood sample or an apheresis is taken from a generally healthy subject who is at risk of developing a disease, but who has not yet developed a disease, and the cells of interest are isolated and frozen for later use. In certain embodiments, the T cells may be expanded, frozen, and used at a later time. In certain embodiments, samples are collected from a patient shortly after diagnosis of a particular disease as described herein but prior to any treatments. In a further embodiment, the cells are isolated from a blood sample or an apheresis from a subject prior to any number of relevant treatment modalities, including but not limited to treatment with agents such as natalizumab, efalizumab, antiviral agents, chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (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 later use in conjunction with (e.g., before, simultaneously or following) bone marrow or stem cell transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cells are isolated prior to and can be frozen for later use for treatment following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan.

[00408] In a further embodiment of the present invention, T cells are obtained from a patient directly following treatment. In this regard, it has been observed that following certain cancer treatments, in particular treatments with drugs that damage the immune system, shortly after treatment during the period when patients would normally be recovering from the treatment, the quality of T cells obtained may be optimal or improved for their ability to expand ex vivo. Likewise, following ex vivo manipulation using the methods described herein, these cells may be in a preferred state for enhanced engraftment and in vivo expansion. Thus, it is contemplated within the context of the present invention to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery phase. Further, in certain embodiments, mobilization (for example, mobilization with GM-CSF) and conditioning regimens can be used to create a condition in a subject wherein repopulation, recirculation, regeneration, and/or expansion of particular cell types is favored, especially during a defined window of time following therapy. Illustrative cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

7. Activation and Expansion of T Cells

[00409] Whether prior to or after genetic modification of the T cells to express a desirable CAR, the T cells can be activated and expanded generally using methods as described, for example, in U.S. Pat. Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; and U.S. Patent Application Publication No. 20060121005.

[00410] Generally, the T cells of the invention are expanded by contact with a surface having attached thereto an agent that stimulates a CD3/TCR complex associated signal and a ligand that stimulates a co-stimulatory molecule on the surface of the T cells. In particular, T cell populations may be stimulated as described herein, such as 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. For costimulation of an accessory molecule on the surface of the T cells, a ligand that binds the accessory molecule is used. For example, a population of T cells can be contacted with an anti-CD3 antibody and an anti-CD28 antibody, under conditions appropriate for stimulating proliferation of the T cells. To stimulate proliferation of either CD4⁺ T cells or CD8⁺ T cells, an anti-CD3 antibody and an anti-CD28 antibody. Examples of an anti-CD28 antibody include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France) can be used as can other methods commonly known in the art (Berg et al., Transplant Proc . 30(8): 3975-7, 1998; Haanen et al., J. Exp. Med. 190(9): 13191328, 1999; Garland et al., J. Immunol Meth. 227(1-2): 53-63, (1999)).

[00411] In certain embodiments, the primary stimulatory signal and the co-stimulatory signal for the T cell may be provided by different protocols. For example, the agents providing each signal may be in solution or coupled to a surface. When coupled to a surface, the agents may be coupled to the same surface (i.e., in “cis” formation) or to separate surfaces (i.e., in “trans” formation). Alternatively, one agent may be coupled to a surface and the other agent in solution. In one embodiment, the agent providing the co-stimulatory signal is bound to a cell surface and the agent providing the primary activation signal is in solution or coupled to a surface. In certain embodiments, both agents can be in solution. In another embodiment, the agents may be in soluble form, and then cross-linked to a surface, such as a cell expressing Fc receptors or an antibody or other binding agent which will bind to the agents. In this regard, see for example, U.S. Patent Application Publication Nos. 20040101519 and 20060034810 for artificial antigen presenting cells (aAPCs) that are contemplated for use in activating and expanding T cells in the present invention.

[00412] In one embodiment, the two agents are immobilized on beads, either on the same bead, i.e., “cis,” or to 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 co-stimulatory signal is an anti-CD28 antibody or antigen-binding fragment thereof; and both agents are co-immobilized to the same bead in equivalent molecular amounts. In one embodiment, a 1 : 1 ratio of each antibody bound to the beads for CD4⁺ T cell expansion and T cell growth is used. In certain aspects of the present invention, a ratio of anti CD3:CD28 antibodies bound to the beads is used such that an increase in T cell expansion is observed as compared to the expansion observed using a ratio of 1 : 1. In one particular embodiment an increase of from about 1 to about 3 fold is observed as compared to the expansion observed using a ratio of 1 : 1. In one embodiment, the ratio of CD3:CD28 antibody bound to the beads ranges from 100: 1 to 1 : 100 and all integer values there between. In one aspect of the present invention, more anti-CD28 antibody is bound to the particles than anti-CD3 antibody, i.e., the ratio of CD3:CD28 is less than one. In certain embodiments of the invention, the ratio of anti CD28 antibody to anti CD3 antibody bound to the beads is greater than 2: 1. In one particular embodiment, a 1 : 100 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :75 CD3:CD28 ratio of antibody bound to beads is used. In a further embodiment, a 1 :50 CD3 :CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :30 CD3 :CD28 ratio of antibody bound to beads is used. In one preferred embodiment, a 1 : 10 CD3:CD28 ratio of antibody bound to beads is used. In another embodiment, a 1 :3 CD3:CD28 ratio of antibody bound to the beads is used. In yet another embodiment, a 3: 1 CD3:CD28 ratio of antibody bound to the beads is used.

[00413] Ratios of particles to cells from 1 :500 to 500: 1 and any integer values in between may be used to stimulate T cells or other target cells. As those of ordinary skill in the art can readily appreciate, the ratio of particles to cells may depend on particle size relative to the target cell. For example, small sized beads could only bind a few cells, while larger beads could bind many. In certain embodiments the ratio of cells to particles ranges from 1 : 100 to 100: 1 and any integer values in-between and in further embodiments the ratio comprises 1 :9 to 9: 1 and any integer values in between, can also be used to stimulate T cells. The ratio of anti-CD3- and anti-CD28- coupled particles to T cells that result in T cell stimulation can vary as noted above, however certain preferred values include 1 : 100, 1 :50, 1 :40, 1 :30, 1 :20, 1 : 10, 1 :9, 1 :8, 1 :7, 1 :6, 1 :5, 1 :4, 1 :3, 1 :2, 1 :1, 2: 1, 3: 1, 4: 1, 5: 1, 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, and 15: 1 with one preferred ratio being at least 1 : 1 particles per T cell. In one embodiment, a ratio of particles to cells of 1 : 1 or less is used. In one particular embodiment, a preferred particle: cell ratio is 1 :5. In further embodiments, the ratio of particles to cells can be varied depending on the day of stimulation. For example, in one embodiment, the ratio of particles to cells is from 1 : 1 to 10: 1 on the first day and additional particles are added to the cells every day or every other day thereafter for up to 10 days, at final ratios of from 1 : 1 to 1 : 10 (based on cell counts on the day of addition). In one particular embodiment, the ratio of particles to cells is 1 : 1 on the first day of stimulation and adjusted to 1 :5 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 :5 on the third and fifth days of stimulation. In another embodiment, the ratio of particles to cells is 2: 1 on the first day of stimulation and adjusted to 1 : 10 on the third and fifth days of stimulation. In another embodiment, particles are added on a daily or every other day basis to a final ratio of 1 : 1 on the first day, and 1 : 10 on the third and fifth days of stimulation. One of skill in the art will appreciate that a variety of other ratios may be suitable for use in the present invention. In particular, ratios will vary depending on particle size and on cell size and type.

[00414] In further embodiments of the present invention, the cells, such as T cells, are combined with agent-coated beads, the beads and the cells are subsequently separated, and then the cells are cultured. In an alternative embodiment, prior to culture, the agent-coated beads and cells are not separated but are cultured together. In a further embodiment, the beads and cells are first concentrated by application of a force, such as a magnetic force, resulting in increased ligation of cell surface markers, thereby inducing cell stimulation.

[00415] By way of example, cell surface proteins may be ligated by allowing paramagnetic beads to which anti-CD3 and anti-CD28 are attached (3x28 beads) to contact the T cells. In one embodiment the cells (for example, 10⁴ to 10⁹ T cells) and beads (for example, DYNABEADS® M-450 CD3/CD28 T paramagnetic beads at a ratio of 1 : 1) are combined in a buffer, preferably PBS (without divalent cations such as, calcium and magnesium). Again, those of ordinary skill in the art can readily appreciate any cell concentration may be used.

[00416] For example, the target cell may be very rare in the sample and comprise only 0.01% of the sample or the entire sample (i.e., 100%) may comprise the target cell of interest. Accordingly, any cell number is within the context of the present invention. In certain embodiments, it may be desirable to significantly decrease the volume in which particles and cells are mixed together (i.e., increase the concentration of cells), to ensure maximum contact of cells and particles. For example, in one embodiment, a concentration of about 2 billion cells/mL is used. In another embodiment, greater than 100 million cells/mL is used. In a further embodiment, a concentration of cells of 10, 15, 20, 25, 30, 35, 40, 45, or 50 million cells/mL is used. In yet another embodiment, a concentration of cells from 75, 80, 85, 90, 95, or 100 million cells/mL is used. In further embodiments, concentrations of 125 or 150 million cells/mL can be used. Using high concentrations can result in increased cell yield, cell activation, and cell expansion. Further, use of high cell concentrations allows more efficient capture of cells that may weakly express target antigens of interest, such as CD28-negative T cells. Such populations of cells may have therapeutic value and would be desirable to obtain in certain embodiments. For example, using high concentration of cells allows more efficient selection of CD8+ T cells that normally have weaker CD28 expression.

[00417] In one embodiment of the present invention, the mixture may be cultured for several hours (about 3 hours) to about 14 days or any hourly integer value in between. In another embodiment, the mixture may be cultured for 21 days. In one embodiment of the invention the beads and the T cells are cultured together for about eight days. In another embodiment, the beads and T cells are cultured together for 2-3 days. Several cycles of stimulation may also be desired such that culture time of T cells can be 60 days or more. Conditions appropriate for T cell culture include an appropriate media (e.g., Minimal Essential Media or RPMI Media 1640 or, X-vivo 15, (Lonza)) that may contain factors necessary for proliferation and viability, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-y, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFP, and TNF-a or any other additives for the growth of cells known to the skilled artisan. Other additives for the growth of cells include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetyl-cysteine and 2- mercaptoethanol. Media can include RPMI 1640, AIM-V, DMEM, MEM, a-MEM, F-12, X- Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and/or an amount of cytokine(s) sufficient for the growth and expansion of T cells. Antibiotics, e.g., penicillin and streptomycin, are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air plus 5% CO2).

[00418] T cells that have been exposed to varied stimulation times may exhibit different characteristics. For example, typical blood or apheresed peripheral blood mononuclear cell products have a helper T cell population (TH, CD4⁺) that is greater than the cytotoxic or suppressor T cell population (Tc, CD8⁺). Ex vivo expansion of T cells by stimulating CD3 and CD28 receptors produces a population of T cells that prior to about days 8-9 consists predominately of TH cells, while after about days 8-9, the population of T cells comprises an increasingly greater population of Tc cells. Accordingly, depending on the purpose of treatment, infusing a subject with a T cell population comprising predominately of TH cells may be advantageous. Similarly, if an antigen-specific subset of Tc cells has been isolated it may be beneficial to expand this subset to a greater degree.

[00419] Further, in addition to CD4 and CD8 markers, other phenotypic markers vary significantly, but in large part, reproducibly during the course of the cell expansion process. Thus, such reproducibility enables the ability to tailor an activated T cell product for specific purposes.

8. Therapeutic Application

[00420] The present invention encompasses a cell (e.g., T cell) transduced with a lentiviral vector (LV). For example, the LV encodes a CAR that combines an antigen recognition domain of a specific antibody with an intracellular domain of CD3-zeta, CD28, 4- IBB, or any combinations thereof. Therefore, in some instances, the transduced T cell can elicit a CAR- mediated T-cell response.

[00421] The invention provides the use of a CAR to redirect the specificity of a primary T cell to a tumor antigen. Thus, the present invention also provides a method for stimulating a T cell- mediated immune response to a target cell population or tissue in a mammal comprising the step of administering to the mammal a T cell that expresses a CAR, wherein the CAR comprises a binding moiety that specifically interacts with a predetermined target, a zeta chain portion comprising for example the intracellular domain of human CD3zeta, and a costimulatory signaling region.

[00422] In one embodiment, the present invention includes a type of cellular therapy where T cells are genetically modified to express a CAR and the CAR T cell is infused to a recipient in need thereof. The infused cell is able to kill tumor cells in the recipient. Unlike antibody therapies, CAR T cells are able to replicate in vivo resulting in long-term persistence that can lead to sustained tumor control.

[00423] In one embodiment, the CAR T cells of the invention can undergo robust in vivo T cell expansion and can persist for an extended amount of time. In another embodiment, the CAR T cells of the invention evolve into specific memory T cells that can be reactivated to inhibit any additional tumor formation or growth.

[00424] Without wishing to be bound by any particular theory, the anti-tumor immunity response elicited by the CAR-modified T cells may be an active or a passive immune response. In addition, the CAR mediated immune response may be part of an adoptive immunotherapy approach in which CAR-modified T cells induce an immune response specific to the antigen binding moiety in the CAR.

[00425] Cancers that may be treated include tumors that are not vascularized, or not yet substantially vascularized, as well as vascularized tumors. The cancers may comprise non-solid tumors (such as hematological tumors, for example, leukemias and lymphomas) or may comprise solid tumors. Types of cancers to be treated with the CARs of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and certain leukemia or lymphoid malignancies, benign and malignant tumors, and malignancies e.g., sarcomas, carcinomas, and melanomas. Adult tumors/cancers and pediatric tumors/cancers are also included. In certain embodiments, CAR T cells can be used therapeutically for patients suffering from non-hematological tumors such as solid tumors arising from breast, CNS, and skin malignancies.

[00426] Hematologic cancers are cancers of the blood or bone marrow. Examples of hematological (or hematogenous) cancers include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin’s disease, nonHodgkin’s lymphoma (indolent and high grade forms), multiple myeloma, Waldenstrom’s macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia and myelodysplasia.

[00427] Solid tumors are abnormal masses of tissue that usually do not contain cysts or liquid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named for the type of cells that form them (such as sarcomas, carcinomas, and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovioma, mesothelioma, Ewing’s tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytomas sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms' tumor, cervical cancer, testicular tumor, seminoma, bladder carcinoma, melanoma, and CNS tumors (such as a glioma (such as brainstem glioma and mixed gliomas), glioblastoma (also known as glioblastoma multiforme) astrocytoma, CNS lymphoma, germinoma, medulloblastoma, Schwannoma craniopharyogioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma and brain metastases).

[00428] In one aspect, CAR T cells may be used for ex vivo immunization. With respect to ex vivo immunization, at least one of the following occurs in vitro prior to administering the cell into a mammal: i) expansion of the cells, ii) introducing a nucleic acid encoding a CAR to the cells, and/or iii) cry opreservation of the cells.

[00429] Ex vivo procedures are well known in the art and are discussed more fully below. Briefly, cells are isolated from a mammal (e.g., a human) and genetically modified (i.e., transduced or transfected in vitro) with a vector expressing a CAR disclosed herein. The CAR- modified cell can be administered to a mammalian recipient to provide a therapeutic benefit. The mammalian recipient may be a human and the CAR-modified cell can be autologous with respect to the recipient. Alternatively, the cells can be allogeneic, syngeneic or xenogeneic with respect to the recipient.

[00430] The procedure for ex vivo expansion of hematopoietic stem and progenitor cells is described in U.S. Pat. No. 5,199,942, incorporated herein by reference, can be applied to the cells of the present invention. Other suitable methods are known in the art, therefore the present invention is not limited to any particular method of ex vivo expansion of the cells. Briefly, ex vivo culture and expansion of T cells comprises: (1) collecting CD34+ hematopoietic stem and progenitor cells from a mammal from peripheral blood harvest or bone marrow explants; and (2) expanding such cells ex vivo. In addition to the cellular growth factors described in U.S. Pat. No. 5,199,942, other factors such as flt3-L, IL-1, IL-3 and c-kit ligand, can be used for culturing and expansion of the cells.

[00431] In addition to using a cell-based vaccine in terms of ex vivo immunization, the present invention also provides compositions and methods for in vivo immunization to elicit an immune response directed against an antigen in a patient.

[00432] The CAR-modified T cells of the present invention may be administered either alone, or as a pharmaceutical composition in combination with diluents and/or with other components such as IL-2 or other cytokines or cell populations. Briefly, pharmaceutical compositions of the present invention may comprise a target cell population as described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives. CAR compositions of the present invention are preferably formulated for intravenous administration.

[00433] Pharmaceutical compositions of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The quantity and frequency of administration will be determined by such factors as the condition of the patient, and the type and severity of the patient’s disease, although appropriate dosages may be determined by clinical trials.

[00434] When “an immunologically effective amount”, “an anti-tumor effective amount”, “an tumor-inhibiting effective amount”, or “therapeutic amount” is indicated, the precise amount of the compositions of the present invention to be administered can be determined by a physician with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (subject). It can generally be stated that a pharmaceutical composition comprising the T cells described herein may be administered at a dosage of 10⁴ to 10⁹ cells/kg body weight, preferably 10⁵ to 10⁶ cells/kg body weight, including all integer values within those ranges. T cell compositions may also be administered multiple times at these dosages. The cells can be administered by using infusion techniques that are commonly known in immunotherapy (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988)). The optimal dosage and treatment regime for a particular patient can readily be determined by one skilled in the art of medicine by monitoring the patient for signs of disease and adjusting the treatment accordingly.

[00435] In certain embodiments, it may be desired to administer activated T cells to a subject and then subsequently redraw blood (or have an apheresis performed), activate T cells therefrom according to the present invention, and reinfuse the patient with these activated and expanded T cells. This process can be carried out multiple times every few weeks. In certain embodiments, T cells can be activated from blood draws of from 10 cc to 400 cc. In certain embodiments, T cells are activated from blood draws of 20 cc, 30 cc, 40 cc, 50 cc, 60 cc, 70 cc, 80 cc, 90 cc, or 100 cc. Not to be bound by theory, using this multiple blood draw/multiple reinfusion protocol may serve to select out certain populations of T cells.

[00436] The administration of the subject compositions may be carried out in any convenient manner, including by aerosol inhalation, injection, ingestion, transfusion, implantation or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In one embodiment, the T cell compositions of the present invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by i.v. injection. The compositions of T cells may be injected directly into a tumor, lymph node, or site of infection.

[00437] In certain embodiments of the present invention, cells activated and expanded using the methods described herein, or other methods known in the art where T cells are expanded to therapeutic levels, are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, or Cytarabine (also known as ARA-C). In further embodiments, the T cells of the invention may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAMPATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. These drugs inhibit either the calcium dependent phosphatase calcineurin (cyclosporine and FK506) or inhibit the p70S6 kinase that is important for growth factor induced signaling (rapamycin) (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 cell compositions of the present invention are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In another embodiment, the cell compositions of the present invention are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in one embodiment, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the expanded immune cells of the present invention. In an additional embodiment, expanded cells are administered before or following surgery.

[00438] The dosage of the above treatments to be administered to a patient will vary with the precise nature of the condition being treated and the recipient of the treatment. The scaling of dosages for human administration can be performed according to art-accepted practices. The dose for CAMPATH, for example, will generally be in the range 1 to about 100 mg for an adult patient, usually administered daily for a period between 1 and 30 days. In certain embodiments, 1 to 10 mg per day is used. In other embodiments, larger doses of up to 40 mg per day may be used (for example as described in U.S. Pat. No. 6,120,766).

F. Diagnostic and Detection Methods and Compositions

[00439] One embodiment of the present invention is directed to a method of determining the presence of a CTHRC1 polypeptide in a sample suspected of containing the CTHRC1 polypeptide, wherein the method comprises exposing the sample to an antibody that binds to the CTHRC1 polypeptide and determining binding of the antibody to the CTHRC1 polypeptide in the sample, wherein the presence of such binding is indicative of the presence of the CTHRC1 polypeptide in the sample. Optionally, the sample may contain cells (which may be cancer cells) suspected of expressing the CTHRC1 polypeptide. The antibody employed in the method may optionally be detectably labeled, attached to a solid support, or the like.

[00440] For example, CTHRC1 polypeptide overexpression may be analyzed by immunohistochemistry (H4C). Parrafin embedded tissue sections from a tumor biopsy may be subjected to the H4C assay and accorded a CTHRC1 protein staining intensity criteria. In a preferred embodiment, determining whether a cancer is amenable to treatment by methods disclosed herein involves detecting the presence of the CTHRC1 tumor epitope in a subject or in a sample from a subject.

[00441] As another example, FISH assays such as the INFORM® (sold by Ventana, Arizona) or PATHVISION® (Vysis, Illinois) may be carried out on formalin-fixed, paraffin-embedded tumor tissue to determine the extent (if any) of CTHRC1 overexpression in the tumor.

[00442] As another example, CTHRC1 overexpression or amplification may be evaluated using an in vivo detection assay, e.g., by administering a molecule (such as an antibody) which binds the molecule to be detected and is tagged with a detectable label (e.g., a radioactive isotope or a fluorescent label) and externally scanning the patient for localization of the label.

[00443] In embodiments, the anti-CTHRCl antibodies of the present invention can be useful for staging of CTHRC1 epitope expressing cancers (e.g., in radioimaging). The antibodies are also useful for purification or immunoprecipitation of CTHRC1 epitope from cells, for detection and quantitation of CTHRC1 epitope in vitro, e.g., in an ELISA or a Western blot, to kill and eliminate CTHRC1 -expressing cells from a population of mixed cells as a step in the purification of other cells. [00444] Another embodiment of the present invention is directed to a method of diagnosing the presence of a tumor in a mammal, wherein the method comprises (a) contacting a test sample comprising tissue cells obtained from the mammal with an antibody that binds to a CTHRC1 polypeptide and (b) detecting the formation of a complex between the antibody and the CTHRC1 polypeptide in the test sample, wherein the formation of a complex is indicative of the presence of a tumor in the mammal. Optionally, the antibody is detectably labeled, attached to a solid support, or the like, and/or the test sample of tissue cells is obtained from an individual suspected of having a cancerous tumor. Antibody detection can be achieved via different techniques as described herein, e.g., IHC and PET imaging.

[00445] The invention also provides kits and articles of manufacture comprising at least one anti-CTHRCl antibody. Kits containing anti-CTHRCl antibodies find use, e.g., for CTHRC1 cell killing assays, for purification or immunoprecipitation of CTHRC1 polypeptide from cells. For example, for isolation and purification of CTHRC1, the kit can contain an anti-CTHRCl antibody coupled to beads (e.g., sepharose beads). Kits can be provided which contain the antibodies for detection and quantitation of CTHRC1 in vitro, e.g., in an ELISA or a Western blot. Such antibody useful for detection may be provided with a label such as a fluorescent or radiolabel.

G. Pharmaceutical Formulations

[00446] The antibodies and/or engineered cells of the invention may be administered by any route appropriate to the condition to be treated. The antibody will typically be administered parenterally, i.e. infusion, subcutaneous, intramuscular, intravenous, intradermal, intrathecal and epidural.

[00447] For treating these cancers, in one embodiment, the antibody is administered via intravenous infusion. The dosage administered via infusion is in the range of about 0.001 mg/kg to about 100 mg/kg per dose by the subject’s body weight, generally one dose per week for a total of one, two, three or four doses. Alternatively, the dosage range is of about 0.01 mg/kg to about 100 mg/kg, about 0.1 mg/kg to about 100 mg/kg, about 1 mg/kg to about 100 mg/kg, about 0.001 mg/kg to about 50 mg/kg, about 0.01 mg/kg to about 50 mg/kg, about 0.1 mg/kg to about 50 mg/kg, about 1 mg/kg to about 50 mg/kg, about 0.001 mg/kg to about 10 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 1 mg/kg to about 10 mg/kg, about 0.001 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 5 mg/kg, about 0.001 mg/kg to about 1 mg/kg, about 0.01 mg/kg to about 1 mg/kg, and about 0.1 mg/kg to about 1 mg/kg. The dose may be administered once per day, once per week, multiple times per week, but less than once per day, multiple times per month but less than once per day, multiple times per month but less than once per week, once per month or intermittently to relieve or alleviate symptoms of the disease. Administration may continue at any of the disclosed intervals until remission of the tumor or symptoms of the cancer being treated. Administration may continue after remission or relief of symptoms is achieved where such remission or relief is prolonged by such continued administration.

[00448] In one aspect, the invention further provides pharmaceutical formulations comprising at least one anti-CTHRCl antibody of the invention. In some embodiments, a pharmaceutical formulation comprises (1) an antibody of the invention, and (2) a pharmaceutically acceptable carrier.

[00449] Therapeutic formulations comprising an anti-CTHRCl antibody used in accordance with the present invention are prepared for storage by mixing the antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington ’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as polysorbate; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG). Pharmaceutical formulations to be used for in vivo administration are generally sterile. This is readily accomplished by filtration through sterile filtration membranes.

[00450] The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington ’s Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[00451] Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2- hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly- D-(-)-3 -hydroxybutyric acid. While polymers such as ethylene-vinyl acetate and lactic acid- glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods. When encapsulated immunoglobulins remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[00452] An antibody may be formulated in any suitable form for delivery to a target cell/tissue. For example, antibodies may be formulated as immunoliposomes. A “liposome” is a small vesicle composed of various types of lipids, phospholipids and/or surfactant which is useful for delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing the antibody are prepared by methods known in the art, such as described in Epstein et al., Proc. Natl. Acad. Set. USA 82:3688 (1985); Hwang et al., Proc. Natl Acad. Set. USA 77:4030 (1980); U.S. Pat. Nos. 4,485,045 and 4,544,545; and WO97/38731 published October 23, 1997. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556.

[00453] Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and PEG- derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to yield liposomes with the desired diameter. Fab' fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem. 257: 286-8 (1982) via a disulfide interchange reaction. A chemotherapeutic agent is optionally contained within the liposome (See Gabizon et al., J. National Cancer Inst. 81(19): 1484 (1989)).

[00454] The formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.

H. Therapeutic Methods and Compositions

[00455] An antibody of the invention may be used in, for example, in vitro, ex vivo, and in vivo therapeutic methods. In one aspect, the invention provides methods for inhibiting cell growth or proliferation, either in vivo or in vitro, the method comprising exposing a cell to an anti-CTHRCl antibody under conditions permissive for binding of the antibody to CTHRC1. “Inhibiting cell growth or proliferation” means decreasing a cell’s growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, and includes inducing cell death. In certain embodiments, the cell is a tumor cell. The anti-CTHRCl antibodies may additionally or alternatively (i) inhibit tumor metastasis in vivo; (ii) inhibit tumor growth in vivo; (iii) decrease tumor size in vivo; (iv) inhibit tumor vascularization in vivo; (v) exhibit cy cotoxic activity activity on tumor cells and cancer associated fibroblasts expressing and/or displaying CTHRC1 in vivo; (vi) exhibit cytostatic activity on tumor cells or cancer associated fibroblasts expressing and/or displaying CTHRC1 in vivo; (vii) enhance infiltration of anti -tumor immune cells in vivo; or (vii) prevent suppression of immune cells in the tumor microenvironment in vivo. [00456] In one aspect, an antibody, or a CAR modified immune cell, of the invention is used to treat or prevent a cell proliferative disorder. In certain embodiments, the cell proliferative disorder is associated with increased expression and/or activity of CTHRC1. For example, in certain embodiments, the cell proliferative disorder is associated with increased expression or display of CTHRC1 on the surface of a cell, either directly or in a complex. In certain embodiments, the cell proliferative disorder is a tumor or a cancer.

[00457] In one aspect, the invention provides methods for treating a cell proliferative disorder comprising administering to an individual an effective amount of an anti-CTHRCl antibody, or an effective amount of a CAR modified immune cell(s) of the invention, thereby effectively treating or preventing said cell proliferative disorder. In one embodiment, said cell proliferative disorder is cancer.

[00458] In one embodiment, an anti-CTHRCl antibody can be used in a method for binding CTHRC1 in an individual suffering from a disorder associated with increased CTHRC1 expression and/or activity, the method comprising administering to the individual the antibody such that CTHRC1 in the individual is bound. In one embodiment, the CTHRC1 is human CTHRC1, and the individual is a human individual. An anti-CTHRCl antibody can be administered to a human for therapeutic purposes. Moreover, an anti-CTHRCl antibody can be administered to a non-human mammal expressing CTHRC1 with which the antibody crossreacts (e.g., a primate, pig, rat, or mouse) for veterinary purposes or as an animal model of human disease. Regarding the latter, such animal models may be useful for evaluating the therapeutic efficacy of antibodies of the invention (e.g., testing of dosages and time courses of administration).

[00459] The invention also provides a method of treating fibrosis and/or fibrotic disease comprising administering to a patient in need thereof a therapeutically effective amount of a CTHRC1 antibody of any one of the preceding embodiments. The antibody will typically be administered in a dosage range of about 0.001 mg/kg to about 100 mg/kg of the subject’s body weight.

[00460] The anti-CTHRCl antibodies of the subject invention can also be advantageously administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities for cancer and/or fibrosis including, e.g., chemotherapy, radiation therapy, or immunotherapy. A patient can also be preconditioned with a therapeutically effective amount of anti-CTHRCl antibodies prior to receiving the chemotherapy, radiation therapy, or immunotherapy. Suitable immunotherapies for use in combination with anti- CTHRCl antibodies include autologous and allogeneic cell therapies, engineered T and NK cells, immune engagers, fusion proteins, or other immune-oncology agents.

[00461] In an exemplary embodiment, the subject antibodies can be administered in conjunction with an appropriate cellular immunotherapy for treating the cancer (e.g. CAR T or CAR NK cells) or fibrosis disease (e.g., Treg therapy). Without being bound by theory, it is expected that the anti-CTHRCl antibodies of the present invention may increase CD8+ T cell recruitment and infiltration in the tumor microenvironment. Thus, in embodiments, a method of treating cancer, fibrosis and/or fibrotic disease according to the present invention can include a conditioning step, e.g. a preconditioning step, of administering a therapeutically effective amount of an anti- CTHRCl antibody to a subject simultaneously or sequentially with administration of a cellular immunotherapy directed against the cancer or fibrosis. In embodiments, the cellular immunotherapy can comprise administering an engineered T cell or NK cell therapy. The antibody will typically be administered in a dosage range of about 0.001 mg/kg to about 100 mg/kg.

[00462] In another exemplary embodiment, the subject antibodies can be administered in conjunction with administering radiation therapy to a patient. Without being bound by theory, it is expected that administration of the anti-CTHRCl antibodies of the present invention may help reduce fibrosis resulting from the radiation, which often presents a dose-limiting side effect. Thus, in some embodiments, a method of treating cancer according to the present invention can include a step of administering a therapeutically effective amount of an anti-CTHRCl antibody to a subject simultaneously or sequentially with radiation therapy. The antibody will typically be administered in a dosage range of about 0.001 mg/kg to about 100 mg/kg.

[00463] In any embodiments of the present disclosure, the fibrotic disease can be selected from the group consisting of idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, pulmonary arterial hypertension, kidney fibrosis, keratinosis, non-alcoholic fatty liver disease (NASH), scleroderma, rheumatoid arthritis, Crohn’s disease, ulcerative colitis, myelofibrosis and systemic lupus erythematosus. [00464] An antibody of the invention (and any additional therapeutic agent or adjuvant) can be administered by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In addition, the antibody is suitably administered by pulse infusion, particularly with declining doses of the antibody. Dosing can be by any suitable route, e.g., by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic.

[00465] Antibodies of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[00466] In general, depending on the stage of the cancer, cancer treatment involves one or a combination of the following therapies: surgery to remove the cancerous tissue, radiation therapy, and chemotherapy. Anti-CTHRCl antibody therapy may be especially desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well and in metastatic disease where radiation therapy has limited usefulness. The tumor targeting anti- CTHRC1 antibodies of the invention are useful to alleviate CTHRC1 -expressing cancers upon initial diagnosis of the disease or during relapse.

[00467] As discussed, the anti-CTHRCl antibodies are administered to a human patient, in accordance with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In some embodiments, intravenous or subcutaneous administration of the antibody is preferred.

[00468] The antibody composition of the invention will be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners.

[00469] For the prevention or treatment of disease, the dosage and mode of administration will be chosen by the physician according to known criteria. The appropriate dosage of antibody will depend on the type of disease to be treated, as defined above, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient’s clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Preferably, the antibody is administered by intravenous infusion or by subcutaneous injections. Depending on the type and severity of the disease, about 1 pg/kg to about 100 mg/kg body weight (e.g., about 0.1-30 mg/kg/dose) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. A dosing regimen can comprise administering an initial loading dose of about 4 mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the anti-CTHRCl antibody. However, other dosage regimens may be useful. A typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art.

[00470] The anti-CTHRCl antibodies of the invention can be in the different forms encompassed by the definition of “antibody” herein. Thus, the antibodies include full length or intact antibody, antibody fragments, native sequence antibody or amino acid variants, humanized, chimeric or fusion antibodies, and functional fragments thereof. In fusion antibodies an antibody sequence is fused to a heterologous polypeptide sequence. The antibodies can be modified in the Fc region to provide desired effector functions. As discussed in more detail in the sections herein, with the appropriate Fc regions, the naked antibody bound on the cell surface can induce cytotoxicity, e.g., via antibody-dependent cellular cytotoxicity (ADCC) or by recruiting complement in complement dependent cytotoxicity, or some other mechanism. Alternatively, where it is desirable to eliminate or reduce effector function, so as to minimize side effects or therapeutic complications, certain other Fc regions may be used. [00471] In one embodiment, the antibody (i) competes for binding to the same epitope, and/or (ii) binds substantially to the same epitope, as the antibodies of the invention. Antibodies having the biological characteristics of the present anti-CTHRCl antibodies of the invention are also contemplated, specifically including the in vivo tumor targeting and any cell proliferation inhibition or cytotoxic characteristics.

[00472] Methods of producing the above antibodies are described in detail herein.

[00473] The present anti-CTHRCl antibodies are useful for treating a CTHRC1 -expressing cancer or alleviating one or more symptoms of the cancer in a mammal. The cancers encompass metastatic cancers of any of the cancers described herein. The antibody is able to bind to at least a portion of the cancer cells that display a CTHRC1 directly or in a complex in the mammal. In a preferred embodiment, the antibody is effective to destroy or kill CTHRC1 -expressing tumor cells or inhibit the growth of such tumor cells, in vitro or in vivo, upon binding to CTHRC1 epitope on the cell. In other preferred embodiments, the antibodies are effective to i) inhibit tumor metastasis in vivo; (ii) inhibit tumor growth in vivo; (iii) decrease tumor size in vivo; (iv) inhibit tumor vascularization in vivo; (v) exhibit cytotoxic activity on tumor cells and cancer associated fibrobalsts expressing and/or displaying CTHRC1 in vivo; (vi) exhibit cytostatic activity on a tumor cells or cancer associated fibroblasts expressing and/or displaying CTHRC1 in vivo; (vii) enhance infiltration of anti-tumor immune cells in vivo; or (vii) prevent suppression of immune-cells in the tumor microenvironment in vivo.

[00474] The present anti-CTHRCl antibodies are additionally or alternatively useful for treating CTHRC1 -expressing fibrotic disease as described herein.

[00475] The invention provides a composition comprising an anti-CTHRCl antibody of the invention, and a carrier. The invention also provides formulations comprising an anti- CTHRC1 antibody of the invention, and a carrier. In one embodiment, the formulation is a therapeutic formulation comprising a pharmaceutically acceptable carrier.

[00476] Another aspect of the invention is isolated nucleic acids encoding the anti-CTHRCl antibodies. Nucleic acids encoding both the H and L chains and especially the hypervariable region residues, chains which encode the native sequence antibody as well as variants, modifications and humanized versions of the antibody, are encompassed. [00477] The invention also provides methods useful for treating a CTHRC1 polypeptide- expressing cancer or alleviating one or more symptoms of the cancer in a mammal, comprising administering a therapeutically effective amount of an anti-CTHRCl antibody to the mammal. The antibody therapeutic compositions can be administered short term (acute) or chronic, or intermittent as directed by physician. Also provided are methods of inhibiting the growth of, and killing a CTHRC1 polypeptide-expressing cell.

6. Articles of Manufacture and Kits

[00478] Another embodiment of the invention is an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of CTHRC1 -expressing cancer. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is effective for treating, preventing and/or diagnosing the cancer condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-CTHRCl antibody of the invention, or a CAR-modified immune cell of the invention, or a nucleic acid of the invention. Optionally, a composition further comprises a carrier, for example a pharmaceutically acceptable carrier. The label or package insert indicates that the composition is used for treating cancer. The label or package insert will further comprise instructions for administering the antibody composition to the cancer patient. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. This disclosure further contemplates similar articles of manufacture useful for the treatment, prevention and/or diagnosis of CTHRC1 -expressing and/or displaying cancer and fibrotic disease.

[00479] Kits are also provided that are useful for various purposes, e.g., for CTHRC1- expressing cell killing assays, for purification or immunoprecipitation of CTHRC1 polypeptide from cells. For isolation and purification of CTHRC1 polypeptide, the kit can contain an anti- CTHRCl antibody coupled to beads (e.g., 131epharose beads). Kits can be provided which contain the antibodies for detection and quantitation of CTHRC 1 polypeptide in vitro, e.g., in an ELISA or a Western blot. As with the article of manufacture, the kit comprises a container and a label or package insert on or associated with the container. The container holds a composition comprising at least one anti-CTHRCl antibody of the invention. Additional containers may be included that contain, e.g., diluents and buffers, control antibodies. The label or package insert may provide a description of the composition as well as instructions for the intended in vitro or detection use.

[00480] For example, a kit can comprise a first container comprising a composition comprising one or more CTHRC 1 antibodies or CAR modified immune cells, such as CAR-T or CAR-NK cells, or CAR macrophages, of the invention; and a second container comprising a buffer. The buffer may be pharmaceutically acceptable.

[00481] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[00482] All patent, patent application, and literature references cited in the present specification are hereby incorporated by reference in their entirety.

EXAMPLES

[00483] In the Examples below, high-affinity monoclonal antibodies (mAbs) were generated that specifically bind CTHRC1. An initial pool of 28 antibody candidates was narrowed to 12 that selectively bound CTHRC1 vs. similar collagen like proteins. A secondary screen then assessed which of these 12 candidates block cell adhesion to CTHRC1 through integrins; this likely a necessary step to enable CTHRC1 dependent signalling. Of the 12 binders, 3 were found that selectively blocked cell adhesion to CTHRC1. The 12 selective binders were also screened for internalization, of these a handful demonstrated robust internalization, including 1 mAb that was also functional, as well as a 2^nd that was the highest affinity binder as measured by ELISA.

[00484] As CTHRC1 has previously been implicated in several diseases and disorders, rationales for using antibodies described herein in different medical applications are laid out in the Examples. In the context of cancer, CTHRC 1 may be a marker, a driver of disease, or a route to targeting cytotoxic, inflammatory, or radiopharmaceuticals to the tumour microenvironment. Data was gathered which supports the role of CTHRC1 as contributing to cancer, as well as data indicating CTHRC1 may be valuable as a means of targeting payloads to the tumor microenvironment, thus making the inhibition of CTHRC1 or use of CTHRC1 mAbs as payload targeting agents a plausible medical intervention.

Example 1. Antibody Discovery - screening, identification, and characterization

Antibody discovery:

[00485] Anti-CTHRCl antibodies were generated from a Humanized transgenic mouse immunized with recombinant human CTHRC1 (rhCTHRCl). Two cohorts of four mice were boosted every 3-4 days for four weeks with O.Olmg of rhCTHRCl protein. One cohort (CTHRC1S) had antigen administered with the Sigma Adjuvant System (Sigma-Aldrich) while the second cohort (CTHRC1A) had antigen administered with a mix of amino bisphosphonate alendronate and muramyl dipeptide (ALD/MDP) (Asensio et al. Mabs, 2019). Serum from mice on day 21 of immunizations was tested for polyclonal binding to CTHRC1, and a positive signal was observed. Following this, splenocytes were harvested, fused to myeloma cells, sorted into single colonies, and expanded as monoclonal hybridomas for binding screens to human and rodent CTHRC1. Positive clones (28 clones) were then expanded and frozen for long-term storage. Following this, a small volume (~5mL) of conditioned media was collected for affinity analysis by bio-layer interferometry (BLI), and l-3mg of antibody was purified from 30mL culture of positive hybridomas. This purified antibody was then delivered to Phenomic for screening in specificity ELISAs and functional activity in a cell adhesion assay. Of the 28 clones, 12 clones showed selective binding to human and/or rat CTHRC1 (Figure 1). The remaining 16 clones were either His tag cross-reactive or showed multi-specificity binding to a number of proteins with 50% or greater sequence identity to CTHRC1 (Figure 1).

[00486] The Antibodies CTHRC1S-M5 (AB987) and CTHRC1S-M23 (AB988) were selected as initial leads, with both antibodies showing cross-reactivity to mouse and rat CTHRC1, and low to sub-nanomolar affinities as measured by BLI. Furthermore, these two antibodies bind to different epitopes as assessed by a BLI-based epitope binning assay. These clones are summarized in Table 2.

Table 2. Summary of binding activity and affinity of top anti-CTHRCl clones

[00487] Table 3 depicts heavy chain variable region complementarity determining regions (CDRs), and Table 4 depicts light chain variable region CDRs, of anti-CTHRCl antibodies disclosed herein. Illustrative heavy chain variable regions are depicted at Table 5, and illustrative light chain variable regions are depicted at Table 6.

Table 3. Complementarity Determining Regions, Heavy Variable Region (IMGT)

Table 4. Complementarity Determining Regions, Light Variable Region (IMGT)

Table 5. Illustrative Immunoglobulin Sequences - Heavy Chain Variable Region

Table 6. Illustrative Immunoglobulin Sequences - Light Chain Variable Region

In vitro assay development: [00488] CTHRC1 has been reported to mediate cell adhesion through interactions with both integrin pi and P3 (Chen et al., PloS One, 2013; Guo et al., J. Ovarian Res., 2017). A cell adhesion assay was set up and optimized with the goal of having a robust, sensitive assay that would allow for the identification of antibodies that functionally block integrin-mediated cell adhesion to CTHRC1. To this end, multiple cell types, including fibroblasts and cancer cells, were assessed for their ability to attach to wells pre-coated with human CTHRC1 protein. Adhesion of these cell lines to CTHRC 1 was compared to adhesion of the ECM proteins periostin and fibronectin. After optimizing several parameters, several cell lines showed 30% or greater cell adhesion to CTHRC1 when compared to fibronectin (Figure 2). Based on these results and previous data reported in the literature, the SKOV3 ovarian cancer cell line was selected for use in this assay.

[00489] Because no tool inhibitors or antibodies exist that block CTHRC 1, commercially available antibodies were utilized against integrin subunits to demonstrate blocking of cell adhesion to CTHRC 1. Treatment of ovarian cancer cells for 1 hour with either lOOnM integrin pi antibody, lOOnM integrin nV antibody or a combination of the two antibodies lead to a 2-fold or greater reduction in cell adhesion (Figure 3). Treatment with an integrin pi antibody blocked cell adhesion to CTHRC 1 by 75%, whereas treatment with an integrin uV antibody reduced cell adhesion to CTHRC 1 by 40%. The ability of these integrin antibodies to block cell adhesion to fibronectin and vitronectin were included as additional controls, and the data is largely consistent with what has been previously reported in literature. Together, this data demonstrates that: 1) cell attachment to CTHRC 1 is mediated by integrins; and (2) Selective integrin blocking mAbs achieve robust inhibition in this assay and can be used as a positive control.

Functional screening:

[00490] The 28 clones identified from the humanized mouse immunization campaign against CTHRC1 were screened in the cell adhesion assay to assess for functional activity. Of the 12 clones identified to be selective CTHRC 1 binders by ELISA, three clones (CTHRC 1S-M5, CTHRC1S-M11 & CTHRC1S-M23) showed functional activity and blocked cell adhesion by 50% or greater. However, CTHRC 1S-M11 (AB989) was human specific and not cross-reactive to mouse or rat CTHRC 1, and therefore was not selected for further development. Importantly, it was demonstrated that the functional activity of antibodies CTHRC1S-M5 (AB987) and CTHRC1S-M23 (AB988) is selective to CTHRC1 as these antibodies do not block cell adhesion to fibronectin and collagen type-1 (Figure 4).

Example 2. CTHRC1 highly selectively localizes to cancer, is associated with poor outcome, and is most upregulated on cancer-associated fibroblasts (CAEs) in immune-cold tumour microenvironments.

[00491] Without being bound to theory, in the context of cancer, it is expected that inhibiting CTHRC1 may confer therapeutic benefit through blocking CAF and Autocrine pro-survival signalling to cancer cells alongside disrupting immune suppression mediated by CTHRC1. Targeting CTHRC1 with antibodies bound to toxins or that engage immune cells may further drive anti -tumor activity. Data was gathered which supports the idea that CTHRC1 is both selectively upregulated in cancer and contributes to cancer progression.

CTHRC1 mRNA is a top ranked marker of CAFs in cancer-rich, immune-cold, tumour samples

[00492] In this example it is demonstrated that CTHRC1 is upregulated specifically in CAFs in cancer rich, immune-cold, samples vs. CAFs in T-cell rich, immune-hot, samples (Figures 5A- 5B). Cancer rich, T-cell poor tumours, i.e., Immune excluded or Immune deserts, are also associated with poor outcome, treatment resistance, and immune-suppression (Gooden et al. British J. of Cancer, 2011), thus, therapeutic targeting of CTHRC1 represents an opportunity to target these challenging tumor types. Cancer cells transform fibroblasts into CAFs, which in turn promote cancer progression, treatment resistance and immune suppression (Sahai et al. Nat. Rev. Cancer, 2020). Targets that associate closely with CAFs in cancer rich vs. T-cell rich samples are therefore an opportunity for therapeutic intervention in these cancers. To show CTHRC1 is associated with CAFs in these tumor types samples were grouped in the scRNA atlas described in Swechha et al. (bioRxiv 2021) into those that comprise at least 50% cancer cells, and less than 25% T-cells (immune-cold) and those that have over 50% T-cells in the sample and less than 25% cancer cells (immune-hot) (Figure 5 A). Four cancers were included in this analysis, namely, Pancreatic, Lung, Breast, and Colorectal, and are representative of the solid tumours in the scRNA atlas (Swechha et al., bioRxiv, 2021). CAF specific gene expression levels in the Cancercell rich, T-cell poor samples were then ranked by the number of samples where expression is significantly greater (P<0.05) than in the T-cell rich, Cancer-cell poor sample; as well as the overall P-value of this finding (Wilcoxon Rank; Navon, Roy, et al., PloS One, 2009). Finally, targets were filtered down to those upregulated by CAFs but not in other cell types, in all or the majority of samples (Top 500 CAF genes as measured by Wilcoxon Rank). This result demonstrates CTHRC1 is ranked as a target highly localized to CAFs in Cancer-rich, T-cell poor tumour samples (Figure 5B), and thus an opportunity for therapeutic intervention.

CTHRC1 mRNA is upregulated in cancer v.v. adjacent tissue and correlates with disease progression

[00493] In this Example it is demonstrated that CTHRC1 expression is highly upregulated in many solid tumors. Specifically, an analysis of bulk-scRNA data taken from the cancer genome atlas (TCGA) demonstrates that CTHRC1 is highly upregulated in cancerous tissue samples vs. normal adjacent tissue samples across numerous solid cancers including Breast, Lung, Ovarian, Pancreatic, Sarcoma, Melanoma, and Uterine Carcinosarcomas (Figure 6). This indicates that within these organs CTHRC1 selectively localizes to cancerous regions. It is also demonstrated that CTHRC1 is a prognostic indicator of survival in many solid cancers based on analysis of TCGA data with the GEPIA online tool (Tang, Z. et al. Nucleic Acids Res, 2017) (Figure 7). Notably poor survival is seen in patients high for CTHRC1 in liver cancer, stomach cancer, and sarcomas. These cancers are all stromal rich, fibrotic cancers, in line with the above example that shows CTHRC1 localizes to CAFs in cancer-rich immune-poor tumor samples. It is also demonstrated that CTHRC1 expression increases with cancer stage in colorectal cancer and liver cancer (Figure 8); this shows that targeting CTHRC1 may be valuable in patients with late-stage aggressive cancers and where prognosis is poor.

CTHRC mRNA has a favourable expression profile in normal tissue

[00494] As well as being upregulated in cancer vs. adjacent tissues, this Example also demonstrates that CTHRC 1 expression is highly selective to cancer tissue and is expressed at comparatively very low levels in normal healthy tissues in the body. This indicates CTHRC1 targeting is accompanied with a significant therapeutic window and can be used to target payloads to the tumor microenvironment. For example, comparing CTHRC1 bulk-RNA expression in pancreatic cancer samples (TCGA) to CTHRC1 expression in normal tissue samples (GTEX data; both reanalyzed by the UCSC Xena project, Goldman et al., Nat. Biotech, 2020) highlights a significant therapeutic window in almost all pancreatic samples analyzed (Figures 9A-9C). At the single-cell level it was seen that CTHRC1 expression localizes to CAFs and epithelial cancer cells in cancer specimens and is not seen in any cell-types in tissue (Figure 10). This level of localization is comparable if not better than previous mAb targets that have been used to target Antibody Drug Conjugates (ADCs) to the tumour microenvironment and shown safe and non-toxic in clinical trials, such as, LRRC15 (Figure 11). Of note, in Breast, Ovarian, Pancreatic, and Lung cancers as well as Melanoma we see cancer epithelial cells expressing CTHRC1. Expression is not seen in normal epithelial cells; this indicates a mesenchymal program is switched on in cancer and further implicates CTHRC1 as playing a pro-vs. anti-tumor role, since there is significant evolutionary pressure for cancer cells to down- regulate anti -tumor targets and mechanisms. Overall, this shows CTHRC1 expression is selective enough to cancer vs. normal tissue that mAbs could be used to target payloads to cancer, and that CTHRC1 expression by cancer cells indicates a pro-tumor role for this protein in humans. Collectively, this example therefore shows the value of CTHRC1 mAbs as a method of treating cancer in humans.

CTHRC1 protein is expressed in cancer-fibroblast co-cultures, and in mouse tumours

[00495] This example also demonstrates that CTHRC1 is upregulated under experimental conditions where fibroblasts are co-cultured with cancer cells versus monocultures of the same cells (Figure 12), indicating induction of CTHRC1 is dependent on fibroblast-cancer cell interactions, showing specificity to cancer tissue. It was also found in vivo that CTHRC1 protein is selectively expressed in tumor sections using mAbs that selectively bind CTHRC1 (Figure 13). Finally it was seen that CTHRC1 is expressed on human cancer samples (Figure 14), either on cancer cells at stromal interfaces (Melanoma and Head and Neck Cancer) or in regions rich in CAFs (Pancreatic Cancer). Overall this analysis shows that at the protein level CTHRC1 expression is seen in cancers similarly to CTHRC1 mRNA.

Example 3. CTHRC1 antibodies internalize and CTHRC1 antibody-drug conjugates selectively kill cancer cells

[00496] In this Example it is demonstrated that a subset of the CTHRC1 mAbs identified with the antibody screening campaign are internalized by human and mouse cancer cell lines, and can be used to develop antibody-drug conjugates. The Cancer Cell Line Encyclopedia

was used to determine the expression of CTHRC1 across human cancer cell lines. High CTHRC1 expression was seen in several cell lines, including SKOV3 ovarian cancer, PANCI pancreatic cancer and HCT116 colorectal cancer (Figure 15 A). The expression of CTHRC1 in human cancer cell lines is consistent with the scRNA analysis in Example 2, where CTHRC1 expression is observed in certain cancer types, including pancreatic and ovarian cancer. A number of these cancer cell lines were selected based on having either low or high CTHRC 1 expression and were used to profile the CTHRC 1 mAbs for cell surface binding by flow cytometry. This analysis identified that CTHRC1S-M14 (AB991) and CTHRC1S-M23 (AB988) bind to a wide range of human cancer cell lines, including SKOV3 ovarian, KP4 pancreatic and HCT116 colorectal cancer, as well as the mouse EMT6 breast cancer line (Figure 15B). Furthermore, the higher affinity antibody CTHRC1S-M14 (AB991) has a greater level of cell surface binding compared to the lower affinity, functional blocking antibody CTHRC 1S- M23 (AB988).

[00497] It was next assessed whether CTHRC1S-M14 (AB991) and CTHRC1S-M23 (AB988) were internalized by cancer cells, and also compared rates of internalization for two human cancer cell lines that had either low or high levels of CTHRC1 expression. For these experiments, antibodies were first labeled with pHrodo, a pH sensitive dye that covalently binds free lysines present within an antibody. pHrodo is non fluorescent outside of the cell (neutral pH) and fluoresces in the acidic environment of phagosomes and endosomes once the antibody has been internalized. SKOV3 ovarian cancer and KP4 pancreatic cancer cell lines were pretreated with or without 50nM CTHRC1 for 30 mins, after which 6.67nM of each pHrodo labeled CTHRC 1 antibody was added for 0, 2, 4 or 24 hours. Cells were then detached using a cell dissociation buffer, washed and the levels of internalization were assessed by flow cytometry as determined by the percentage of pHrodo iFL Red positive cells. Both CTHRC1 mAbs were rapidly internalized by human cancer cell lines with or without the addition of exogenous CTHRC1 and showed maximal levels of internalization by 24 hours (Figures 16A-16D). The rate of antibody internalization occurs much faster in SKOV3 ovarian cancer cells, which express high levels of CTHRC1, when compared to the internalization rates in KP4 pancreatic cancer cells (Figures 16A-16D). Furthermore, the addition of exogenous CTHRC1 had less of an effect on the rates of internalization for CTHRC1S-M14 and CTHRC1S-M23 in SKOV3 cells when compared to KP4 cells, which have low levels of CTHRC1. Consistent with higher levels of antibody binding, CTHRC1S-M14 had the highest levels of internalization after 24 hours (Figures 16A-16D). Similar experiments were performed in the mouse breast cancer cell lines EMT6 and 4T1 (Figure 17). Internalization of both CTHRC1S-M14 (left) and CTHRC1S-M23 (right) was observed in these mouse cancer cell lines and occurs without the addition of exogenous CTHRC1. Again, the expression levels of CTHRC1 correlated with the rate of antibody internalization. For example, the EMT6 cell line has high levels of CTHRC1 and showed rapid rates of internalization when compared to 4T1, which has little to no expression of CTHRC1 (Figure 17). Similar to human cancer cell lines, a greater rate of internalization was observed for the CTHRC1S-M14 antibody compared to CTHRC1S-M23 in both mouse cancer cell lines. Based on this data, CTHRC1S-M14 was selected for the development of an ADC.

[00498] Figure 18 illustrates selective killing of SKOV3 cells via CTHRC1S-M14 conjugated to MMAE (Vedotin) as measured by caspace 3/7 MFI (left) and LDH release (right). Anti- CTHRC1 antibody M14 was conjugated to MMAE with a VC linker via a light-activated sitespecific conjugation reaction (oYo-Link® conjugation kit, AlphaThera). Conjugated M14, naked monoclonal antibody, and relevant isotype controls were incubated with indicated cells for 24 to 72 hours with killing assessed by either quantifying LDH release by standard colorimetric assays or assessment of Caspase activity via flow cytometry.

Example 4. In vivo models

[00499] The efficacy of anti-CTHRCl was tested in syngeneic mouse breast tumor model, EMT6. Briefly, 100,000 EMT6 cells were injected into the mammary fat pad (MFP) of female Balb/c mice. Mice were grouped out according to tumor volume once size reached 120-250 mm³ range, 10 days post inoculation. Following group out, mice were dosed with 2.5mg/kg anti- CTHRCl or Isotype control, 5mg/kg aPD-1, and/or lOmg/kg a-TGFb (SR) according to group treatment. Tumor volume was assessed twice weekly following caliper measurement and calculated as (length x width²)/2. Initial dose was given intravenous (iv), and remaining doses were administered intraperitoneal (ip) three times a week for three weeks. Mice were euthanized when tumor size exceeds 1500 mm3 or due to tumor ulceration. Figure 19 illustrates the tumorgrowth curves obtained from measurements for 31 days post-tumor cell inoculation. As shown in Figure 19 A, anti-CTHRCl clone M5 in combination with anti-PD-1 resulted in tumor growth inhibition comparable to that obtained with anti-PD-1 in combination with anti-TGFb. Similar results were obtained for anti-CTHRCl clone M23 (Figure 19B) whereas only modest combination activity was observed with anti-CTHRCl clone M14 in combination with anti-PD- 1 (Figure 19C). Control data are identical for Figures 19A-C. Data are plotted as mean +/- standard deviation for each data point across 9 mice per group.

Example 5. In vivo model.

[00500] Female C57BL/6J mice were inoculated s.c. with Pan02 cells in Matrigel. Tumors were measured, and mice were randomized to treatment group when tumors reached an average volume of 100mm³. Treatment with lOmg/kg of isotype control or anti-CTHRCl mAh (clone M5) began 24 hours post-randomization and continued for 3 doses/week for the indicated treatment duration. Mice were monitored for tumor growth (Figure 20A) and overall survival (Figure 20B). Tumor grown inhibition was deemed significantly significant by ANOVA. As shown in Figure 20A, anti-CTHRCl clone M5 resulted in tumor growth inhibition relative to isotype control. Data are plotted as mean tumor volume +/- standard deviation for each data point across 9 mice per group. As shown in Figure 20B, anti-CTHRCl clone M5 resulted in improved survival relative to isotype control.

Example 6. Anti-CTHRCl Pretreatment Results in Recruitment of CD8 T cells in the tumor microenvironment. [00501] Balb/c mice were inoculated orthotopically with EMT6 tumor cells in Matrigel. When tumors reached an average volume of 200 mm³, animals were assigned to treatment group. Mice were treated with isotype or anti-CTHRCl mAb (M5 clone) at 10 mg/kg for 1 week (3 doses). Following dosing, tumors were isolated and processed to slides. Slides were stained with an anti-CD8 antibody, and level of CD8 infiltration into tumor nests was quantified by HALO image analysis software. Data were plotted as number of infiltrating CD8 T cells versus distance from tumor margin (FIGURE 21). As shown in FIGURE 21, pretreatment of cells with an anti- CTHRCl antibody resulted in improved CD8 T cell recruitment in the tumor microenvironment. These results suggest that preconditioning treatment with an anti-CTHRCl antibody of the invention can result in CD8 T cell recruitment that could be advantageous for cellular immunotherapies such as CAR-T cell therapy.

Table 7. Additional Biological Sequences

[0001] While the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be clear to one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention. For example, all the techniques and apparatus described above can be used in various combinations. All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes.

Claims

WHAT IS CLAIMED IS:

1. An anti-CTHRCl antibody that binds to human CTHRC1.

2. The anti-CTHRCl antibody according to claim 1, that (i) selectively binds CTHRC1, (ii) blocks cell adhesion to CTHRC1, and/or (iii) is internalized upon binding to cells that express CTHRC1.

3. The anti-CTHRCl antibody according to claim 1 or claim 2, comprising a heavy chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 1, 3, 5, 7, and 9.

4. The anti-CTHRCl antibody according to any claim 2, comprising a heavy chain variable region comprising an amino acid sequence selected from Table 3.

5. The anti-CTHRCl antibody according to claim 2, comprising a light chain variable region comprising an amino acid sequence selected from the group consisting of: SEQ ID NOs: 2, 4, 6, 8, and 10.

6. The anti-CTHRCl antibody according to claim 2, comprising a light chain variable region comprising an amino acid sequence selected from Table 4.

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

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

9. The anti-CTHRCl antibody according to claim 2, comprising a heavy chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 150; a CDR2 sequence comprising SEQ ID NO: 180; and a CDR3 sequence comprising SEQ ID NO: 210 and a light chain variable region comprising a CDR1 sequence comprising SEQ ID NO: 240; a CDR2 sequence comprising SEQ ID NO: 270; and a CDR3 sequence comprising SEQ ID NO: 300. The anti-CTHRCl antibody according to any one of claims 1-9, having a binding affinity (KD) for CTHRC 1 of less than 10 nM, preferably less than 5 nM, more preferably less than 1 nM. The anti-CTHRCl antibody according to any one of claims 1-9, comprising a heavy chain variable region comprising SEQ ID NO: 1 and a light chain variable region comprising SEQ ID NO: 2. The anti-CTHRCl antibody according to any one of claims 1-8, comprising a heavy chain variable region comprising SEQ ID NO: 3 and a light chain variable region comprising SEQ ID NO: 4. The anti-CTHRCl antibody according to any one of claims 1-8, comprising a heavy chain variable region comprising SEQ ID NO: 5 and a light chain variable region comprising SEQ ID NO: 6. The anti-CTHRCl antibody according to any one of claims 1-8, comprising a heavy chain variable region comprising SEQ ID NO: 7 and a light chain variable region comprising SEQ ID NO: 8. The anti-CTHRCl antibody according to any one of claims 1-8, comprising a heavy chain variable region comprising SEQ ID NO: 9 and a light chain variable region comprising SEQ ID NO: 10. The anti-CTHRCl antibody according to any one of claims 1-16, wherein the anti- CTHRCl antibody is a chimeric, humanized, or human antibody. The anti-CTHRCl antibody according to any one of claims 1-15, wherein the anti- CTHRCl antibody is a monoclonal antibody. The anti-CTHRCl antibody according to any one of claims 1-15, wherein the anti- CTHRCl antibody is an antibody fragment. The anti-CTHRCl antibody according to any one of claims 1-15, wherein the anti- CTHRCl antibody comprises a single-chain antibody. The anti-CTHRCl antibody according to any one of claims 3-4, or 7, wherein the anti-CTHRCl antibody is a heavy-chain only antibody (single domain antibody). A modified immune cell, comprising a chimeric antigen receptor (CAR), wherein said CAR comprises the anti-CTHRCl antibody according to claim 19. The modified immune cell according to claim 21, wherein said modified immune cell is a modified T cell. The modified immune cell according to claim 21, wherein said modified immune cell is a modified NK cell. The modified immune cell according to claim 21, wherein said modified immune cell is a modified macrophage. An antibody-drug conjugate (ADC), comprising the antibody according to any one of claims 1-15. The ADC of claim 25, wherein said ADC is a radioconjugate. A method of inhibiting the growth of a cell that displays a CTHRC1 epitope, comprising contacting said cell with the anti-CTHRCl antibody according to any one of claims 1-15, the modified immune cell according to claim 25, or the ADC according to claim 25 or claim 26. A method of treating a subject having cancer, comprising administering to said subject the anti-CTHRCl antibody according to any one of claims 1-15, the modified immune cell according to claim 21, or the ADC according to claim 25 or claim 26. The method according to claim 28, wherein said subject is a human. The method according to claim 28, wherein said cancer is selected from the group consisting of adrenocortical cancer, bladder cancer, breast cancer, cervical cancer, cholangiocarcinoma, colon adenocarcinoma, B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, head and neck cancer, kidney clear cell cancer, kidney papillary cell cancer, myeloid leukemia, liver hepatocellular cancer, lung adenocarcinoma, lung squamous cancer, ovarian cancer, pancreatic adenocarcinoma, prostate adenocarcinoma, rectal adenocarcinoma, sarcoma, melanoma, stomach adenocarcinoma, testicular germ cell cancer, thymoma, uterine corpus, and uterine carcinosarcoma. A method of treating fibrosis and/or fibrotic disease in a subject in need thereof, comprising administering to said subject the anti-CTHRCl antibody according to any one of claims 1-15, the modified immune cell according to claim 21, or the ADC according to claim 25 or claim 26. The method according to claim 31, wherein said subject is a human.

149 A pharmaceutical composition comprising an antibody according to any one of claims 1-15, and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the modified immune cell according to claim 21, and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the ADC according to claim 25 or claim 26, and a pharmaceutically acceptable carrier. Use of the antibody according to any one of claims 1-15; the modified immune cell according to claim 21; or the ADC according to claim 26 or claim 27 in the preparation of a medicament for the treatment of cancer. Use of the antibody according to any one of claims 1-16; the modified immune cell according to claim 22; or the ADC according to claim 26 or claim 27 in the preparation of a medicament for the treatment of fibrosis and/or fibrotic disease.

150