Anti-CTHRC1 fusion proteins and methods of use thereof

Abstract

The present invention relates to anti-CTHRC1 fusion proteins, compositions comprising same, and methods of using such anti-CTHRC1 fusion proteins and compositions for the prevention and / or treatment of cancer, wherein the anti-CTHRC1 fusion protein comprises a first domain comprising a CTHRC1 binding moiety and a second domain comprising a T cell engager, preferably the T cell engager comprises a ligand for a receptor expressed on T cells.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 63 / 298,194, filed January 10, 2022, which is incorporated by reference in its entirety. [Background technology]

[0002] Lymphocyte activation typically requires the convergence of two signals. In T cells, the first signal results from activation of the antigen-specific T cell receptor triggered by the binding of the TCR to an MHC receptor presenting a neoantigen (in the context of cancer, these are mutated proteins) (Allison, McIntyre and Block, J. Immunology 129(5):2293-2300 (1982)). Following initial T cell receptor ligation and activation or priming, lymphocytes upregulate a series of surface receptors considered costimulatory molecules. These surface proteins bind to ligands on accessory cells to further enhance and sustain T cell function. CD28 was the first T cell costimulatory molecule discovered and remains the prototype of an immune activator (June et al., Molecular and Cellular Biology 7(12):4472-81 (1987)). CD28 is an activating receptor that binds to B7.1 and B7.2 ligands on antigen-presenting cells (APCs) (Hatchock, et al., J. Exp. Med. https: / / doi.org / 10.1084 / jem.180.2.631 (1994)). In combination with T cell receptor activation via neoantigen-loaded MHC class 2 receptors on APCs, this signal then induces differentiation of naive T cells into proliferating effector populations and release of proinflammatory cytokines that enhance the response.

[0003] A conceptually similar sequence of events occurs to drive NK cell activation. In NK cells, MHC class-1 receptors inhibit the activation of NK cell receptors independently of antigen. Thus, cells with low expression of MHC class-1 induce the first activation signal in NK cells. (Ljunggren and Karre, Immunology Today 11(7):237-44(1990)). Alternatively, the first signal can result from activation of the NKG2D receptor via MHC-like molecules, MICA / B and ULBP1-6 protein classes. (Schmiedel and Mandelboim, Frontiers in Immunology 9(September):2040(2018)). The second signal results from convergence of signaling from NK cell costimulatory receptors, e.g., KIR or NKP30 / NKP46, respectively, which can be either inhibitory or activating. (Chen and Flies, Nature Reviews. Immunology 13(4):227-42(2013)). Taken together, both signals induce antitumor immune responses leading to immune-mediated elimination of tumors, but sufficient signals from T / NK cell costimulatory receptors alone can induce a response. (Chen and Flies(2013)).

[0004] Costimulatory pathways are also conserved across multiple cell types, including, for example, the expression of the IgG receptor FcGRIIIA, whose binding leads to a strong inflammatory response (Bryceson et al., Immunological Reviews 214(December):73-91(2006)). Although generally considered a receptor on NK cells, this receptor is also expressed on macrophages, B cells, and CD8 T cells (Mellor, et al., Journal of Hematology & Oncology 6(January):1(2013)). Additional examples of costimulatory receptors with broad cell specificity are the TNF family receptors, such as GITR and 4-1-BB. These receptors induce activation of both T cells and NK cells (Barao, Frontiers in Immunology 3:402(2012)). On the other hand, the TNF family receptor CD27 is expressed on NK cells, T cells, and B cells. (Buchan, Rogel, and Al-Shamkhani, Blood 131(1):39-48(2018)). Thus, delivery of costimulatory agents can affect the activity of various cells of the immune system.

[0005] To prevent overactivation, innate feedback mechanisms induce the upregulation of inhibitory T cell costimulatory receptors, including PD1 and CTLA4, which suppress costimulatory signals and reduce the cell killing capacity of T cell populations (Chen and Flies (2013)), but may also facilitate the evasion of transformed tumor cells from immune-mediated destruction. Tumor cells also shed ligands for NK cell receptors, which have been suggested to downregulate costimulatory molecules and reduce cytotoxic function. Thus, while mutations leading to tumorigenesis have the potential to induce neoantigens capable of eliciting immune responses, tumors as a whole have been shown to downregulate the expression of costimulatory molecule ligands at either the transcript or protein level to evade immune surveillance. CD28, the prototype of the T cell costimulatory receptor, has been shown to be a primary target of CTLA-4 as well as PD-1-mediated T cell exhaustion, either by direct competition for its ligand CD80 / 86 by CTLA-4 or via PD-1-mediated signaling suppression to limit activation. (Hui, et al., Science 355(6332):1428-33(2017)). Signaling through other T cell costimulatory molecules, notably 4-1BB and ICOS, has been suggested to remain intact even in the context of PD-1-mediated exhaustion. (Habib-Agahi, Jaberipour, and Searle, Cellular Immunology 256(1-2):39-46(2009)).

[0006] Providing additional costimulation has long been considered a promising therapeutic strategy for treating cancer as well as several other indications. Across multiple costimulatory molecules, activation of 4-1BB has been suggested as a feasible therapeutic strategy since 4-1BBL treatment has been shown to restore CD28 expression in an in vitro system (Habib-Agahi, Jaberipour, and Searle (2009)). Agonistic antibodies against 4-1BB as well as other TNFα family members (OX40, GITR, CD40) have been developed and undergone preclinical testing, with several of these progressing into clinical development. Additional preclinical and clinical development programs target the costimulatory molecules ICOS, CD27, and CD28. Unfortunately, however, preclinical success has not translated to the clinic for these antibodies, which may be due to a narrow window for maximizing therapeutic efficacy while limiting the systemic toxicity associated with global immune activation. Various promising clinical trials have been unsuccessful, many of which were due to the development of treatment-related AEs. For example, in a study of healthy volunteers treated with a superagonist against the B7.1 and 2 receptor CD28, cytokine release syndrome was observed in all patients who received this therapy, indicating severe toxicity. (Hunig, The FEBS Journal 283(18):3325-34(2016)). Agonists against OX40, CD40, and 4-1BB also suffered from a high incidence of treatment-related adverse events, which negatively affected their clinical development (Segal, et al., Clinical Cancer Research: An Official Journal of the American Association for Cancer Research 23(8):1929-36(2017)). For example, CD40 agonists were observed to have high AE rates in Phase 1 safety studies at some subeffective doses. (Calvo, et al., Journal of Clinical Orthodontics: JCO 37(15_suppl):2527-2527(2019)).

[0007] Taken together, this indicates that the natural window for lymphocyte-specific activation in tumors may be higher than that which induces systemic effects of costimulatory molecule activation, and suggests that targeted delivery may be a necessary route in the future to accumulate drugs and deliver stimulation at tumor sites. Tethering of costimulatory molecules via tumor antigens may also provide the additional cross-linking required to drive costimulatory activity that may be limited when relying on Fc interactions in the context of agonistic mAbs.

[0008] Tumor stroma represents a major barrier for antitumor immunity and therapeutic targeting, and the stroma is primarily considered a major immunosuppressive component that promotes resistance to immune checkpoint inhibitors. Tumor-targeting ligands offer an innovative solution to alleviate stroma-mediated immunosuppression and may offer several advantages. For example, cancer cells can often rapidly evolve to downregulate / upregulate many specific targets, while the genetic information of fibroblasts / macrophages is stable. As a result, fibroblast activation protein, a highly selective stromal target, has been pioneered as a means to target T-cell and NK-cell costimulatory receptor agonists to the tumor microenvironment.

[0009] Yet, to date, readouts from FAP-targeted IO therapy trials have been mixed, with simlukafusp alpha (Fap-targeted IL-2) failing to demonstrate clear tumor regression as a single agent or provide much additive activity to anti-PD-L1 alone ("ASCO 2021: Randomized Phase Ib Study To Evaluate Safety, Pharmacokinetics and Therapeutic Activity of Simlukafusp α in Combination With Atezolizumab ± Bevacizumab in Patients With Unresectable Advanced / Metastatic Renal Cell Carcinoma (Rcc) (NCT03063762)" (undated)). Although future studies exploring alternative combination treatments may improve outcomes, the limitations associated with the activity of FAP blockade alone, coupled with the fact that FAP-targeted agents likely only bind to CAFs, indicate that new inventions are needed to effectively alleviate stromal-mediated immune suppression while simultaneously targeting cancer cells. Successful relief of stromal-mediated immunosuppression in concert with activation of immune cells to directly attack cancer cells addresses the challenge of targeting solid tumors and therefore presents an opportunity to improve outcomes for patients with these diseases. The present invention addresses this and other unmet needs. Summary of the Invention

[0010] As demonstrated for the first time herein, antibodies developed against the stromal target CTHRC1 show activity in reprogramming the tumor microenvironment, as well as efficacy in combination with PD1 inhibitors. CTHRC1 mRNA expression is also highly selective to tumors, with expression seen in both CAFs and, to a lesser extent, cancer cells. Given the selective localization of CTHRC1 to tumors, the use of CTHRC1-based agents to target costimulatory ligands to the tumor microenvironment provides a uniquely effective solution to the above-mentioned problems in the prior art.

[0011] To this end, the present disclosure provides anti-CTHRC1 fusion proteins and compositions thereof, the anti-CTHRC1 fusion proteins comprising a collagen triple helical repeat containing 1 (CTHRC) binding moiety and a T cell engager, preferably the T cell engager comprising a ligand for a receptor expressed on a T cell. In some embodiments, the T cell engager comprises a ligand for a costimulatory receptor, or a functional fragment, variant, or mutant thereof. In some embodiments, the costimulatory receptor is a tumor necrosis factor superfamily (TNFSF) member or an immunoglobulin superfamily (IgSF) member. In some embodiments, the costimulatory receptor is selected from the group consisting of 4-1BB (CD137), OX40 (CD134), CD40, CD27, CD28, herpes virus entry mediator (HVEM), ICOS, CD2, and GITR. The present disclosure further provides methods for making the same and uses thereof in the treatment of cancer.

[0012] In one aspect, an anti-collagen triple helix repeat containing 1 (anti-CTHRC1) fusion protein comprises (a) a first domain comprising a CTHRC1 binding moiety, and (b) a second domain comprising a T cell engager, preferably the T cell engager comprises a ligand for a receptor expressed on T cells.

[0013] In embodiments, the T cell engager comprises a ligand for a costimulatory receptor expressed on the T cell, or a functional fragment, variant, or mutant thereof, preferably where the costimulatory receptor is a member of the tumor necrosis factor superfamily (TNFSF).

[0014] In some embodiments, the costimulatory receptor is selected from the group consisting of 4-1BB (CD137), OX40 (CD134), CD40, CD27, CD28, herpes virus entry mediator (HVEM), ICOS, CD2, and GITR.

[0015] In several embodiments, when the costimulatory receptor is 4-1BB (CD137), preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1-10, or a functional fragment thereof.

[0016] In several embodiments, when the costimulatory receptor is OX40 (CD134), preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 11-13, or a functional fragment thereof.

[0017] In embodiments, when the costimulatory receptor is CD40, preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 14-16, or a functional fragment thereof.

[0018] In several embodiments, when the costimulatory receptor is GITR, preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 17-18, or a functional fragment thereof.

[0019] In several embodiments, when the costimulatory receptor is CD27, preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 19-20, or a functional fragment thereof.

[0020] In several embodiments, when the costimulatory receptor is HVEM, preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 21-23, or a functional fragment thereof.

[0021] In several embodiments, when the costimulatory receptor is ICOS, preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 30-31, or a functional fragment thereof.

[0022] In several embodiments, when the costimulatory receptor is CD28, preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 32-35, or a functional fragment thereof.

[0023] In several embodiments, when the costimulatory receptor is CD2, preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 39-40, or a functional fragment thereof.

[0024] In embodiments, the anti-CTHRC1 fusion protein further comprises a linker positioned between the CTHRC1 binding moiety and the ligand, preferably the linker is (G4S) n or (GSG) n (wherein n is 1 to 4).

[0025] In embodiments, the anti-CTHRC1 binding moiety comprises an anti-CTHRC1 antibody that binds to CTHRC1, preferably the CTHRC1 antibody binds to CTHRC1 with a binding affinity of less than 10 nM.

[0026] In some embodiments, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising the sequence of SEQ ID NO: 46, a CDR2 sequence comprising the sequence of SEQ ID NO: 51, and a CDR3 sequence comprising the sequence of SEQ ID NO: 56, and a light chain variable region comprising a CDR1 sequence comprising the sequence of SEQ ID NO: 61, a CDR2 sequence comprising the sequence of SEQ ID NO: 66, and a CDR3 sequence comprising the sequence of SEQ ID NO: 71.

[0027] In embodiments, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO:77 and a light chain variable region comprising SEQ ID NO:78.

[0028] In some embodiments, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising the sequence of SEQ ID NO: 45, a CDR2 sequence comprising the sequence of SEQ ID NO: 50, and a CDR3 sequence comprising the sequence of SEQ ID NO: 55, and a light chain variable region comprising a CDR1 sequence comprising the sequence of SEQ ID NO: 60, a CDR2 sequence comprising the sequence of SEQ ID NO: 65, and a CDR3 sequence comprising the sequence of SEQ ID NO: 70.

[0029] In embodiments, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO:75 and a light chain variable region comprising SEQ ID NO:76.

[0030] In embodiments, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 75, 77, 79, 81, and 83.

[0031] In embodiments, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from Table 4.

[0032] In embodiments, the anti-CTHRC1 antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 78, 80, 82, and 84.

[0033] In embodiments, the anti-CTHRC1 antibody comprises a light chain variable region comprising an amino acid sequence selected from Table 5.

[0034] In some embodiments, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 45-49, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 50-54, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 55-59.

[0035] In several embodiments, the anti-CTHRC1 antibody comprises a light chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 60-64, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 65-69, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 70-74.

[0036] In embodiments, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO:75 and a light chain variable region comprising SEQ ID NO:76.

[0037] In embodiments, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO:79 and a light chain variable region comprising SEQ ID NO:80.

[0038] In embodiments, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO:81 and a light chain variable region comprising SEQ ID NO:82.

[0039] In embodiments, the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO:83 and a light chain variable region comprising SEQ ID NO:84.

[0040] In embodiments, the anti-CTHRC1 antibody is a chimeric, humanized, or human antibody.

[0041] In embodiments, the anti-CTHRC1 antibody is a monoclonal antibody.

[0042] In embodiments, the anti-CTHRC1 antibody is an antibody fragment.

[0043] In embodiments, the anti-CTHRC1 antibody comprises a single chain antibody.

[0044] In embodiments, the anti-CTHRC1 antibody is a heavy chain only antibody (single domain antibody).

[0045] In embodiments, the method of activating T cells in a tumor microenvironment comprises contacting the tumor with an anti-CTHRC1 fusion protein or any of the above-described embodiments.

[0046] In embodiments, a method of inhibiting the growth of a cell displaying a CTHRC1 tumor epitope comprises contacting the cell with an anti-CTHRC1 fusion protein of any of the above embodiments.

[0047] In embodiments, a method of treating a subject having cancer comprises contacting a cell with an anti-CTHRC1 fusion protein of any of the above embodiments.

[0048] In some embodiments, the cancer is selected from the group consisting of breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, renal cancer, kidney cancer, liver cancer, head and neck cancer, colorectal cancer, melanoma, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, and myeloma.

[0049] In embodiments, the methods described above may further include administering an allogeneic or autologous T cell therapy in combination with the anti-CTHRC1 fusion protein of any of the above embodiments.

[0050] In embodiments, a pharmaceutical composition comprises an anti-CTHRC1 fusion protein of any of the above embodiments and a pharma- ceutical acceptable carrier.

[0051] In embodiments, the use of the pharmaceutical composition of the above embodiments may be in the preparation of a medicament for the treatment of a cell proliferative disorder, preferably cancer. [Brief description of the drawings]

[0052] [Figure 1A] Illustrated is a sample from a patient with colorectal cancer stained with FAP showing the tumor and stroma. [Figure 1B] Illustrates a sample from a patient with colorectal cancer stained with anti-CTHRC1 antibody showing the tumor and stroma. [Figure 2A] Illustrates FACS data for staining of cancer cell lines with anti-CTHRC1 antibody. [Figure 2B] Illustrates FACS data for staining of cancer cell lines with anti-CTHRC1 antibody. [Figure 3A]The extent of proliferation is shown for Jurkat T cells stained with CFSE. Two-way ANOVA, *p<0.5, ****p<0.001. All h41BBL vs. M23-h41BBL, not significant. [Figure 3B] Illustrates flow cytometry data for Jurkat T cells. [Figure 4] Illustrates tumor volume over time for mice treated with control (Dengue-41BBL) or anti-CTHRC1 fusion protein (M23-41BBL). [Diagram 5] Survival curves are depicted for mice treated with control (Dengue-41BBL), anti-CTHRC1 fusion protein (M23-41BBL) or anti-CTHRC1 fusion protein (M5-41BBL (low dose)). [Figure 6] Illustrates tumor volume over time for mice treated with control (Dengue-41BBL), anti-CTHRC1 fusion protein (M23-41BBL) or anti-CTHRC1 fusion protein (M5-41BBL). [Figure 7] Illustrates tumor volume over time for mice treated with control (isotype control) or various doses of anti-CTHRC1 fusion protein (M23-41BBL). [Figure 8A] Figure 1 illustrates that CTHRC1 mRNA is a top marker for cancer-associated fibroblasts (CAFs) in cancer-rich and immune-cold tumor samples. Aggregated tumor samples were profiled across cancer scRNA studies to generate a large scRNA atlas. Samples were then grouped into T cell-rich and cancer-poor samples (immune-hot), and cancer-rich and T cell-poor samples (immune-cold). [Figure 8B]Figure 1 illustrates that CTHRC1 mRNA is a top marker for cancer-associated fibroblasts (CAFs) in cancer-enriched and immunocold tumor samples. Genes expressed by CAFs were compared between the two groups to determine which were most associated with cancer-enriched samples (Wilcoxon rank). These genes were then narrowed down to genes specifically expressed by CAFs in all samples (top 500; Wilcoxon rank). CTHRC1 was the top 11 gene in this analysis. [Figure 9] Figure 1 illustrates that CTHRC1 is highly upregulated in cancer versus normal adjacent tissues as determined in bulk RNA measurements analyzed from The Cancer Genome Atlas. Across the set of profiled indications, the highest levels of CTHRC1 expression were found in solid cancers, particularly breast, lung, ovarian, pancreatic, sarcoma, melanoma, and uterine carcinosarcoma (P<0.001 in all cases tested). Taken together, this indicates that CTHRC1 expression is highest in cancers rich in desmoplastic stroma, consistent with the idea that it is a target primarily secreted by CAFs. [Figure 10] 1 illustrates a series of survival plots showing CTHRC1 survival curves for several solid tumors where CTHRC1 is associated with poor survival. Values ​​were obtained from The Cancer Genome Atlas. Survival curves were calculated and plotted using the online tool GEPIA. [Figure 11] Illustrates that CTHRC1 levels increase with disease 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 stages I and IV. Stages II and III show intermediate CTHRC1 expression levels. [Figure 12A]12A-12C are histograms showing bulk-RNA expression levels of known stromal targets FAP (FIG. 12A) and LRRC15 (FIG. 12B) aligned with CTHRC1 (FIG. 12C) in pancreatic cancer samples (The Cancer Genome Atlas) versus 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 the known / explored stromal targets FAP and LRRC15. [Figure 12B] 12A-12C are histograms showing bulk-RNA expression levels of known stromal targets FAP (FIG. 12A) and LRRC15 (FIG. 12B) aligned with CTHRC1 (FIG. 12C) in pancreatic cancer samples (The Cancer Genome Atlas) versus 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 the known / explored stromal targets FAP and LRRC15. [Figure 12C] 12A-12C are histograms showing bulk-RNA expression levels of known stromal targets FAP (FIG. 12A) and LRRC15 (FIG. 12B) aligned with CTHRC1 (FIG. 12C) in pancreatic cancer samples (The Cancer Genome Atlas) versus 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 the known / explored stromal targets FAP and LRRC15. [Figure 13]This data set illustrates that very high levels of CTHRC1 expression are seen in CAFs across many solid cancers, as well as in cancer epithelium expression in breast, pancreatic, lung, ovarian, and skin cancers. In contrast, minimal CTHRC1 expression is seen in normal tissues. The data was obtained based on a previously generated large-scale integrated single-cell RNA sequencing atlas to enable probing of gene expression at the single-cell level across 11 toxic cancer and normal tissue samples (Swechha, 2021). The data highlight the potentially large therapeutic window for blocking CTHRC1 and the value of using mAbs against CTHRC1 to target payloads, such as ADCs, to the tumor microenvironment. [Figure 14] A dataset using the same atlas discussed with respect to FIG. 10 was performed against LRRC15, a known non-toxic stromal target, where an antibody ADC has been engineered and shown to be safe in clinical settings. Low levels of LRRC15 were found in normal tissues compared to CTHRC1. In a cancer single cell RNA (scRNA) dataset, it was observed that LRRC15 expression, unlike CTHRC1, was preferentially localized by certain CAFs in certain breast cancers, and low levels of expression were also seen on sarcoma cancer cells. [Figure 15] Graph showing quantitative ELISA of CTHRC1 in monocultures and cocultures. Supernatants from different sets of monocultures (fibroblasts or cancer cells) and cocultures (fibroblasts and cancer cells) were profiled for CTHRC1 levels. CTHRC1 was expressed at low levels in fibroblast monocultures (BJ, CCD18-Co) and upregulated in cocultures, indicating that interactions between fibroblasts and cancer cells drive CTHRC1 expression. [Figure 16] A series of images of tissues from three mouse models probed with CTHRC1-specific mAb are shown. Extensive staining was observed within the tumor area, indicating that CTHRC1 protein localizes to cancer areas in vivo. [Figure 17] A series of images of tissues from three human cancers showing variations in CTHRC1 expression patterns at the protein level, indicating different expression kinetics. In head and neck and melanoma cancer samples, CTHRC1 is localized at the border between cancer and stromal tissue. In pancreatic cancer, CTHRC1 is seen to be expressed broadly over areas of high CAF / stromal tissue density. [Figure 18A] 1 illustrates the efficacy of CTHRC1 tested in the syngenic mouse breast tumor model EMT6. Results are shown for anti-CTHRC1 antibodies, specifically M5. [Figure 18B] Illustrates the efficacy of CTHRC1 tested in the syngenic mouse breast tumor model EMT6. Results are shown for anti-CTHRC1 antibodies, specifically M14. [Figure 18C] Illustrates the efficacy of CTHRC1 tested in the syngenic mouse breast tumor model EMT6. Results are shown for anti-CTHRC1 antibodies, specifically M14. [Figure 19A] Illustrates efficacy of anti-CTHRC1 (clone M5) in a PD-1 resistant Pan02 pancreatic cancer model. [Figure 19B] Illustrates efficacy of anti-CTHRC1 (clone M5) in a PD-1 resistant Pan02 pancreatic cancer model. [Figure 20] 1 illustrates the effect of pretreatment of cells with anti-CTHRC1 antibody on CD8 T cell infiltration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] The present disclosure provides anti-CTHRC1 fusion proteins and compositions thereof, the anti-CTHRC1 fusion proteins comprising a collagen triple helix repeat containing 1 (CTHRC) binding moiety and a T cell engager, e.g., a ligand for a receptor expressed on a T cell. In some embodiments, the T cell engager comprises a ligand for a costimulatory receptor, or a functional fragment, variant, or mutant thereof. In some embodiments, the T cell engager comprises a cytokine, e.g., IL-2, or a functional fragment, variant, or mutant thereof. The present disclosure further provides methods for making the same and uses thereof in the treatment of cancer.

[0054] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of a person of ordinary skill in the art. Such techniques are fully explained in such publications as "Molecular Cloning: A Laboratory Manual", 2nd edition (Sambrook et al., 1989), "Oligonucleotide Synthesis" (MJ Gait, ed., 1984), "Animal Cell Culture" (RI Freshney, ed., 1987), "Methods in Enzymology" (Academic Press, Inc.), "Current Protocols in Molecular Biology" (FMA Usubel et al., eds., 1987, and periodically updated versions), "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994), "A Practical Guide to Molecular Cloning" (Perbal Bernard V., 1988), and "Phage Display: A Laboratory Manual" (Barbas et al., 2001).

[0055] 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 as follows.

[0056] definition For purposes of interpreting this specification, the following definitions shall apply, and whenever appropriate, terms used in the singular shall also include the plural and vice versa. In the event that any definition set forth conflicts with any document incorporated herein by reference, the definition set forth below shall control.

[0057] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0058] Use of the term "or" in the claims and this disclosure is intended to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive.

[0059] The use of the term "about" when used in conjunction with a numerical value is intended to include + / - 10%. By way of non-limiting example, if a number of amino acids are specified as "about 100", this would include 90 to 110 (plus or minus 10%).

[0060] As used herein, "comprising," "including," "containing," "having," and the like are intended to be open-ended and inclusive so as not to exclude additional unrecited elements, unless otherwise noted.

[0061] The term "collagen triple helix repeat containing 1 (CTHRC1)" as used herein, unless otherwise indicated, refers to any naturally occurring CTHRC1 from any vertebrate source, including mammals such as primates (e.g., humans, primates, and rodents (e.g., mice and rats). The term encompasses several isoforms (see, e.g., SEQ ID NOs: 85-87). Human CTHRC1 is encoded by a nucleotide sequence corresponding to GenBank Accession No. NG031985.

[0062] The term "collagen triple helix repeat-containing 1" encompasses "full-length" unprocessed CTHRC1 as well as any form of CTHRC1 resulting from processing in cells. The term encompasses naturally occurring variants of CTHRC1, such as splice variants, allelic variants, and isoforms. The CTHRC1 polypeptides described herein may be isolated from various sources, such as from human tissue types or from another source, or may be prepared by recombinant or synthetic methods. A "native sequence CTHRC1 polypeptide" includes a polypeptide having the same amino acid sequence as the corresponding CTHRC1 polypeptide derived from nature. Such native sequence CTHRC1 polypeptides may be isolated from nature or produced by recombinant or synthetic means. The term "native sequence CTHRC1 polypeptide" specifically encompasses naturally occurring truncated or secreted forms of a particular CTHRC1 polypeptide (e.g., extracellular domain sequences), naturally occurring variant forms of the polypeptide (e.g., alternatively spliced ​​forms), and naturally occurring allelic variants. In certain embodiments of the invention, the native sequence CTHRC1 polypeptides disclosed herein are mature or full-length native sequence polypeptides comprising the full-length amino acid sequence set forth in the accompanying disclosure.

[0063] As used herein, "costimulatory receptor" refers to a cognate binding partner on an immune cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the cell, such as, but not limited to, proliferation. Costimulatory receptors are cell surface molecules other than antigen receptors or their ligands that are required for an efficient immune response. Costimulatory receptors include MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDS, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB (CD137), B7-H3, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, I TGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(C D229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD48, STING, and ligands that specifically bind to CD83.

[0064] As used herein, "ligand" refers to an agent that binds to a native receptor under normal physiological conditions (eg, 4-1BBL).

[0065] A "fragment" is a portion of a polypeptide that includes a region that contains at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or any range therebetween of a reference sequence and retains at least a portion of the binding properties of the reference sequence. For example, a fragment of 4-1BB ligand (4-1BBL) can still bind to the 4-1BB receptor with at least some, the same, or greater affinity than the original 4-1BBL sequence.

[0066] A "variant" is a polypeptide that contains substitutions, insertions, or deletions compared to a reference sequence and retains at least a portion of the binding properties of the reference sequence. For example, a variant of 4-1BBL can still bind to the 4-1BB receptor with at least some, the same, or greater affinity than the original 4-1BBL sequence.

[0067] The "identity" percentage between an amino acid sequence and a reference sequence is defined as the percentage of amino acid residues in a polypeptide sequence that are identical to the amino acid residues in the reference sequence after aligning the sequences and introducing gaps, if necessary, to obtain the maximum sequence identity percentage. Alignment for the purpose of determining amino acid sequence identity percentage can be achieved in various ways that are within the skill of the person skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, or MUSCLE software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithm required to obtain maximum alignment over the entire length of the sequences being compared. In certain embodiments, default parameters are used.

[0068] As used herein, "modification" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, where the modification results from a change in the sequence that includes the amino acid residue / position. For example, typical modifications include substitution of the residue (or at the position) with another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more (usually less than 5 or 3) amino acids adjacent to the residue / position, and deletion of the residue / position. An "amino acid substitution" or variant thereof refers to the replacement of an existing amino acid residue in a given (starting) amino acid sequence with a different amino acid residue. Generally, the modification results in a change in at least one physico-biochemical activity of the variant polypeptide compared to the polypeptide that includes the starting (or "wild type") amino acid sequence. For example, in the case of an antibody, the physico-biochemical activity that is changed can be binding affinity, binding ability, and / or binding effect for a target molecule.

[0069] The term "antibody" is used in the broadest sense and specifically encompasses, for example, single anti-CTHRC1 monoclonal antibodies (including agonist, antagonist, neutralizing antibodies, full-length or intact monoclonal antibodies), anti-CTHRC1 antibody compositions with polyepitopic specificity, polyclonal antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies, so long as they exhibit the desired biological activity), single-chain anti-CTHRC1 antibodies, as well as fragments of anti-CTHRC1 antibodies, including Fab, Fab', F(ab')2, and Fv fragments (see below), diabodies, single domain antibodies (sdAbs), so long as they exhibit the desired biological or immunological activity. Also included among the anti-CTHRC1 antibodies, and in particular among the fragments, are portions of the anti-CTHRC1 antibodies (and combinations of portions of the anti-CTHRC1 antibodies, e.g., scFvs) that can be used as targeting arms, e.g., directed to the CTHRC1 tumor epitope, in the chimeric antigen receptor of CAR-T cells, CAR-NK cells, or CAR-macrophages. Such fragments are not necessarily proteolytic fragments, but rather portions of a polypeptide sequence that can confer affinity to a target. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein. The antibody can be, for example, a human antibody, a humanized antibody, and / or an affinity matured antibody.

[0070] The terms "anti-CTHRC1 antibody," "CTHRC1 antibody," and "antibody that binds CTHRC1" are used interchangeably. The anti-CTHRC1 antibody, whether isolated or as part of a fusion protein, cell, or cell composition, is preferably capable of binding with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent.

[0071] In one embodiment, a CTHRC1 antibody is used herein to specifically refer to an anti-CTHRC1 monoclonal antibody that (i) comprises a heavy chain variable domain of any one of SEQ ID NOs: 75, 77, 79, 81, and 83, and / or a light chain variable domain of any one of SEQ ID NOs: 76, 78, 80, 82, and 84, or (ii) comprises one, two, three, four, five, or six of the CDRs shown in Table 2 or Table 3.

[0072] An "isolated antibody" is one that has been identified, separated and / or recovered from components of its natural environment that are materials that would interfere with therapeutic use of the antibody, and which may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

[0073] An "intact" antibody is one that comprises an antigen-binding site as well as a CL and at least heavy chain constant domains CH1, CH2, and CH3. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the intact antibody has one or more effector functions.

[0074] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies constituting the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they may be synthesized without contamination by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma technique first described by Kohler et al., Nature, 256:495 (1975), or may be made using recombinant DNA methods (e.g., 4,816,567) in bacterial, eukaryotic, or plant cells. 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.

[0075] The basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. For IgG, the four-chain unit is generally about 150,000 daltons. Each L chain is linked to the H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced interchain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus, followed by three constant domains (CH) for each of the α and γ chains, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end. The VL is aligned with the VH, and the CL is aligned with the first constant domain (CH1) of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain. The pairing of VH and VL together forms a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, page 71 and chapter 6 of Basic and Clinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr and Tristram G. Parslow (eds.), Appleton & Lange, Norwalk, CT, 1994.

[0076] L chains from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the constant domain (CH) of their heavy chains, immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated α, δ, ε, γ, and μ, respectively. The γ and α classes are further divided into subclasses based on relatively minor differences in CH sequence and function, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0077] 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 is designated "VH" or "V H The variable domain of the light chain is sometimes referred to as a "VL" or "V L These domains are generally the most variable parts of the antibody and contain the antigen-binding sites.

[0078] The term "variable" refers to the fact that certain segments of the variable domains differ significantly in sequence among antibodies. The V domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across the 110 amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches of 15-30 amino acids called framework regions (FRs) separated by shorter regions of extreme variability called "hypervariable regions", each 9-12 amino acids long. Naturally occurring heavy and light chain variable domains each contain four FRs that adopt a predominantly β-sheet configuration, connected by three hypervariable regions that form loops connecting, and in some cases forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by 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)).

[0079] An "antibody fragment" includes a portion of an intact antibody, preferably an antigen-binding region or one or more variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-62 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment includes the antigen-binding site of the intact antibody and thus retains the ability to bind to antigen. Anti-CTHRC1 antibody fragments also include portions of anti-CTHRC1 antibodies (and combinations of portions of anti-CTHRC1 antibodies, e.g., scFvs) that can be used as targeting arms, e.g., directed to the CTHRC1 tumor epitope, in the chimeric antigen receptor of CAR-T cells or CAR-NK cells, or CAR macrophages. Such fragments are not necessarily proteolytic fragments, but rather are portions of a polypeptide sequence that may confer affinity for a target.

[0080] Papain digestion of an antibody results in 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 the entire L chain plus the variable region domain of the H chain (VH) and the first constant domain (CH1) of one heavy chain. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of an antibody produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of cross-linking antigen. The Fab' fragment differs from the Fab fragment by having a few additional residues at the carboxy terminus of the CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments which have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0081] The Fc fragment contains the carboxy-terminal portions of both H chains held together by disulfides. The effector functions of the antibody are determined by sequences in the Fc region, which is also the region recognized by Fc receptors (FcRs) found on certain types of cells.

[0082] An "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. This fragment consists of a dimer of one heavy chain variable region domain and one light chain variable region domain in tight non-covalent association. In a single chain Fv (scFv) species, one heavy chain variable domain and one light chain variable domain can be covalently linked by a flexible peptide linker so that the light and heavy chains can associate in a "dimeric" structure similar to that in a two-chain Fv species. The folding of these two domains results in six hypervariable loops (three loops each from the H and L chains) that provide the amino acid residues for antigen binding to the antibody and confer antigen binding specificity. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to an antigen, albeit with a lower affinity than the complete binding site.

[0083] A "single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. For a review of sFv, see, for example, 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 scFv derived from an anti-CTHRC1 antibody is used as a targeting arm of a CAR-T cell, CAR-NK cell, or CAR-macrophage disclosed herein.

[0084] The term "hypervariable region", "HVR" or "HV" as used herein refers to a region of an antibody variable domain that is highly variable in sequence and / or forms structurally defined loops. Generally, antibodies contain six hypervariable regions, three in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Several hypervariable region demarcations are in use and are encompassed herein. The Kabat complementarity determining regions (CDRs) are the most commonly used, based on sequence variability (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). Chothia instead refers to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The ends of the Chothia CDR-H1 loops, when numbered using the Kabat numbering convention, vary between H32 and H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable regions are based on an analysis of the available complex crystal structures. Residues from each of these hypervariable regions are listed below. [Table 1]

[0085] The hypervariable regions may include "extended hypervariable regions" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 (L3) in VL, and 26-35B (H1), 50-65, 47-65, or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in VH. The variable domain residues are numbered according to Kabat et al. (supra) for each of these definitions.

[0086] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0087] The terms "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat", and variations thereof, refer to the numbering system used for the heavy or light chain variable domains of the collection 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, the FRs or CDRs of the variable domain. For example, a heavy chain variable domain may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c, etc. according to Kabat). The Kabat numbering of residues may be determined for a given antibody by alignment of the antibody's sequence with the "standard" Kabat numbered sequence at the regions of homology.

[0088] The Kabat numbering system is generally used when referring to residues in the variable domain (approximately residues 1-107 in the light chain and residues 1-113 in the heavy chain) (e.g., Kabat et al., supra). The "EU numbering system" or "EU index" is generally used when referring to residues in an immunoglobulin heavy chain constant region (e.g., the EU index as reported in Kabat et al., supra). "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody. Unless otherwise specified herein, reference to residue numbers in the variable domain of an antibody means residue numbering according to the Kabat numbering system.

[0089] An antibody that "binds" to an antigen or epitope of interest is one that binds to the antigen or epitope with sufficient affinity that it is measurably different from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule (which is generally a molecule of similar structure that has no binding activity).

[0090] An antibody that inhibits tumor cell growth is an antibody that results in measurable growth inhibition of cancer cells. In one embodiment, an anti-CTHRC1 antibody can inhibit the growth of cancer cells presenting a CTHRC1 tumor epitope. As referred to herein, a CTHRC1 tumor epitope includes a CTHRC1 epitope to which an anti-CTHRC1 antibody as disclosed herein, or a fragment thereof, can bind, or to which an antibody or other molecule that competes with an anti-CTHRC1 antibody as disclosed herein for binding to the epitope can at least partially bind. A preferred growth-inhibitory anti-CTHRC1 antibody inhibits the growth of CTHRC1-presenting tumor cells by more than 20%, preferably about 20% to about 50%, and even more preferably more than 50% (e.g., about 50% to about 100%), compared to a suitable control, which is typically a tumor cell that is not treated with the antibody being tested.

[0091] Anti-CTHRC1 antibodies may (i) inhibit tumor metastasis in vivo, (ii) inhibit tumor growth in vivo, (iii) reduce tumor size in vivo, (iv) inhibit tumor angiogenesis in vivo, (v) exhibit cytotoxic activity against tumor cells and cancer-associated fibroblasts presenting CTHRC1 in vivo, (vi) exhibit cytostatic activity against tumor cells or cancer-associated fibroblasts presenting CTHRC1 in vivo, (vii) enhance the infiltration of anti-tumor immune cells in vivo, or (viii) prevent the suppression of immune cells in the tumor microenvironment in vivo.

[0092] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. A "tumor" contains one or more cancerous cells. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), skin cancer, melanoma, lung cancer, including small cell lung cancer, non-small cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous cell carcinoma of the lung, cancer of the peritoneum, hepatocellular carcinoma, gastric cancer, including gastrointestinal cancer, pancreatic cancer (e.g., pancreatic ductal adenocarcinoma), glioblastoma, cervical cancer, ovarian cancer (e.g., high-grade serous ovarian cancer), liver cancer (e.g., hepatocellular carcinoma (HCC)), bladder cancer (e.g., urothelial bladder cancer), testicular (germ cell tumor) cancer, hepatocellular carcinoma, breast cancer, brain cancer (e.g., astrocytoma), colon cancer, rectal cancer, colorectal cancer, endometrial cancer or uterine cancer, salivary gland cancer, kidney cancer or renal cancer, and the like. Cancers include, but are not limited to, adrenal cortical carcinoma, cholangiocarcinoma, colon adenocarcinoma, B-cell lymphoma, esophageal carcinoma, glioblastoma multiforme, renal clear cell carcinoma, papillary renal cell carcinoma, myeloid leukemia, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, rectal adenocarcinoma, sarcoma, gastric adenocarcinoma, thymoma, uterine corpus, and uterine carcinosarcoma.

[0093] The term "metastatic cancer" refers to a cancer condition in which cancer cells from a primary tissue spread by blood or lymphatic vessels from the primary site to one or more other sites in the body and form one or more secondary tumors in one or more organs in addition to the primary tissue. A prominent example is metastatic breast cancer.

[0094] As used herein, a "CTHRC1-associated cancer" is a cancer associated with overexpression of the CTHRC1 gene or gene product and / or associated with presentation of a CTHRC1 epitope. Suitable control cells can be, for example, cells from an individual not afflicted with cancer, or non-cancerous cells from a subject with cancer.

[0095] Anti-CTHRC1 fusion proteins may (i) inhibit tumor metastasis in vivo, (ii) inhibit tumor growth in vivo, (iii) reduce tumor size in vivo, (iv) inhibit tumor angiogenesis in vivo, (v) exhibit cytotoxic activity against tumor cells and cancer-associated fibroblasts presenting CTHRC1 in vivo, (vi) exhibit cytostatic activity against tumor cells or cancer-associated fibroblasts presenting CTHRC1 in vivo, (vii) enhance the infiltration of anti-tumor immune cells in vivo, or (viii) prevent the suppression of immune cells in the tumor microenvironment in vivo.

[0096] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer.

[0097] "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.

[0098] The terms "patient," "subject," "individual," and the like are used interchangeably herein and refer to any animal amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0099] The term "therapeutically effective amount" or simply "effective amount" refers to an amount of an agent or composition (e.g., a composition containing an agent) that elicits a biological or medical response in a tissue, system, or subject that is desired by a researcher, veterinarian, physician, or other clinician. The term "therapeutically effective amount" includes an amount of an agent or a composition containing an agent that, when administered, is sufficient to prevent or alleviate to some extent one or more of the manifestations of the signs or symptoms of the disorder or disease (e.g., hematological or solid tumor) being treated. The therapeutically effective amount will vary depending on the composition, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0100] As used herein, the term "administration" means providing or giving to a subject, by any effective route, one or more agents, such as agents to treat one or more signs or symptoms associated with a condition / disorder or disease, including, but not limited to, cancer (e.g., lymphoma), viral infection, bacterial infection, etc. Exemplary routes of administration include, but are not limited to, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes. Administration "in combination with" one or more additional therapeutic agents includes simultaneous (concurrent) administration and sequential administration in any order.

[0101] The term "pharmaceutical acceptable" as used herein refers to a material, including but not limited to salts, carriers, or diluents, that does not abrogate the biological activity or properties of a compound and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesired biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. Pharmaceutically acceptable carriers (vehicles) useful in the present disclosure are conventional. Remington's Pharmaceutical Sciences, 19th Edition (1995), by E. W. Martin, Mack Publishing Co., Easton, Pa., describes compositions and formulations suitable for pharmaceutical delivery of one or more agents, such as one or more modulators. In general, the nature of the carrier will depend on the particular mode of administration being used. For example, parenteral formulations may include injectable solutions that contain medicamentarily and physiologically acceptable fluids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, and the like, as a vehicle. In addition to biologically neutral carriers, the administered pharmaceutical preparation may contain minor amounts of nontoxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents, for example, sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate.

[0102] "Encoding" refers to the inherent property of a particular nucleotide sequence in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either having a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA.

[0103] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may contain introns.

[0104] In one aspect of the invention, the anti-CTHRC1 fusion protein provides an activation signal to T cells and NK cells present in the tumor microenvironment.

[0105] In another aspect of the invention, the T cell and NK cell activation signals induce T cell and / or NK cell dependent cytotoxicity directed against cancer cells.

[0106] In another aspect, anti-CTHRC1 fusion proteins bring lymphocytes, including T cells and NK cells, into proximity with CAFs or cancer cells in a CTHRC1-dependent manner.

[0107] In another aspect, it enhances T cell and NK cell mediated killing of CAFs and cancer cells.

[0108] In one aspect of the invention, the anti-CTHRC1 fusion protein provides a pro-inflammatory signal to a broader set of immune cells expressing the receptor, which may include macrophages, dendritic cells, monocytes, B cells, plasma cells, neutrophils, mast cells, and other blood-borne immune cells, as well as antigen-presenting cells.

[0109] In another aspect of the invention, the immune activation signal induces an inflammatory immune response in the tumor microenvironment that will result in the killing of tumor cells.

[0110] In another embodiment of the invention, the anti-CTHRC1 fusion protein brings inflammatory macrophages into close proximity with CAFs or cancer cells, inducing direct macrophage-mediated cell killing, e.g., via the complement system and complement receptor-mediated phagocytosis.

[0111] In another aspect of the invention, the anti-CTHRC1 fusion protein brings inflammatory macrophages, dendritic cells, or other antigen-presenting cells into close proximity with CAFs and cancer cells to induce enhanced presentation of antigens associated with CAFs or cancer cells and an associated indirect adaptive immune response.

[0112] In another aspect of the invention, the proximity of immune cells induces a broad immune response, including the release of inflammatory cytokines.

[0113] In some embodiments, an anti-CTHRC1 fusion protein is provided, which may comprise a first domain comprising a CTHRC1 binding moiety and a second domain comprising a ligand for a receptor expressed on T cells, preferably human T cells. In some embodiments, the second domain comprises a ligand for a costimulatory receptor. In some embodiments, the second domain comprises a ligand for a cytokine receptor. In some embodiments, the anti-CTHRC1 fusion protein further comprises a peptide linker located between the first domain and the second domain.

[0114] The subject matter of the present disclosure encompasses antibody fusions. For example, proteins can be linked together by either chemical or genetic engineering using methods known in the art. See, e.g., Gillies et al., Proc. Nat'l Acad. Sci. USA 89:1428-1432 (1992) and U.S. Patent No. 5,650,150.

[0115] In one example, the present disclosure encompasses an anti-CTHRC1 antibody-cytokine fusion protein. In principle, the anti-CTHRC1 antibody as disclosed herein can be fused to any cytokine by using recombinant molecular biology techniques. In a preferred embodiment, the anti-CTHRC1 antibody is fused to IL-2 (Gillies, S., Protein Engineering, Design and Selection 26(10):561-569(2013); Klein, C. et al., OncoImmunology 6:3(2017).

[0116] In another example, the disclosure encompasses an anti-CTHRC1 antibody-T cell engager fusion protein. As discussed herein, an anti-CTHRC1 antibody-T cell engager fusion protein comprises a fusion between an anti-CTHRC1 antibody and a ligand for a receptor expressed on a T cell. Examples of such ligands include, but are not limited to, CD40L, OX40L, 4-1BBL, CD80 / 86, ICOSL, and the like. In some embodiments, the ligand is fused to the Fc portion of the anti-CTHRC1 antibody. In some embodiments, the ligand is fused to the C-terminus of the light chain of the anti-CTHRC1 antibody. Such an approach has been described for 4-1BBL (Dafne M. et al., Journal of Immunotherapy 38(8):714-722(2008) and similar approaches can be used to generate other antibody-T cell engager fusion proteins.

[0117] Costimulatory Receptor Ligands In any of the above-mentioned embodiments, the second domain comprises a ligand for a costimulatory receptor. In certain aspects, the costimulatory receptor is a member of the immunoglobulin superfamily (IgSF). In other aspects, the costimulatory receptor is a member of the tumor necrosis factor superfamily (TNFSF). In certain aspects, the costimulatory receptor is on an immune cell. By way of example and not limitation, the immune cell can be a T cell, a NK cell, a B cell, a dendritic cell, or an antigen-presenting cell.

[0118] In any of the above-described embodiments, the costimulatory receptor may be selected from the group consisting of 4-1BB (CD137), OX40 (CD134), CD40, GITR, CD27, herpes virus entry mediator (HVEM), CD30, DR3, DR5, ICOS, CD28, TIM1, SLAMF1 (CDw150), CD2, SLAMF6, SLAMF8, CD48, and STING. Preferably, the costimulatory receptor may be selected from the group consisting of 4-1BB (CD137), OX40 (CD134), CD40, CD27, CD28, herpes virus entry mediator (HVEM), ICOS, CD2, and GITR.

[0119] In any of the above-mentioned embodiments, the ligand may be a natural ligand for the costimulatory receptor, a fragment thereof, a mutant thereof, or a conservatively substituted variant of the natural ligand or fragment thereof. Alternatively, the ligand may be an engineered ligand, for example, a non-natural ligand that can bind to and stimulate the costimulatory receptor. Exemplary non-natural ligands are disclosed in Suo, et al., Pharmaceutics 14(1):181 (2022), Zwolak, et al., Scientific Reports 12:20538 (2022), and U.S. Patent Application Publication No. 2021 / 0188995, each of which is incorporated herein by reference in its entirety.

[0120] An exemplary list of costimulatory receptors and their natural ligands is provided in Table 1. [Table 2]

[0121] 4-1BB receptor In some embodiments, the costimulatory receptor may be 4-1BB (CD137). In some embodiments, the ligand may be a natural ligand of 4-1BB, such as 4-1BBL, a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. In other embodiments, the ligand may be a non-natural ligand of 4-1BB.

[0122] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 1 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of any one of SEQ ID NOs: 2-10. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 1-10.

[0123] OX40 receptor In some embodiments, the costimulatory receptor can be OX40.In some embodiments, the ligand can be the natural ligand of OX40, such as OX40L, its fragment, its full-length or fragmentary sequence conservatively substituted variant, or its mutant of full-length or fragmentary sequence.In other embodiments, the ligand can be the non-natural ligand of OX40.

[0124] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 11 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of any one of SEQ ID NOs: 12-13. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 11-13.

[0125] CD40 receptor In some embodiments, the costimulatory receptor may be CD40. In some embodiments, the ligand may be a natural ligand of CD40, such as CD40L, a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant thereof of the full-length or fragmentary sequence. In other embodiments, the ligand may be a non-natural ligand of CD40.

[0126] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 14 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of any one of SEQ ID NOs: 15-16. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 14-16.

[0127] GITR Receptor In some embodiments, the costimulatory receptor can be GITR.In some embodiments, the ligand can be the natural ligand of GITR, such as GITRL, its fragment, its full-length or fragmentary sequence conservatively substituted variant, or its mutant of full-length or fragmentary sequence.In other embodiments, the ligand can be the non-natural ligand of GITR.

[0128] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 17 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of SEQ ID NO: 18. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 17-18.

[0129] CD27 receptor In some embodiments, the costimulatory receptor may be CD27. In some embodiments, the ligand may be a natural ligand of CD27, such as CD70, a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant thereof of the full-length or fragmentary sequence. In other embodiments, the ligand may be a non-natural ligand of CD27.

[0130] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 19 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of SEQ ID NO: 20. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 19-20.

[0131] HVEM Receptor In some embodiments, the costimulatory receptor can be HVEM. In some embodiments, the ligand can be the natural ligand of HVEM, such as LIGHT, its fragment, its full-length or fragmentary sequence conservatively substituted variant, or its full-length or fragmentary sequence mutant. In other embodiments, the ligand can be the non-natural ligand of LIGHT.

[0132] In some embodiments, the ligand may comprise the sequence of SEQ ID NO:21 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of any one of SEQ ID NOs:22-23. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs:21-23.

[0133] CD30 receptor In some embodiments, the costimulatory receptor may be CD30. In some embodiments, the ligand may be a natural ligand of CD30, such as CD30L, a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. In other embodiments, the ligand may be a non-natural ligand of CD30.

[0134] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 24 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of SEQ ID NO: 25. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 24-25.

[0135] DR3 receptor In some embodiments, the co-stimulatory receptor can be DR3. In some embodiments, the ligand can be the natural ligand of DR3, such as TRAIL, its fragment, a conservatively substituted variant of full-length or fragmentary sequence, or its mutant of full-length or fragmentary sequence. In other embodiments, the ligand can be a non-natural ligand of DR3.

[0136] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 26 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of SEQ ID NO: 27. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 26-27.

[0137] DR5 receptor In some embodiments, the costimulatory receptor can be DR5.In some embodiments, the ligand can be the natural ligand of DR5, such as TL1A, its fragment, its full-length or fragmentary sequence conservatively substituted variant, or its mutant of full-length or fragmentary sequence.In other embodiments, the ligand can be the non-natural ligand of DR5.

[0138] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 28 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of SEQ ID NO: 29. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 28-29.

[0139] ICOS receptor In some embodiments, the costimulatory receptor can be ICOS.In some embodiments, the ligand can be the natural ligand of ICOS, such as ICOSL, its fragment, the conservatively substituted variant of full-length or fragmentary sequence, or its mutant of full-length or fragmentary sequence.In other embodiments, the ligand can be the non-natural ligand of ICOS.

[0140] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 30 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of SEQ ID NO: 31. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 30-31.

[0141] CD28 receptor In some embodiments, the costimulatory receptor may be CD28. In some embodiments, the ligand may be a natural ligand of CD28, such as CD80 or CD86, a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. In other embodiments, the ligand may be a non-natural ligand of CD28.

[0142] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 32 or SEQ ID NO: 34 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of SEQ ID NO: 33 or SEQ ID NO: 35. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 32-35.

[0143] TIM-1 receptor In some embodiments, the costimulatory receptor can be TIM-1. In some embodiments, the ligand can be a natural ligand of TIM-1, such as TIM-4, a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. In other embodiments, the ligand can be a non-natural ligand of TIM-1.

[0144] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 36 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 36 or a fragment thereof.

[0145] SLAM receptor In some embodiments, the costimulatory receptor can be SLAM.In some embodiments, the ligand can be the natural ligand of SLAM, for example, SLAM, its fragment, the conservatively substituted variant of full-length or fragmentary sequence, or its mutant of full-length or fragmentary sequence.In other embodiments, the ligand can be the non-natural ligand of SLAM.

[0146] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 37 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of SEQ ID NO: 38. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 37-38, or a fragment thereof.

[0147] CD2 receptor In some embodiments, the costimulatory receptor may be CD2. In some embodiments, the ligand may be a natural ligand of CD2, such as CD58, a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. In other embodiments, the ligand may be a non-natural ligand of CD2.

[0148] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 39 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of SEQ ID NO: 40. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 39-40.

[0149] SLAMF6 receptor In some embodiments, the costimulatory receptor can be SLAMF6. In some embodiments, the ligand can be a natural ligand of SLAMF6, such as SLAMF6, a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. In other embodiments, the ligand can be a non-natural ligand of SLAMF6.

[0150] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 41 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of SEQ ID NO: 42. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 41-42, or a fragment thereof.

[0151] SLAMF8 Receptor In some embodiments, the costimulatory receptor may be SLAMF8. In some embodiments, the ligand may be a natural ligand of SLAMF8, such as SLAMF8, a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. In other embodiments, the ligand may be a non-natural ligand of SLAMF8.

[0152] In some embodiments, the ligand may comprise the sequence of SEQ ID NO: 43 or a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant of the full-length or fragmentary sequence. By way of non-limiting example, the fragment may comprise the sequence of SEQ ID NO: 44. In some embodiments, the ligand may comprise a sequence having at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 43-44, or a fragment thereof.

[0153] CD48 In some embodiments, the costimulatory receptor may be CD48. In some embodiments, the ligand is a natural ligand of CD48, such as CD244, a fragment thereof, a conservatively substituted variant of the full-length or fragmentary sequence, or a mutant or full-length or fragmentary sequence thereof. In other embodiments, the ligand may be a non-natural ligand of CD48.

[0154] STING In some embodiments, the costimulatory receptor may be STING. In some embodiments, the ligand may be a natural ligand of STING, such as cyclic GAMP-AMP, or other small molecule ligand that binds to STING. In other embodiments, the ligand may be a non-natural ligand of STING. Exemplary ligands of STING useful in the present invention are disclosed in Amouzegar, et al., Cancers 2021, 13, 2695, which is incorporated herein by reference in its entirety.

[0155] Other receptors Additional costimulatory receptors, ligands (including variants thereof) and sequences thereof are described in WO2018 / 144514, U.S. Patent Application Publication No. 2019 / 0016771, U.S. Patent Application Publication No. 2021 / 0169932, WO2019 / 089921, WO2017 / 211321, U.S. Patent No. 9,534,056, U.S. Patent Application Publication No. 2021 / 0253706, U.S. Patent No. 8,440,192, U.S. Patent Application Publication No. 2020 / 0758901, WO2020 / 172189, WO2017 / 060144, U.S. Patent No. 11,053,293, Chen, Lieping and Flies, Dallas B., “Molecular mechanisms of T cell co-stimulation and co-inhibition,”Nat.Rev.Immunol.13(4):227-242(2013), Dostert,C.,et al.,“The TNF Family of Ligands and Receptors:Communication Modules in the Immune System and Beyond,”Physiol.Rev.99:115-60(2018),Li,et al.,“A novel tumor-homing TRAIL variant eradicates tumor xenografts of refractory colorectal cancer cells in combination with tumor cell-targeted photodynamic therapy,”Drug Delivery 29(1):1698-1711(2022), Meylan,et al.,“TL1A and DR3,a TNF-family ligand-receptor pair that promotes lymphocyte costimulation,mycosal hyperplasia and autoimmune inflammation,”Immunol Rev.244(1)(2011), McArdel, et al., “Roles of CD48 in regulating immunity and tolerance,” Clin. Immunol.164:10-20(2016), Mavaddat, et al., “Signaling Lymphocytic Activation Molecule (CDw150) is Homophilic but Self-Associates with Very Low Affinity,” J. Biol. Chem. 275(36):28100-28109(2000), Suo, et al., Pharmaceutics 14(1):181(2022), Zwolak, et al., Scientific Reports 12:20538(2022), and U.S. Patent Application Publication No. 2021 / 0188995, each of which is incorporated herein by reference in its entirety.

[0156] It is to be understood that the ligands of the present disclosure can be directed to any costimulatory receptor on any type of cell. Also contemplated herein are cognate ligands for costimulatory receptors found on NK cells, e.g., NKG2D ligands such as MICA / MICB and ULBPs.

[0157] CTHRC1 binding part In embodiments of the disclosure, an anti-CTHRC1 fusion protein of the disclosure comprises a CTHRC1 binding moiety.

[0158] In any of the above-mentioned embodiments, the CTHRC1 binding moiety can be an antibody as defined herein that can bind to CTHRC1. In some embodiments, the anti-CTHRC1 antibodies of the present invention further comprise a human subgroup III heavy chain framework consensus sequence. In one embodiment of these antibodies, these antibodies further comprise a human κI light chain framework consensus sequence.

[0159] It should be understood that the CTHRC1 binding moiety may further comprise a peptide linker as described herein. By way of example and not limitation, when the CTHRC1 binding moiety comprises an scFv, the VH and VL moieties may be linked by any suitable linker, such as those described in this disclosure.

[0160] Exemplary CDRs and antibody sequences are provided in the table below. [Table 3] [Table 4] [Table 5] [Table 6]

[0161] Thus, in any of the above-mentioned embodiments, the CTHRC1 binding portion may comprise an antibody portion comprising the HCDR1, HCDR2, and HCDR3 of AB987, AB988, AB989, AB990, or AB991, and / or the LCDR1, LCDR2, and LCDR3 of AB987, AB988, AB989, AB990, or AB991 (from Tables 2 and 3). In any of the above-mentioned embodiments, the CTHRC1 binding portion may comprise an antibody portion comprising the VH and / or VL of any of AB987, AB988, AB989, AB990, or AB991 (from Tables 4 and 5).

[0162] Preferably, the CTHRC1 binding portion of the invention is an scFv or scFab, wherein the nucleic acid sequence of the scFv comprises a nucleic acid sequence(s) encoding one or more light chain CDRs and one or more heavy chain CDRs as disclosed herein for an anti-CTHRC1 antibody, and the nucleic acid sequence of the scFab comprises a nucleic acid sequence(s) encoding one or more light chain CDRs and one or more heavy chain CDRs as disclosed herein for an anti-CTHRC1 antibody.

[0163] Preferably, the CTHRC1-binding portion of the present invention is an scFv or scFab comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs:75-84.

[0164] Preferably, the CTHRC1 binding moiety 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: 75, 77, 79, 81, and 83, more preferably an scFv or scFab comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 77 and 83. Even more preferably, the scFv or scFab comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 75 and 77, even more preferably SEQ ID NO: 77.

[0165] Preferably, the CTHRC1 binding moiety 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: 76, 78, 80, 82, and 84, more preferably an scFv or scFab comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 87 and 84. Even more preferably, the scFv or scFab comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 76 and 78, even more preferably SEQ ID NO: 78.

[0166] Preferably, the CTHRC1 binding portion 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: 75, 77, 79, 81, and 83, and any one of SEQ ID NOs: 76, 78, 80, 82, and 84. More preferably, the portion of the antigen binding portion of the CAR of the invention is an scFv or scFab comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 77 and 83, and any one of SEQ ID NOs: 78 and 84. Even more preferably, the scFv or scFab comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 75 and 77, and any one of SEQ ID NOs: 76 and 78, even more preferably SEQ ID NOs: 77 and SEQ ID NO: 78.

[0167] In some embodiments, the CTHRC1 binding portion of the invention is an scFv or scFab that comprises amino acids encoded by a nucleotide sequence selected from the group consisting of SEQ ID NOs: 88 to 97. In some embodiments, the CTHRC1 binding portion of the invention is an scFv or scFab that comprises amino acids encoded by a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 88, 90, 92, 94, and 96.

[0168] In embodiments, the CTHRC1 binding portion of the invention is an scFv or scFab comprising amino acids encoded by a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 89, 91, 93, 95, and 97.

[0169] In several embodiments, the CTHRC1 binding portion of the invention is an scFv or scFab comprising an amino acid encoded by a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 100, 102, 104, 106, and 108, and a nucleotide sequence selected from the group consisting of any one of SEQ ID NOs: 101, 103, 105, 107, and 109.

[0170] In embodiments, the CTHRC1 binding portion of the invention is an scFv or scFab comprising an amino acid sequence selected from the group consisting of any of the CDR sequences in Tables 4 and 5.

[0171] Preferably, the CTHRC1-binding portion of the present invention is an scFv or scFab comprising an amino acid sequence selected from the group consisting of any one of the CDR sequences in Tables 4 and 5, and further comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 75 to 84. More preferably, the CTHRC1-binding portion of the present invention is an scFv or scFab comprising a heavy chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 45 to 49, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 50 to 54, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 55 to 59, and a light chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 60 to 64, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 65 to 69, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 70 to 74, and further comprising an amino acid sequence selected from the group consisting of any one of SEQ ID NOs: 75 to 84.

[0172] Preferably, the CTHRC1 binding portion of the present invention is an scFv or scFab comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 102, 104, 106, and 108, more preferably selected from the group consisting of SEQ ID NOs: 102 and 106, most preferably SEQ ID NO: 102.

[0173] In a preferred embodiment, the CTHRC1 binding portion comprises an HCDR1 of SEQ ID NO:46, an HCDR2 of SEQ ID NO:51, an HCDR3 of SEQ ID NO:56, an LCDR1 of SEQ ID NO:61, an LCDR2 of SEQ ID NO:66, and an LCDR3 of SEQ ID NO:71.

[0174] In another preferred embodiment, the CTHRC1 binding portion comprises an HCDR1 of SEQ ID NO:45, an HCDR2 of SEQ ID NO:50, an HCDR3 of SEQ ID NO:55, an LCDR1 of SEQ ID NO:60, an LCDR2 of SEQ ID NO:65, and an LCDR3 of SEQ ID NO:70.

[0175] In some embodiments, the CTHRC1 binding portion comprises an HCDR1 of SEQ ID NO:47, an HCDR2 of SEQ ID NO:52, an HCDR3 of SEQ ID NO:57, an LCDR1 of SEQ ID NO:62, an LCDR2 of SEQ ID NO:67, and an LCDR3 of SEQ ID NO:72.

[0176] In some embodiments, the CTHRC1 binding portion comprises an HCDR1 of SEQ ID NO:48, an HCDR2 of SEQ ID NO:53, an HCDR3 of SEQ ID NO:58, an LCDR1 of SEQ ID NO:63, an LCDR2 of SEQ ID NO:68, and an LCDR3 of SEQ ID NO:73.

[0177] In some embodiments, the CTHRC1 binding portion comprises an HCDR1 of SEQ ID NO:49, an HCDR2 of SEQ ID NO:54, an HCDR3 of SEQ ID NO:59, an LCDR1 of SEQ ID NO:64, an LCDR2 of SEQ ID NO:69, and an LCDR3 of SEQ ID NO:74.

[0178] In a preferred embodiment, the CTHRC1 binding portion comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:77 and a light chain variable domain comprising the sequence of SEQ ID NO:78.

[0179] In another preferred embodiment, the CTHRC1 binding portion comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:75 and a light chain variable domain comprising the sequence of SEQ ID NO:76.

[0180] In some embodiments, the CTHRC1 binding portion comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:79 and a light chain variable domain comprising the sequence of SEQ ID NO:80.

[0181] In some embodiments, the CTHRC1 binding portion comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:81 and a light chain variable domain comprising the sequence of SEQ ID NO:82.

[0182] In some embodiments, the CTHRC1 binding portion comprises a heavy chain variable domain comprising the sequence of SEQ ID NO:83 and a light chain variable domain comprising the sequence of SEQ ID NO:84.

[0183] In any of the above-mentioned embodiments, the anti-CTHRC1 fusion protein may have a binding affinity to CTHRC1 of less than 10 nM, preferably less than 5 nM, more preferably less than 1 nM. The binding affinity of the anti-CTHRC1 fusion protein can be determined, for example, by Scatchard analysis as described in Munson et al., Anal.Biochem.107:220 (1980).

[0184] In some embodiments, when the CTHRC1 binding moiety is an antibody, it can be a chimeric, humanized, or human antibody. In some embodiments, when the CTHRC1 binding moiety is an antibody, it can be a full-length antibody fragment, a single-chain antibody, a single-domain antibody (e.g., a heavy chain only antibody), a single-chain variable fragment (scFv), or any other type of "antibody" as defined herein. Exemplary antibodies include monoclonal, chimeric, humanized, and human antibodies.

[0185] In embodiments, the CTHRC1 binding moiety is a compound selected from the group consisting of those disclosed in WO2010 / 047448, U.S. Patent Application Publication No. 2005 / 0147602, U.S. Patent Application Publication No. 2016 / 0000866, WO2014 / 200134, U.S. Patent No. 9,050,296, U.S. Patent Application Publication No. 2013 / 0190357, U.S. Patent Application Publication No. 2018 / 0313846, U.S. Patent No. 9,718, 878, U.S. Patent Application Publication No. 2022 / 0204599, WO2021 / 063972, U.S. Patent Application Publication No. 2018 / 0221442, WO2010 / 047448, CN110257389A, and JPWO2007123010, each of which is incorporated by reference in its entirety.Additional CTHRC1 binding moieties include, by way of example and not limitation, 10G07 (Duarte et al., 2014 PLOS ONE, 9(6):e100449, incorporated herein by reference), 13D11, and 19C07, clone 13E09, anti-CTHRC1 antibody, H-213, incorporated herein by reference, anti-CTHRC1 antibody, T-19, incorporated herein by reference (Santa Cruz Biosciences, Inc., (Sigma-Aldrich®, St. Louis, Mo.), and anti-CTHRC1 antibody: SAB1102667, HPA059806, SAB2107469, and SAB1402656 (each of which is incorporated by reference herein) (Thermo and antibodies or fragments thereof comprising HCDR1, HCDR2, and HCDR3 and LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 9-11 and 13-15, respectively, of WO2014 / 200134, and antibodies or fragments thereof comprising HCDR1, HCDR2, and HCDR2 and LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 1-3 or 11-13 (HCDRs) and 4-6 or 14-16 (LCDRs), respectively, of U.S. Patent Application Publication No. 2022 / 0204599, and / or any of SEQ ID NOs: 7, 17, 21, or 25 heavy chain variable regions and / or any of SEQ ID NOs: 9, 19, 23, or 27 light chain variable regions (including clones cCMAb45, cCMAb96, hCMAb45, and hCMab96, 6C1, and 1D1).

[0186] Monoclonal antibodies Monoclonal antibodies (mAbs) against an antigen of interest can be prepared by using any technique known in the art, including, but not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Selected Lymphocyte Antibody Method (SLAM) (Babcook, JS, et al., A novel strategy for generating monoclonal antibodies from single, isolated lymphocytes producing antibodies of defined specificities. Proc Natl Acad Sci USA, 1996.93(15):p.7843-8.) and (McLean G et al., 2005, J Immunol.174(8):4768-78). Such antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. Hybridomas for producing mAbs useful in the present invention can be cultured in vitro or in vivo.

[0187] 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).

[0188] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes can be immunized in vitro. After immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0189] The hybridoma cells thus prepared are seeded and grown in a suitable medium which may contain one or more substances that inhibit the growth or survival of the unfused parental myeloma cells (also referred to as the fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective medium for the hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which substances inhibit the growth of HGPRT-deficient cells.

[0190] 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 unfused parent cells. Preferred myeloma cell lines are mouse 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 available from the American Type Culture Collection, Manassas, Va. USA, such as X63-Ag8-653 cells. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984), and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp.51-63 (Marcel Dekker, Inc., New York, 1987)).

[0191] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0192] 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).

[0193] Once hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and / or activity, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in animals, for example, by intraperitoneal injection of the cells into mice.

[0194] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using Protein A or Protein G-Sepharose) or ion exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, or the like.

[0195] DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). 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 that do not otherwise produce antibody protein, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding antibodies include Skerra et al., Curr. Opinion in Immunol. 5:256-62 (1993) and Pluckthun, Immunol. Rev. 130:151-88 (1992).

[0196] In further embodiments, 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 mouse 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)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0197] DNA encoding an antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy and light chain constant domains (CH and CO sequences) for the homologous murine sequences (U.S. Pat. No. 4,816,567, and Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing an immunoglobulin coding sequence with all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide). The non-immunoglobulin polypeptide sequences can be substituted for the constant domains of an antibody, or they can be substituted for the variable domains of one antigen-binding site of an antibody to create a chimeric, bivalent antibody having one antigen-binding site with specificity for one antigen and another antigen-binding site with specificity for a different antigen.

[0198] Chimeric, Humanized, and Human Antibodies In some embodiments, the CTHRC1 binding portion is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567, and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-5 (1984). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate, such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, the chimeric antibody is a "class-switched" antibody, in which the class or subclass is changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0199] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans while retaining the specificity and affinity of the non-human parent antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof) are derived from a non-human antibody and the FRs (or portions thereof) are derived from a human antibody sequence. The humanized antibody also optionally comprises at least a portion of a human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from the non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0200] The anti-CTHRC1 antibodies of the present invention may include humanized or human antibodies. Humanized forms of non-human (e.g., mouse or rabbit) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies) that contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibodies) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit that has the desired specificity, affinity, and capacity. In some cases, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are 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)).

[0201] A humanized antibody as a CTHRC1 binding moiety of the present 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 in the human and / or human consensus non-hypervariable region sequences. In one embodiment, the heavy chain variable domain of the antibody of the present invention comprises a human consensus framework sequence, which in one embodiment is a subgroup III consensus framework sequence. In one embodiment, the antibody of the present invention comprises a variant subgroup III consensus framework sequence modified at at least one amino acid position.

[0202] As known in the art and described in more detail herein, the amino acid positions / boundaries that define the hypervariable regions of an antibody can vary depending on the context and the various definitions known in the art (described below). Some positions within a variable domain can be considered hybrid hypervariable positions, in that these positions can be considered to be within a hypervariable region under one set of criteria, while being considered to be outside a hypervariable region under a different set of criteria. One or more of these positions can also be found in an extended hypervariable region (defined further below). The present invention provides antibodies that contain modifications at these hybrid hypervariable positions. In one embodiment, these hypervariable positions include one or more of positions 26-30, 33-35B, 47-49, 57-65, 93, 94, and 101-102 in the heavy chain variable domain. In one embodiment, these hybrid hypervariable positions include one or more of positions 24-29, 35-36, 46-49, 56, and 97 in the light chain variable domain. In one embodiment, an antibody of the invention comprises a human variant human subgroup consensus framework sequence altered at one or more hybrid hypervariable positions.

[0203] The CTHRC1 binding portion of the present invention may comprise any suitable human or human consensus light chain framework sequence, so long as the antibody exhibits the desired biological properties (e.g., the desired binding affinity). In one embodiment, the antibody of the present invention comprises at least a portion (or all) of the framework sequence of a human κ light chain. In one embodiment, the CTHRC1 binding portion of the present invention comprises at least a portion (or all) of the human κ subgroup I framework consensus sequence.

[0204] Methods for humanizing non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, and these residues are typically taken from an "import" variable domain. Humanization is essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). Such "humanized" antibodies are thus essentially chimeric antibodies (U.S. Pat. No. 4,816,567) in which less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0205] The choice of human variable domains, both light and heavy, used in making the humanized antibody is very important to reduce antigenicity and HAMA responses (human anti-mouse antibodies) if the antibody is intended for human therapeutic use. Reduction or elimination of the HAMA response is an important aspect of the clinical development of suitable therapeutic agents (see, e.g., Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al., Transplantation (1986), 41:572; Shawler et al., J. Immunol. (1985), 135:1530; Sears et al., J. Biol. Response Mod. (1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352; Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res. (1990), 50:1495). As described herein, the present invention provides antibodies that are humanized to reduce or eliminate HAMA response. Variants of these antibodies can be further obtained using conventional methods known in the art, some of which are further described below. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human V-domain sequence that is closest to the rodent V-domain sequence is identified, and the human framework region (FR) therein is 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 specific framework region derived from the consensus sequence of all human antibodies of a specific subgroup of light or heavy chains.The same framework can be used for several different humanized antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol. 151:2623 (1993)).

[0206] For example, an amino acid sequence from an antibody as described herein may serve as the starting (parent) sequence for diversification of the framework and / or hypervariable sequence(s). The selected framework sequence to which the starting hypervariable sequence is linked is referred to herein as the acceptor human framework. The acceptor human framework may be from or derived from a human immunoglobulin (its VL and / or VH regions), but preferably the acceptor human framework is from or derived from a human consensus framework sequence, as such frameworks have been demonstrated to have minimal or no immunogenicity in human patients.

[0207] If the acceptor is derived from a human immunoglobulin, optionally the human framework sequence may be selected based on its homology with the donor framework sequence by aligning the donor framework sequence with various human framework sequences in a population of human framework sequences and selecting the framework sequence that is most homologous to the acceptor.

[0208] In one embodiment, the human consensus framework herein is from or derived from a VH subgroup III and / or VL kappa subgroup I consensus framework sequence.

[0209] Although the acceptor may be identical in sequence to the selected human framework sequence, whether derived from a human immunoglobulin or a human consensus framework, the present invention contemplates that the acceptor sequence may contain pre-existing amino acid substitutions compared to the human immunoglobulin sequence or the human consensus framework sequence. These pre-existing substitutions are preferably minimal, usually no more than four, three, two, or one amino acid difference compared to the human immunoglobulin sequence or the consensus framework sequence.

[0210] Hypervariable region residues of a non-human antibody are incorporated into the VL and / or VH acceptor human framework. For example, residues corresponding to Kabat CDR residues, Chothia hypervariable loop residues, Abm residues, and / or contact residues may be introduced. Optionally, extended hypervariable region residues are incorporated as follows: 24-34 (L1), 50-56 (L2), and 89-97 (L3), 26-35B (H1), 50-65, 47-65, or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3).

[0211] While the "incorporation" of hypervariable region residues is discussed herein, it will be understood that this can be accomplished in a variety of ways. For example, a nucleic acid encoding a desired amino acid sequence can be generated by mutating a nucleic acid encoding a murine variable domain sequence such that its framework residues are changed to acceptor human framework residues, or by mutating a nucleic acid encoding a human variable domain sequence such that the hypervariable domain residues are changed to non-human residues, or by synthesizing a nucleic acid encoding the desired sequence, etc.

[0212] Hypervariable region grafted variants may be generated by Kunkel mutagenesis of nucleic acid encoding the human acceptor sequence, using separate oligonucleotides for each hypervariable region, as described herein. Kunkel et al., Methods Enzymol. 154:367-382 (1987). Using routine techniques, appropriate changes can be introduced within the framework and / or hypervariable regions to modify and re-establish proper hypervariable region-antigen interactions.

[0213] Phage(mid) display (also referred to herein in some contexts as phage display) can be used as a convenient and rapid method for generating and screening many different potential variant antibodies in libraries generated by sequence randomization. However, other methods for making and screening altered antibodies are available to those of skill in the art.

[0214] Phage(mid) display technology provides a powerful tool for generating and selecting novel proteins that bind to ligands such as antigens. The use of phage(mid) display techniques allows the generation of large libraries of protein variants that can be rapidly screened for sequences that bind to target molecules with high affinity. The nucleic acid encoding the variant polypeptide is generally fused to a nucleic acid sequence encoding a viral coat protein such as gene III protein or gene VIII protein. Monovalent phagemid display systems have been developed in which a nucleic acid sequence encoding a protein or polypeptide is fused to a nucleic acid sequence encoding a portion of the gene III protein (Bass, S., Proteins, 8:309 (1990); Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3:205 (1991)). In monovalent phagemid display systems, gene fusions are expressed at low levels and wild-type gene III protein is also expressed so that the infectivity of the particles is maintained. Methods for generating peptide libraries and screening those libraries are disclosed in many patents (e.g., U.S. Patent No. 5,723,286, U.S. Patent No. 5,432,018, U.S. Patent No. 5,580,717, U.S. Patent No. 5,427,908, and U.S. Patent No. 5,498,530).

[0215] Libraries of antibodies or antigen-binding polypeptides have been prepared in several 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 are described in U.S. Patent Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727.The libraries are then screened for the expression of antibodies or antigen-binding proteins with desired properties.

[0216] Methods for substituting selected amino acids 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)).

[0217] The sequence of the oligonucleotide comprises one or more of the codon sets designed for the hypervariable region residues to be changed. A codon set is a set of different nucleotide triplet sequences used to code the desired variant amino acid. A codon set can be represented using symbols that designate a particular nucleotide or equimolar mixture of nucleotides, as shown below in the IUB code: IUB Code G Guanine A Adenine T Thymine C Cytosine R (A or G) Y (C or T) M (A or C) K (G or T) S (C or G) W (A or T) H (A or C or T) B (C ​​or G or T) V (A or C or G) D(A or G or T)H N (A or C or G or T)

[0218] For example, in the codon set DVK, D can be the nucleotide A or G or T, V can be A or G or C, and K can be G or T. This codon set can represent 18 different codons and can code for the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

[0219] Oligonucleotide or primer sets can be synthesized using standard methods. A set of oligonucleotides containing sequences that represent all possible combinations of nucleotide triplets provided by the codon set and will code for the desired amino acid group can be synthesized, for example, by solid-phase synthesis. The synthesis of oligonucleotides with selected nucleotide "degeneracy" at a particular position is well known in the art. Such a set of oligonucleotides with a particular codon set can be synthesized using a commercial nucleic acid synthesizer (e.g., available from Applied Biosystems, Foster City, Calif.) or can be obtained commercially (e.g., from Life Technologies, Rockville, Md.). Thus, a set of synthesized oligonucleotides with a particular codon set typically includes multiple oligonucleotides with different sequences, the difference being established by the codon set in the overall sequence. Oligonucleotides as used by the present invention have sequences that allow hybridization to a variable domain nucleic acid template and may also include restriction enzyme sites for cloning purposes.

[0220] In one method, the nucleic acid sequence encoding variant amino acid can be generated by oligonucleotide-mediated mutagenesis. This technique is well known in the art, as described by Zoller et al. Nucleic Acids Res. 10:6487-6504 (1987). Briefly, the nucleic acid sequence encoding variant amino acid is generated by hybridizing the oligonucleotide set encoding desired codon set to DNA template, where the template is a plasmid in single-stranded form containing the nucleic acid template sequence of variable region. After hybridization, DNA polymerase is used to synthesize the entire second complementary strand of the template, so that it incorporates the oligonucleotide primer and contains the codon set as provided by the oligonucleotide set.

[0221] Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide will have 12-15 nucleotides that are perfectly complementary to the template on each side of the nucleotide(s) that code for the mutation(s). This ensures that the oligonucleotide hybridizes properly to the single-stranded DNA template molecule. Oligonucleotides are easily synthesized using techniques known in the art, such as those described by Crea et al., Proc. Nat'l. Acad. Sci. USA, 75:5765 (1978).

[0222] The DNA template is generated by either a vector derived from a bacteriophage M13 vector (the commercially available M13 mp 18 and M13 mp 19 vectors are suitable) or a vector containing a single-stranded phage origin of replication as described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus, to generate a single-stranded template, the DNA to be mutated can be inserted into one of these vectors. Production of single-stranded templates is described in Sections 4.21-4.41 of Sambrook et al., supra.

[0223] To alter the native DNA sequence, an oligonucleotide is hybridized to a single-stranded template under suitable hybridization conditions. A DNA polymerizing enzyme, usually T7 DNA polymerase or the Klenow fragment of DNA polymerase I, is then added to synthesize the complementary strand of the template using the oligonucleotide as a primer for synthesis. In this way, a heteroduplex molecule is formed in which one DNA strand encodes the mutated version of gene 1 and the other strand (the original template) encodes the unchanged native sequence of gene 1. This heteroduplex molecule is then transformed into a suitable host cell, usually a prokaryote such as E. coli JM101. After the cells are grown, they are plated on agarose plates and screened using an oligonucleotide primer radiolabeled with 32 phosphate to identify bacterial colonies containing the mutated DNA.

[0224] The method just described may be modified so that homoduplex molecules are created in which both strands of the plasmid contain the mutation(s). The modification is as follows: a single-stranded oligonucleotide is annealed to a single-stranded template as described above. A mixture of the three deoxyribonucleotides deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTT) is combined with a modified thiodeoxyribocytosine called dCTP-(aS), which is available from Amersham. This mixture is added to the template-oligonucleotide complex. When DNA polymerase is added to this mixture, a DNA strand identical to the template except for the mutated base is generated. In addition, this new DNA strand will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion. After the double-stranded heteroduplex template strand is nicked with an appropriate restriction enzyme, the template strand can be digested with ExoIII nuclease or another appropriate nuclease beyond the region containing the site(s) to be mutagenized. The reaction is then stopped, leaving a molecule that is only partially single-stranded. A complete double-stranded DNA homoduplex is then formed using DNA polymerase in the presence of all four deoxyribonucleotide triphosphates, ATP, and DNA ligase. This homoduplex molecule can then be transformed into a suitable host cell.

[0225] As indicated above, the sequence of the oligonucleotide set is of sufficient length to hybridize to the template nucleic acid and may, but does not necessarily, contain a restriction site. The DNA template may be generated by either a vector derived from a bacteriophage M13 vector or a vector containing a single-stranded phage origin of replication as described by Viera et al., Meth. Enzymol., 153:3 (1987). Thus, to generate a single-stranded template, the DNA to be mutated must be inserted into one of these vectors. The production of single-stranded templates is described in Sections 4.21-4.41 of Sambrook et al. (supra).

[0226] According to another method, antigen binding can be restored during antibody humanization by selection of restored hypervariable regions (see, e.g., U.S. Application No. 11 / 061,841, filed February 18, 2005). This method involves incorporating a non-human hypervariable region onto an acceptor framework and further introducing one or more amino acid substitutions in one or more hypervariable regions without modifying the acceptor framework sequence. Alternatively, introduction of one or more amino acid substitutions may be accomplished by modification in the acceptor framework sequence.

[0227] According to another method, a library can be generated by providing upstream and downstream oligonucleotide sets, each set having a plurality of oligonucleotides with different sequences, the different sequences being established by the codon sets provided within the sequences of the oligonucleotides. The upstream and downstream oligonucleotide sets can be used in a polymerase chain reaction with a variable domain template nucleic acid sequence to generate a "library" of PCR products. The PCR products can be referred to as "nucleic acid cassettes" because they can be fused to other related or unrelated nucleic acid sequences, such as viral coat proteins and dimerization domains, using established molecular biology techniques.

[0228] The sequences of the PCR primers contain one or more of the codon sets designed for the solvent accessible and diverse positions in the hypervariable regions. As discussed above, a codon set is a set of different nucleotide triplet sequences used to encode the desired variant amino acids.

[0229] Antibody selectants that meet the desired criteria, as selected by appropriate screening / selection steps, can be isolated and cloned using standard recombinant techniques.

[0230] It is further important that antibodies be humanized with retention of high binding affinity to 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 conformations of selected candidate immunoglobulin sequences. Inspection of these displays permits analysis of the possible role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences such 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.

[0231] Various forms of humanized anti-CTHRC1 antibodies are contemplated. For example, the humanized antibody may be an antibody fragment, such as a Fab. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

[0232] As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, the homozygous deletion of the antibody heavy-chain joining region (JH) gene in chimeric and germ-line mutant mice has been described, resulting in complete inhibition of endogenous antibody production. Transfer of the human germ-line immunoglobulin gene array into such germ-line mutant mice results in the production of human antibodies upon antigenic stimulation (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-8 (1993); Bruggemann et al., Year in Immuno. 7:33 (1993); U.S. Patent Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807, and WO 97 / 17852).

[0233] Alternatively, phage display technology (McCafferty et al., Nature 348:552-53 (1990)) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in frame into either the major or minor coat protein genes 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, for example, as reviewed in Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a wide variety of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the pancreas of immunized mice. Essentially following the techniques described by Marks et al., J. Mol. Biol. 222:581-97 (1991), or Griffith et al., EMBO J. 12:725-34 (1993) (see also U.S. Pat. Nos. 5,565,332 and 5,573,905), a repertoire of V genes from non-immunized human donors can be constructed and antibodies against a wide variety of antigens (including self-antigens) can be isolated.

[0234] As discussed above, human antibodies may also be generated by in vitro activated B cells (see, eg, US Pat. Nos. 5,567,610 and 5,229,275).

[0235] In another embodiment, the anti-CTHRC1 fusion proteins of the present disclosure may include a human monoclonal antibody as the CTHRC1 binding moiety. Such human monoclonal antibodies directed against CTHRC1 can be generated using transgenic or transchromosomic mice that carry parts of the human immune system rather than the mouse system. These transgenic and transchromosomic mice include those referred to herein as HuMAb Mice™ and KM Mice™, respectively, and are collectively referred to herein as "human Ig mice."

[0236] The HuMAb Mouse™ (Medarex, Inc.) contains a human immunoglobulin gene miniloci encoding unrearranged human heavy (μ and γ) and κ light chain immunoglobulin sequences together with targeted mutations that inactivate the endogenous μ and κ chain loci (see, e.g., Lonberg, et al. (1994) Nature 368(6474):856-9). Thus, the mice exhibit reduced mouse IgM or κ expression, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to give rise to high affinity human IgGκ monoclonal antibodies (Lonberg, N. et al. (1994) supra; reviewed in Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13:65-93; and Harding, F. and Lonberg, N. (1995) Ann. NY Acad. Sci. 764:536-46). The preparation and use of HuMAb Mice™, and the genomic modifications carried by such mice, are described in Taylor, L. et al. (1992) Nucleic Acids Research 20:6287-6295; Chen, J. et al. (1993) International Immunology 5:647-656; Tuaillon et al. (1993) Proc. Natl. Acad. Sci. USA 90:3720-4; Choi et al. (1993) Nature Genetics 4:117-23; Chen, J. et al. (1993) EMBO J. 12:21-830; Tuaillon et al., (1994) J. Immunol. 152:2912-20; Taylor, L. et al. (1994) International Immunology 6:579-91, and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-51, the entire contents of which are expressly incorporated herein by reference in their entireties.Further, U.S. Patent Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,789,650, 5,877,397, 5,661,016, 5,814,318, 5,874,299, and 5,770,429 No. 5,545,807, PCT Publication Nos. WO 92 / 03918, WO 93 / 12227, WO 94 / 25585, WO 97 / 13852, WO 98 / 24884, and WO 99 / 45962, and PCT Publication No. WO 01 / 14424.

[0237] In another embodiment, human antibodies of the disclosure can be produced using a mouse carrying human immunoglobulin sequences on a transgene and a transchromosome, such as a mouse carrying a human heavy chain transgene and a human light chain transchomosome, referred to herein as a "KM Mouse™," and described in detail in PCT Publication No. WO 02 / 43478.

[0238] Still further, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-CTHRC1 antibodies of the present disclosure. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used, and such mice are described, for example, in U.S. Patent Nos. 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963.

[0239] Moreover, alternative transchromosomic animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-CTHRC1 antibodies of the present disclosure. For example, mice carrying both human heavy chain transchromosomes and human light chain transchromosomes, referred to as "TC mice", can be used, and such mice are described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-7. As another example, cattle carrying human heavy and light chain transchromosomes have been described in the art (e.g., Kuroiwa et al. (2002) Nature Biotechnology 20:889-94 and PCT Application WO2002 / 092812) and can be used to produce the anti-CTHRC1 antibodies of the present disclosure. Additional examples of transgenic animals that can be used to produce anti-CTHCR1 antibodies include OmniRat™ and OmniMouse™ (see, for example, Osborn M., et al. (2013) Journal of Immunology 190:1481-90; Ma B., et al. (2013) Journal of Immunological Methods 400-401:78-86; Geurts A., et al. (2009) Science 325:433; U.S. Patent No. 8,907,157; ​​European Patent No. 2152880B1; European Patent No. 2336329B1). Yet another example includes the use of VELOCIMMUNE® technology (see, for example, U.S. Patent No. 6,596,541; Regeneron Pharmaceuticals, VELOCIMMUNE®). Briefly, VELOCIMMUNE® technology involves the generation of transgenic mice having a genome comprising human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mice produce antigen binding proteins, e.g., antibodies, comprising human variable regions and mouse constant regions in response to antigenic challenge. DNA encoding the antibody heavy and light chain variable regions is isolated and operably linked to DNA encoding the human heavy and light chain constant regions.This DNA is then expressed in a cell capable of expressing fully human antibodies.

[0240] antibody fragment In certain circumstances, there are advantages to using antibody fragments rather than whole antibodies: the smaller size of the fragments allows for rapid clearance and may lead to improved access to solid tumors.

[0241] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments were obtained via proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-7 (1992), and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv, and scFv antibody fragments can all be expressed in and secreted from E. coli, allowing for the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries mentioned 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 directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-life containing salvage receptor binding epitope residues are described in US Patent 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv) (see WO 93 / 16185, US Patent 5,571,894, and US Patent 5,587,458). Fv and sFv are the only species with intact binding sites that are devoid of constant regions, and therefore, they are suitable for reducing non-specific binding during in vivo use. sFv fusion proteins can be constructed to generate fusion of effector proteins at either the amino or carboxy terminus of sFv (see Antibody Engineering, ed. Borrebaeck, supra). An antibody fragment may also be a "linear antibody", eg, as described in US Pat. No. 5,641,870.

[0242] Antibody Variants and Modifications Substitution, insertion, and deletion variants In addition to the anti-CTHRC1 antibodies described herein, it is contemplated that anti-CTHRC1 antibody variants can be prepared. Anti-CTHRC1 antibody variants can be prepared by introducing appropriate nucleotide changes into the coding DNA and / or by synthesis of the desired antibody or polypeptide. Those skilled in the art will understand that amino acid changes can alter post-translational processes of anti-CTHRC1 antibodies, such as changing the number or position of glycosylation sites or changing membrane anchoring properties.

[0243] The changes in the anti-CTHRC1 antibodies described herein can be made using any of the techniques and guidelines for conservative and non-conservative mutations described, for example, in U.S. Pat. No. 5,364,934. The changes can be substitutions, deletions, or insertions of one or more codons encoding the antibody or polypeptide resulting in a change in the amino acid sequence compared to the native sequence antibody or polypeptide. Optionally, the changes are by substitution of at least one amino acid with any other amino acid in one or more of the domains of the anti-CTHRC1 antibody. Guidance in determining which amino acid residues can be inserted, substituted, or deleted without adversely affecting the desired activity can be found by comparing the sequence of the anti-CTHRC1 antibody with the sequence of a known protein molecule of the same species and minimizing the number of amino acid sequence changes made in areas 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, for example, replacing a leucine with a serine, i.e., a conservative amino acid substitution. The insertion or deletion can optionally range from about 1 to 5 amino acids. Permissible changes may be determined by systematically making insertions, deletions, or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

[0244] Anti-CTHRC1 antibody fragments are provided herein.Such fragments may, for example, be N-terminally or C-terminally shortened or lack internal residues when compared with full-length native antibody or protein.Certain fragments lack amino acid residues that are not essential for the desired biological activity of anti-CTHRC1 antibody.

[0245] Anti-CTHRC1 antibody fragments may be prepared by any of several conventional techniques. The desired peptide fragment may be chemically synthesized. An alternative approach involves generating antibody or polypeptide fragments by enzymatic digestion, for example by treating the protein with an enzyme known to cleave the protein at sites defined by specific amino acid residues, or by digesting the DNA with a suitable restriction enzyme and isolating the desired fragment. Yet another suitable technique involves isolating a DNA fragment encoding the desired antibody or polypeptide fragment and amplifying it by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are used at the 5' and 3' primers of the PCR. Preferably, the anti-CTHRC1 antibody fragment shares at least one biological and / or immunological activity with the native anti-CTHRC1 antibody disclosed herein.

[0246] In certain embodiments, conservative substitutions of interest are set forth under the heading of preferred substitutions in Table 6. If such substitutions result in a change in biological activity, more substantial changes, either designated as exemplary substitutions in Table 6 or as further described below with reference to amino acid classes, are introduced and the products screened. [Table 7]

[0247] Substantial alterations in the function or immunological properties of anti-CTHRC1 antibodies are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the region of the substitution, e.g., as a sheet or helix structure, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into the following groups based on common side chain properties: (1) Hydrophobic: norleucine, met, ala, val, leu, ile, (2) Neutral hydrophilicity: cys, ser, thr, (3) Acidic: asp, glu, (4) Basic: asn, gln, his, lys, arg, (5) Residues that affect chain orientation: gly, pro, and (6) Aromatic: trp, tyr, phe

[0248] Non-conservative substitutions involve exchanging a member of one of these classes for another class. Such substituted residues may also be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

[0249] It is understood that conservatively substituted versions of the anti-CTHRC1 fusion proteins or the first or second domains of the anti-CTHRC1 fusion proteins of the present disclosure are included within the scope of the present disclosure.

[0250] The changes can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter et al., Nucl. Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487 (1987)), cassette mutagenesis (Wells et al., Gene, 34:315 (1985)), restriction enzyme selection mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)) or other known techniques can be performed on the cloned DNA to produce anti-CTHRC1 antibody variant DNA.

[0251] Scanning amino acid analysis can also be used 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 the preferred scanning amino acid within this group because it excludes the side chain beyond the beta-carbon and is unlikely to alter the main-chain conformation of the variant (Cunningham and Wells, Science, 244:1081-5 (1989)). Alanine is also typically preferred because it is the most common amino acid. Moreover, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (WHNY); Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not produce a sufficient amount of variants, an isosteric amino acid can be used.

[0252] Any cysteine ​​residues not involved in maintaining the proper conformation of the anti-CTHRC1 antibody may also be substituted, typically with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine ​​bond(s) may be added to the anti-CTHRC1 antibody to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[0253] A particularly preferred type of substitution variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient method for generating such substitution 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 M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or in addition, it may be beneficial to analyze the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the CTHRC1 polypeptide. Such contact and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, a panel of variants may be subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays may be selected for further development.

[0254] Nucleic acid molecules encoding amino acid sequence variants of anti-CTHRC1 antibodies are prepared by a variety of methods known in the art, including, but not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of previously prepared variant or non-variant versions of anti-CTHRC1 antibodies.

[0255] qualification Covalent modification of anti-CTHRC1 antibody is included within the scope of the present invention.One type of covalent modification comprises reacting the targeted amino acid residue of anti-CTHRC1 antibody with an organic derivatizing agent that can react with selected side chain or N-terminal or C-terminal residue of anti-CTHRC1 antibody.For example, derivatization with bifunctional agents is useful for crosslinking anti-CTHRC1 antibody to water-insoluble support matrix or surface for use in the method for purifying anti-CTHRC1 antibody, and vice versa. Commonly used cross-linking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, e.g., esters with 4-azidosalicylic acid, homobifunctional imidoesters including disuccinimidyl esters such as 3,3′-dithiobis(succinimidyl propionate), bifunctional maleimides such as bis-N-maleimido-1,8-octane, and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0256] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0257] Another type of covalent modification of anti-CTHRC1 antibodies that falls within the scope of the present invention includes altering the native glycosylation pattern of the antibody or polypeptide. For the purposes of this specification, "altering the native glycosylation pattern" is intended to mean deleting one or more carbohydrate moieties found in the native sequence anti-CTHRC1 antibody (either by removing the underlying glycosylation site or by deleting glycosylation by chemical and / or enzymatic means) and / or adding one or more glycosylation sites that are not present in the native sequence anti-CTHRC1 antibody. In addition, this phrase includes a qualitative change in the glycosylation of the native protein, involving a change in the nature of the various carbohydrate moieties present and their proportions.

[0258] Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0259] Addition of glycosylation sites to an anti-CTHRC1 antibody is conveniently accomplished by altering the amino acid sequence so that it contains one or more of the above-mentioned tripeptide sequences (for N-linked glycosylation sites). Alterations may also be made by adding or substituting one or more serine or threonine residues to the original anti-CTHRC1 antibody sequence (for O-linked glycosylation sites). Optionally, the anti-CTHRC1 antibody amino acid sequence may be altered through alterations at the DNA level, in particular by mutating the DNA encoding the anti-CTHRC1 antibody at preselected bases so that a codon that translates into the desired amino acid is generated.

[0260] Another means of increasing the number of carbohydrate moieties on an anti-CTHRC1 antibody is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, for example, in WO 87 / 05330 published Sep. 11, 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0261] Removal of carbohydrate moieties present on the anti-CTHRC1 antibody may be accomplished chemically or enzymatically, or by mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and are described, for example, by Hakimuddin, et al., Arch. Biochem. Biophys., 259:52 (1987), and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of various endo- and exo-glycosidases, as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

[0262] Fc region variants It may be desirable to modify the antibody of the invention with respect to effector function, for example to enhance antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This may be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively, or additionally, cysteine ​​residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability, and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (see Caron et al., J. Exp Med. 176:1191-5 (1992); Shopes, BJ Immunol. 148:2918-22 (1992)). Homodimeric antibodies with enhanced antitumor activity may also be prepared using heterobifunctional crosslinkers, as described in Wolff et al., Cancer Research 53:2560-5 (1993). Alternatively, antibodies may be engineered to have dual Fc regions, thereby enhancing complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-30 (1989). To increase the serum half-life of an antibody, a salvage receptor binding epitope may be incorporated into the antibody (especially an antibody fragment), as described, for example, in U.S. Pat. No. 5,739,277. As used herein, the term "salvage receptor binding epitope" refers to an epitope in the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.

[0263] Cysteine ​​Engineered Antibody Variants In certain embodiments, it may be desirable to create cysteine ​​engineered antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are replaced with cysteine ​​residues. In certain embodiments, the replaced residues occur at accessible sites of the antibody. By replacing those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create immunoconjugates as further described herein. Cysteine ​​engineered antibodies can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0264] Certain methods for producing antibodies Screening for anti-CTHRC1 antibodies with desired properties Techniques for generating antibodies that bind to CTHRC1 polypeptides are described above. If desired, antibodies with certain biological properties may be further selected.

[0265] The growth inhibitory effect of the anti-CTHRC1 antibodies of the invention may be evaluated by methods known in the art, for example, using cells expressing CTHRC1 polypeptide, either endogenously or after transfection with the CTHRC1 gene. For example, appropriate tumor cell lines and CTHRC1 transfected cells may be treated with various concentrations of the anti-CTHRC1 monoclonal antibodies of the invention for several days (e.g., 2-7 days) and analyzed by staining with crystal violet or MTT or some other colorimetric assay. Another method of measuring proliferation would be by 3H-thymidine incorporation by cells treated in the presence or absence of the anti-CTHRC1 antibodies of the invention. After treatment, cells are harvested and the amount of radioactivity incorporated into DNA is quantified in a scintillation counter. A suitable positive control includes treatment of a selected cell line with a growth inhibitory antibody known to inhibit the growth of that cell line. Growth inhibition of tumor cells in vivo can be determined in a variety of ways known in the art. The tumor cells may overexpress and / or present CTHRC1 polypeptide. In one embodiment, the anti-CTHRC1 antibody inhibits cell proliferation of CTHRC1-presenting tumor cells in vitro or in vivo by about 25-100%, more preferably about 30-100%, even more preferably about 50-100% or 70-100% compared to untreated tumor cells at an antibody concentration of about 0.5-30 μg / mL. Growth inhibition can be measured at an antibody concentration of about 0.5-30 μg / mL or about 0.5 nM-200 nM in cell culture, where growth inhibition is determined 1-10 days after exposure of tumor cells to the antibody. An antibody is growth inhibitory in vivo if administration of an anti-CTHRC1 antibody at about 1 μg / kg to about 100 mg / kg body weight results in a reduction in tumor size or a reduction in tumor cell proliferation within about 5 days to 3 months, preferably within about 5-30 days, of the first administration of the antibody.

[0266] To select for anti-CTHRC1 antibodies that induce cell death, loss of membrane integrity, as indicated by, for example, propidium iodide (PI), trypan blue, or 7AAD uptake, can be evaluated compared to controls. PI uptake assays can be performed in the absence of complement and immune effector cells. CTHRC1 polypeptide-presenting tumor cells are incubated with medium alone or medium containing the appropriate anti-CTHRC1 antibody (e.g., at about 10 μg / mL). Cells are incubated for a period of 3 days. Following each treatment, cells are washed and aliquoted into 12×75 tubes with 35 mm strainer caps (1 mL per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes are then loaded with PI (10 μg / mL). Samples can be analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). An anti-CTHRC1 antibody that induces a statistically significant level of cell death as determined by PI uptake can be selected as a cell death-inducing anti-CTHRC1 antibody.

[0267] To screen for antibodies that bind to the epitope on the CTHRC1 polypeptide bound by the antibody of interest, a conventional cross-blocking assay can be carried out, such as that described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988). This assay can be used to determine whether the test antibody binds to the same site or epitope as a known anti-CTHRC1 antibody. Alternatively, or in addition, epitope mapping can be carried out by methods known in the art. For example, mutations can be induced in the antibody sequence, for example by alanine scanning, to identify contact residues. Mutant antibodies are first tested for binding with polyclonal antibodies to ensure proper folding. In a different method, peptides corresponding to different regions of the CTHRC1 polypeptide can be used in a competitive assay with the test antibody or with the test antibody and an antibody with a characterized or known epitope.

[0268] In addition, candidate antibodies may also be screened for functionality using one or more of the following: in vivo screening for inhibition of metastasis, inhibition of chemotaxis by in vitro methods (e.g., US2010 / 0061978, incorporated herein by reference in its entirety), inhibition of angiogenesis, inhibition of tumor growth, and reduction in tumor size.

[0269] Certain Library Screening Methods The anti-CTHRC1 antibodies of the present invention can be generated by using combinatorial libraries to screen for antibodies with desired activity(ies). For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with desired binding properties. Such methods are generally described in Hoogenboom et al. (2001) 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.

[0270] In principle, synthetic antibody clones are selected by screening a phage library containing phages displaying various antibody variable region fragments (Fv) fused to a phage coat protein. Such a phage library is panned by affinity chromatography against a desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones in the library. Binding clones are then eluted from the antigen and can be further enriched by additional antigen adsorption / elution cycles. Any of the anti-CTHRC1 antibodies of the present invention can be obtained by designing a suitable antigen screening procedure to select for the phage clone of interest, followed by constructing a full-length anti-CTHRC1 antibody clone using the Fv sequence from the phage clone of interest and the suitable constant region (Fc) sequence described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols.1-3.

[0271] 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 a light (VL) chain and a heavy (VH) chain, each displaying three hypervariable loops (HVRs) or complementarity determining regions (CDRs). The variable domains can be functionally displayed on phage either as single-chain Fv (scFv) fragments in which VH and VL are covalently linked via 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."

[0272] Repertoires of VH and VL genes can be cloned separately by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be searched for antigen-binding clones, as described by Winter et al., Ann. Rev. Immunol., 12:433-55 (1994). Libraries from immunized sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, naive repertoires can be cloned to provide a single source of human antibodies to a wide range of non-self antigens and also self antigens, without any immunization, as described by Griffiths et al., EMBO J, 12:725-34 (1993). Finally, naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and perform in vitro rearrangements, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-8 (1992).

[0273] In certain embodiments, filamentous phage are used to display antibody fragments by fusion to the minor coat protein pill. Antibody fragments can be displayed as single chain Fv fragments in which the VH and VL domains are connected on the same polypeptide chain by a flexible polypeptide spacer, 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 and the assembly of the Fab coat protein structure becomes displayed on the phage surface by replacing a portion of the wild-type coat protein, e.g., as described by Hoogenboom et al., Nucl. Acids Res., 19:4133-7 (1991).

[0274] Generally, nucleic acids encoding antibody gene fragments are obtained from immune cells taken from humans or animals. If a library biased in favor of anti-CTHRC1 clones is desired, subjects are immunized with CTHRC1 to generate an antibody response, and spleen cells and / or circulating B cells, other peripheral blood lymphocytes (PBLs), are collected for library construction. In some embodiments, a human antibody gene fragment library biased in favor of anti-CTHRC1 clones is obtained by generating an anti-CTHRC1 antibody response in a transgenic mouse carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system), such that CTHRC1 immunization generates B cells that produce human antibodies against CTHRC1. The generation of human antibody-producing transgenic mice is described below.

[0275] Further enrichment of the anti-CTHRC1-reactive cell population can be obtained by isolating B cells expressing CTHRC1-specific membrane-bound antibodies using a suitable screening procedure, for example by cell separation using CTHRC1 affinity chromatography or adsorption of cells to fluorescent dye-labeled CTHRC1 followed by flow-activated cell sorting (FACS).

[0276] Alternatively, the use of spleen cells and / or B cells or other PBLs from non-immunized donors provides a better representation of possible antibody repertoires and allows the construction of libraries of antibodies using any animal (human or non-human) species for which CTHRC1 is not antigenic. For libraries incorporating in vitro antibody gene constructs, stem cells are harvested from the subject to provide nucleic acids encoding unrearranged antibody gene segments. Immune cells of interest can be obtained from a variety of animal species, including human, mouse, rat, rabbit, wolf, dog, cat, pig, cow, horse, and avian species.

[0277] Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered and amplified from the cells of interest. In the case of 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) using primers that match the 5' and 3' ends of the rearranged VH and VL genes, as described in Orlandi et al., Proc. Natl. Acad. Sci. (USA), 86:3833-7 (1989), thereby generating a diverse V gene repertoire for expression.

[0278] V genes can be amplified from cDNA and genomic DNA using a reverse primer at the 5' end of the exon encoding the mature V domain and a forward primer placed within the J segment, as described in Orlandi et al. (1989) and Ward et al., Nature, 341:544-6 (1989). However, when amplifying from cDNA, the reverse primer can also be placed in the leader exon as described in Jones et al., Biotechnol., 9:88-9 (1991) and the forward primer can be within the constant region as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86:5728-32 (1989). To maximize complementarity, degeneracy can be built into the primers, as described in Orlandi et al. (1989) or Sastry et al. (1989). In certain embodiments, library diversity is maximized by using PCR primers targeted to each V gene family to amplify all available VH and VL configurations present in the immune cell nucleic acid sample, for example, as described in the method of Marks et al., J. Mol. Biol., 222:581-97 (1991) or Orum et al., Nucleic Acids Res., 21:4491-98 (1993). When cloning the amplified DNA into an expression vector, rare restriction sites can be introduced into the PCR primers as tags at one end, as described in Orlandi et al. (1989), or further PCR amplification can be performed with tagged primers, as described in Clackson et al., Nature, 352:624-628 (1991).

[0279] A repertoire of synthetically rearranged V genes can be obtained 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 major conformations of the H1 and H2 loops) can be used to generate diverse VH gene repertoires using PCR primers encoding H3 loops of diverse sequences and lengths, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). VH repertoires can also be generated by concentrating all sequence diversity in a single long H3 loop, as described by Barbas et al., Proc. Natl. Acad. Sci. USA, 89:4457-61 (1992). Human Vκ and Vλ segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23:1456-61 (1993)) and can be used to generate synthetic light chain repertoires. Synthetic V gene repertoires based on various VH and VL folds and L3 and H3 lengths will encode antibodies with considerable structural diversity. Following amplification of V gene coding DNA, germline V gene segments can be rearranged in vitro according to the method of Hoogenboom and Winter, J. Mol. Biol., 227:381-8 (1992).

[0280] Repertoires of antibody fragments can be constructed by combining VH and VL gene repertoires together in several ways. Each repertoire can be produced in a different vector, and the vectors can be recombined in vitro as described in Hogrefe et al., Gene, 128:119-26 (1993), or in vivo by combinatorial infection, e.g., the loxP system, as described in Waterhouse et al., Nucl. Acids Res., 21:2265-66 (1993). The in vivo recombination approach takes advantage of the double-chain nature of Fab fragments to overcome the limitations on library size imposed by the transformation efficiency of E. coli. Naive VH and VL repertoires are cloned separately, one into a phagemid and the other into a phage vector. These two libraries are then combined by phage infection of phagemid-containing bacteria, such that each cell contains a different combination, with the library size limited only by the number of cells present (approximately 1012 clones). Both vectors contain in vivo recombination signals, which allow the VH and VL genes to be recombined on a single replicon and copackaged into phage virions. These large libraries provide a large number of diverse antibodies with good affinities (Kd-1 of approximately 10-8M).

[0281] 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 may be assembled together by PCR and then cloned, e.g., as described in Clackson et al., Nature, 352:624-628 (1991). PCR assembly can also be used to link VH and VL DNA with DNA encoding a flexible peptide spacer to form single-chain Fv (scFv) repertoires. In yet another technique, VH and VL genes are combined in lymphocytes by PCR and the repertoire of linked genes is then cloned, using "in-cell PCR assembly," as described in Embleton et al., Nucl. Acids Res., 20:3831-3837 (1992).

[0282] Antibodies (either natural or synthetic) produced by naive libraries have moderate affinity (approximately 10 6 ~10 7The affinity maturation can also be mimicked in vitro by constructing a secondary library as described in Winter et al. (1994) (above) and reselecting from them, although affinity maturation can also be mimicked in vitro. For example, mutations can be randomly introduced in vitro by using error-prone polymerases (Leung et al., Technique, 1:11-5 (1989)), in the manner of Hawkins et al., J. Mol. Biol., 226:889-96 (1992), or in the manner 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, for example using PCR with primers carrying random sequences spanning the CDRs of interest in the selected individual Fv clones, and screening for higher affinity clones. WO9607754 described a method for inducing mutagenesis in the complementarity determining regions of immunoglobulin light chains to generate a library of light chain genes. Another effective approach is to recombine VH or VL domains selected by phage display with a repertoire of naturally occurring V domain variants obtained from non-immunized donors and screen for higher affinity by 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.

[0283] Screening of the library can be accomplished by various techniques known in the art.For example, CTHRC1 can be used to coat the well of an adsorption plate, can be expressed on a host cell attached to an adsorption plate, can be used in cell sorting, can be conjugated to biotin for capture with streptavidin-coated beads, or can be used in any other way to pan a phage display library.

[0284] The phage library sample is contacted with the immobilized CTHRC1 under conditions suitable for binding of at least a portion of the phage particles by the adsorbent. Usually, conditions including pH, ionic strength, temperature, etc. are selected to mimic physiological conditions. The phages bound to the solid phase are washed and then eluted by acid, 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 20-1,000-fold in a single selection round. Furthermore, the enriched phages can be grown in bacterial culture and subjected to further rounds of selection.

[0285] The efficiency of selection depends on many factors, including the dissociation rate during washing and whether multiple antibody fragments on a single phage can bind to the antigen simultaneously. Antibodies with fast dissociation rates (and weak binding affinity) can be retained by using short washing times, multivalent phage display, and high coating density of antigen on the solid phase. High density not only stabilizes phage through multivalent interactions, but also favors rebinding of dissociated phage. Selection of antibodies with slow dissociation rates (and good binding affinity) can be promoted by using long washing times and monovalent phage display, as described in Bass et al., Proteins, 8:309-314 (1990) and WO92 / 09690, and by using low coating density of antigen, as described in Marks et al., Biotechnol., 10:779-783 (1992).

[0286] It is possible to select between phage antibodies with different affinities, even if they have slightly different affinities for CTHRC1. However, random mutation of selected antibodies (e.g., as performed in some affinity maturation techniques) can generate many mutants, most of which bind to the antigen and a few with higher affinities. Limiting CTHRC1 can result in the rare high affinity phages being competed out. To retain all higher affinity mutants, phages can be incubated with excess biotinylated CTHRC1, but at a molar concentration lower than the target molar affinity constant of CTHRC1. High affinity binding phages can then be captured by streptavidin-coated paramagnetic beads. Such "equilibrium capture" allows antibodies to be selected according to their binding affinity with a sensitivity that allows the isolation of mutant clones with only 2-fold higher affinities from a large excess of phages with lower affinities. The conditions used in washing the phages bound to the solid phase can also be manipulated to differentiate based on dissociation rates.

[0287] Anti-CTHRC1 clones may be selected based on activity.In certain embodiments, the present invention provides anti-CTHRC1 antibodies that bind to living cells that naturally express CTHRC1.In one embodiment, the present invention provides anti-CTHRC1 antibodies that block the binding between CTHRC1 ligand and CTHRC1, but do not block the binding between CTHRC1 ligand and a second protein. Fv clones corresponding to such anti-CTHRC1 antibodies can be selected by (1) isolating anti-CTHRC1 clones from the phage library described above and, optionally, amplifying the population of isolated phage clones by growing the population in a suitable bacterial host, (2) selecting for CTHRC1 and a second protein for which blocking and non-blocking activity, respectively, is desired, (3) adsorbing the anti-CTHRC1 phage clones to immobilized CTHRC1, (4) using an excess of the second protein to elute any undesired clones that recognize CTHRC1 binding determinants that overlap or are shared with the binding determinants of the second protein, and (5) eluting the clones that remain adsorbed following step (4). Optionally, clones with the desired blocking / non-blocking properties can be further enriched by repeating the selection procedure described herein one or more times.

[0288] DNA encoding the hybridoma-derived monoclonal antibodies or phage-displayed Fv clones of the present 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 a 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, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, resulting in the synthesis of the desired monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol. 5:256 (1993) and Pluckthun, Immunol. Rev. 130:151 (1992).

[0289] DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding heavy and / or light chain constant regions (e.g., suitable DNA sequences can be obtained from Kabat et al., supra) to form clones encoding full-length or partial-length heavy and / or light chains. It will be understood that constant regions of any isotype can be used for this purpose, including IgG, IgM, IgA, IgD, and IgE constant regions, and such constant regions can be derived from any human or animal species. Fv clones derived from variable domain DNA of one animal (such as human) species and then fused to constant region DNA of another animal species to form the coding sequence(s) for a "hybrid" full-length heavy and / or light chain are included in the definition of "chimeric" and "hybrid" antibodies as used herein. In certain embodiments, Fv clones derived from human variable DNA are fused to human constant region DNA to form the coding sequence(s) for a full-length or partial-length human heavy and / or light chain.

[0290] DNA encoding the hybridoma-derived anti-CTHRC1 antibody can also be modified, for example, by substituting coding sequences for human heavy and light chain constant domains in place of the 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 the hybridoma- or Fv-derived antibody or fragment can be further modified by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this way, "chimeric" or "hybrid" antibodies having the binding specificity of the antibody derived from the Fv clone or hybridoma clone of the invention are prepared.

[0291] Generation of antibodies using CAR-T cells The anti-CTHRC1 antibody of the present invention can be generated by screening for antibodies with the desired activity(ies) using a CAR T cell platform. Chimeric antigen receptors (CARs) are composed of an extracellular antigen recognition domain (usually a single-chain variable fragment (scFv) antibody) linked to a transmembrane domain and a cytoplasmic signaling domain. Alvarez-Vallina, L, Curr Gene Ther 1:385-97 (2001). CAR-mediated recognition transforms tumor-associated antigens (TAA) expressed on the cell surface into recruitment points for effector function, addressing the goal of effector cell activation independent of major histocompatibility complexes. First generation CARs were typically constructed by fusing scFv-based TAA binding domains to cytoplasmic signaling domains derived from either the zeta chain of the T cell receptor (TCR) / CD3 complex or the gamma chain associated with some Fc receptors (Gross, G. et al., Proc Natl Acad Sci USA 86:10024-8 (1989)). Second generation CARs (CARv2) have also been developed that contain the signaling region of TCRζ in tandem with signaling domains derived from the T cell costimulatory receptors CD28, 4-1BB (CD137), or OX40 (CD134) (Sanz, L. et al., Trends Immunol 25:85-91 (2004)). Third generation CARs further combine the signaling potential of two costimulatory domains (e.g., both CD28 and 4-1BB) (Subklewe, M., et al., Transfus Med Hemother 46(1):15-24 (2019)).

[0292] Upon encountering an antigen, the interaction of the transfected CAR can trigger effector functions and mediate cytolysis of tumor cells. The utility and efficacy of the CAR approach has been demonstrated in various animal models and in ongoing clinical trials using CAR-based engineered T lymphocytes for the treatment of cancer patients. Lipowska-Bhalla, G. et al., Cancer Immunol Immunother 61:953-62 (2012). CARs allow the targeting of effector cells to any natural 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)).

[0293] Current methods for the generation of recombinant antibodies are mainly based on the use of purified proteins. Hoogenboom, HR et al., Nat Biotechnol 23:1105-1116 (2005). However, a mammalian cell-based antibody display platform has recently been described that exploits the functional capabilities of T lymphocytes. Alonso-Camino et al., Molecular Therapy Nucleic Acids (2013) 2, e93. Presentation of antibodies on the surface of T lymphocytes as part of CAR-mediated signaling could ideally link antigen-antibody interactions to demonstrable changes in cell phenotype due to surface expression of activation markers. Alonso-Camino, V. et al., PLoS ONE 4:e7174 (2009). By using an scFv-based CAR that recognizes a TAA, CAR-mediated activation combined with fluorescence-activated cell sorting (FACS) of CD69+ T cells resulted in at least 10 mAbs after two rounds. 3It has been demonstrated that a 3-fold enrichment factor makes it possible to isolate binders to surface TAAs, resulting in a homogenous population of T cells expressing a TAA-specific CAR. Alonso-Camino, V, et al., PLoS ONE 4:e7174 (2009).

[0294] Preparation of anti-CTHRC1 antibody The following description is mainly directed to the production of anti-CTHRC1 antibodies by culturing cells transformed or transfected with a vector containing anti-CTHRC1 antibody-encoding nucleic acid. Of course, it is contemplated that anti-CTHRC1 antibodies may be prepared using alternative methods well known in the art. For example, the appropriate amino acid sequence, or a portion thereof, may be produced by direct peptide synthesis using solid-phase techniques (e.g., Stewart et al., Solid-Phase Peptide Synthesis, WH 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 example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) using the manufacturer's instructions. Various portions of the anti-CTHRC1 antibody may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-CTHRC1 antibody.

[0295] Isolation of DNA encoding anti-CTHRC1 antibody DNA encoding an anti-CTHRC1 antibody may be obtained from a cDNA library prepared from tissues believed to harbor and express anti-CTHRC1 antibody mRNA at detectable levels. Thus, human anti-CTHRC1 antibody DNA can be conveniently obtained from a cDNA library prepared from human tissue. Anti-CTHRC1 antibody-encoding genes may also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0296] Libraries can be screened using 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 of cDNA or genomic libraries with selected probes can be performed using standard procedures, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative means for isolating genes encoding anti-CTHRC1 antibodies is to use PCR techniques (Sambrook et al., supra; Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[0297] Techniques for screening cDNA libraries are well known in the art. The oligonucleotide sequence selected as a probe should be of sufficient length and sufficiently unambiguous to minimize false positives. The oligonucleotide is preferably labeled so that it can be detected upon hybridization to DNA in the library being screened. Labeling methods are well known in the art and include the use of radioactive labels such as 32P-labeled ATP, biotinylation, or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al. (supra).

[0298] 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.

[0299] Nucleic acids having protein-coding sequences may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequences disclosed herein for the first time, and, if necessary, 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.

[0300] Selection and transformation of host cells Host cells are transfected or transformed with the expression or cloning vectors described herein for producing anti-CTHRC1 antibodies and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify genes encoding desired sequences. Culture conditions such as media, temperature, pH, etc. can be selected by those skilled in the art without undue experimentation. In general, principles, protocols, and practical techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al. (supra).

[0301] Linker In any of the above-mentioned embodiments, the anti-CTHRC1 fusion protein may further comprise a peptide linker located between the first domain and the second domain. In certain aspects, the peptide linker directly connects the first domain to the second domain. It should be understood that the first domain and the second domain may be located at either end of the peptide linker. For example, the anti-CTHRC1 fusion protein may comprise (first domain-linker-second domain) or (second domain-linker-first domain).

[0302] In any of the above-described embodiments, the peptide linker can be any suitable linker. By way of example and not limitation, the peptide linker can be (G4S) n , (GSG) n , (SG4) n , G4(SG4) n It may include a peptide linker, where n is 1 to 4. Preferably, the linker has the amino acid sequence of SEQ ID NO: 100 ((G4S)3).

[0303] Additional linkers are also described in U.S. Patent No. 11,053,293 and U.S. Patent Application Publication No. 2019 / 0016771, each of which is incorporated herein by reference in its entirety. Further exemplary linkers can include GGGGSGGGGS (SEQ ID NO: 109), SGGGGSGGGG (SEQ ID NO: 110), GGGGSGGGGSGGGG or G4(SG4)2 (SEQ ID NO: 111), and (G4S)4 or GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 112), GSPGSSSSGS (SEQ ID NO: 113), GSGSGSGS (SEQ ID NO: 114), GSGSGNGS (SEQ ID NO: 115), GGSGSGSG (SEQ ID NO: 116), GGSGSG (SEQ ID NO: 117), GGSG (SEQ ID NO: 118), GGSGNGSG (SEQ ID NO: 119), GGNGSGSG (SEQ ID NO: 120), and GGNGSG (SEQ ID NO: 121).

[0304] Alternative methods and designs for constructing anti-CTHRC1 fusion proteins of the present disclosure are provided in US Pat. No. 10,392,445, which is incorporated herein by reference.

[0305] Pharmaceutical Compositions In embodiments, the anti-CTHRC1 fusion proteins of the disclosure may be formulated into a pharma- ceutically acceptable composition, which may include an anti-CTHRC1 fusion protein and a pharma- ceutically acceptable carrier.

[0306] The anti-CTHRC1 fusion proteins and compositions thereof of the invention may be administered by any route appropriate to the condition to be treated. Fusion proteins will typically be administered parenterally, i.e., by injection, subcutaneously, intramuscularly, intravenously, intradermally, intrathecally, and epidurally.

[0307] For the treatment of these cancers, in one embodiment, the antibody is administered via intravenous infusion. The dosage administered via infusion ranges from about 0.001 mg / kg to about 100 mg / kg per dose based on the subject's body weight, generally spanning one dose per week for a total of 1, 2, 3, or 4 doses. Alternatively, the dosage ranges are from 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. ... 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. Doses may be administered once a day, once a week, multiple times a week but less than once a day, multiple times a month but less than once a day, multiple times a month but less than once a week, once a month, or intermittently to reduce or alleviate symptoms of the disease. Administration may continue at any of the disclosed intervals until symptoms of the tumor or cancer being treated are in remission. Administration may be continued after remission or alleviation of symptoms has been achieved, if such remission or alleviation is prolonged by such continued administration.

[0308] Therapeutic formulations containing the anti-CTHRC1 fusion proteins used according to the present invention are prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the anti-CTHRC1 fusion protein having the desired degree of purity with optional pharma- ceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). 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; protein complexes such as serum albumin, gelatin, or immunoglobulins. Proteins; 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 dextrin; chelating agents such as EDTA; tonicifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose, or sorbitol; surfactants such as polysorbates; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONIC®, or polyethylene glycol (PEG). Pharmaceutical formulations used for in vivo administration are generally sterile. This is readily accomplished by filtration through sterile filtration membranes.

[0309] The active ingredient may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, such as hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1980).

[0310] Sustained release preparations may be prepared. Suitable examples of sustained release preparations include semipermeable matrices of hydrophobic solid 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 (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (such as 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 allow the release of molecules for more than 100 days, certain hydrogels release proteins for shorter periods of time. If encapsulated immunoglobulins remain in the body for extended periods of time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Depending on the mechanism involved, rational strategies for stabilization can be implemented. For example, if the aggregation mechanism is found to be the formation of intermolecular S-S bonds by thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

[0311] The anti-CTHRC1 fusion protein of the present disclosure may be formulated in any form suitable for delivery to target cells / tissues.For example, the anti-CTHRC1 fusion protein may be formulated as an immunoliposome.A "liposome" is a small vesicle composed of various types of lipids, phospholipids, and / or surfactants that is useful for delivery of drugs to mammals.The components of the liposome are generally arranged in a bilayer formation similar to the lipid arrangement of biological membranes. Liposomes containing antibodies are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985), Hwang et al., Proc. Natl Acad. Sci. USA 77:4030 (1980), U.S. Patent Nos. 4,485,045 and 4,544,545, and WO 97 / 38731 published October 23, 1997. Liposomes with improved circulation time are disclosed in U.S. Patent No. 5,013,556.

[0312] Particularly useful liposomes can be generated by the reverse phase evaporation method using 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 invention can be conjugated to liposomes via a disulfide interchange reaction as described in Martin et al., J. Biol. Chem. 257:286-8 (1982). Optionally, a chemotherapeutic agent is included within the liposome (see Gabizon et al., J. National Cancer Inst. 81(19):1484 (1989)).

[0313] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0314] Manufacturing method Conventional methods for the production of fusion proteins are known in the art.

[0315] Eukaryotic cell transfection and prokaryotic cell transformation methods, which refer to the introduction of DNA into a host so that it can replicate, either extrachromosomally or by chromosomal integrant, are known to those skilled in the art, such as CaCl2, CaPO4, liposome-mediated, polyethylene-glycol (gycol) / DMSO, and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate for such cells. Calcium treatment using calcium chloride as described by Sambrook et al. (supra), or electroporation, are commonly 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 WO89 / 05859 published June 29, 1989. For mammalian cells that do not have such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be used. General aspects of mammalian cell host system transfection are described in U.S. Patent No. 4,399,216. Transformation into yeast is typically performed according to the methods of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao et al., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, other methods for introducing DNA into cells may also be used, such as by intranuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

[0316] In some embodiments, the polynucleotide of the present disclosure is provided that encodes the anti-CTHRC1 fusion protein.In some embodiments, the polynucleotide of the present disclosure that encodes the anti-CTHRC1 fusion protein can be incorporated into a suitable vector for expressing the anti-CTHRC1 fusion protein in host cell.

[0317] The vector may be in the form of, for example, 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 a suitable vector containing one or more of these components employs standard ligation techniques known to those skilled in the art.

[0318] The anti-CTHRC1 fusion protein may be recombinantly produced and may further comprise a heterologous polypeptide, which may be a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature protein or polypeptide, or an affinity tag such as a 6xHis tag. In general, the signal sequence may be a component of the vector, or it may be part of the anti-CTHRC1 fusion protein-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence, for example, selected from the group of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence may be, for example, the yeast invertase leader, alpha factor leader (including the Saccharomyces and Kluyveromyces α-factor leaders, the latter of which is described in U.S. Pat. No. 5,010,182), or the acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published April 4, 1990), or a signal described in WO 90 / 13646 published November 15, 1990. In expression in mammalian cells, mammalian signal sequences, such as signal sequences from secreted polypeptides of the same or related species, may be used to direct secretion of the protein, as well as viral secretory leaders.

[0319] For recombinant production of the fusion protein of the present 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. The DNA encoding the fusion protein is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the antibody). Many vectors are available. The choice of vector depends, in part, on the host cell to be used. In general, preferred host cells are of either prokaryotic or eukaryotic (generally mammalian) origin. Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryotic, yeast, or higher eukaryotic cells.

[0320] The vector may be in the form of, for example, 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 a suitable vector containing one or more of these components employs standard ligation techniques known to those skilled in the art.

[0321] Anti-CTHRC1 fusion proteins can be produced recombinantly not only directly but also as fusion polypeptides with heterologous polypeptides, which may be signal sequences or other polypeptides with specific cleavage sites 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-CTHRC1 antibody-encoding DNA that is inserted into the vector. The signal sequence may be a prokaryotic signal sequence, for example, selected from the group of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the signal sequence may be, for example, the yeast invertase leader, alpha factor leader (including the Saccharomyces and Kluyveromyces α-factor leaders, the latter of which is described in U.S. Pat. No. 5,010,182), or the acid phosphatase leader, the C. albicans glucoamylase leader (EP 362,179 published April 4, 1990), or the signals described in WO 90 / 13646 published November 15, 1990. In expression in mammalian cells, a mammalian signal sequence, such as a signal sequence from a secreted polypeptide of the same or a closely related species, may be used in conjunction with a viral secretory leader to direct secretion of the protein. In some embodiments, the anti-CTHRC1 fusion protein may be produced with a tag, such as a 6×His tag.

[0322] vector Nucleic acid sequences encoding the desired molecules can be obtained using recombinant methods known in the art, e.g., by screening libraries from cells which express the gene, by deriving the gene from a vector known to contain the gene, or by directly isolating it from cells and tissues containing the gene, using standard techniques, etc. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0323] The present invention also provides a vector into which the DNA of the present invention is inserted.Vector derived from retroviruses such as lentiviruses is a suitable tool for achieving long-term gene transfer, since it allows long-term stable integration of transgenes and their proliferation in daughter cells.Compared to vector derived from oncoretroviruses such as mouse leukemia virus, lentivirus vectors have the additional advantage that they can transduce non-proliferating cells such as hepatocytes.They also have the additional advantage of being less immunogenic.

[0324] In brief summary, expression of natural or synthetic nucleic acid encoding CAR is typically carried out by operably linking the nucleic acid encoding the anti-CTHRC1 fusion protein or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. A typical cloning vector contains transcription and translation terminators, initiation sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0325] In addition to the methods described above, the following methods may be used.

[0326] The expression constructs of the present invention may also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art (e.g., U.S. Patent Nos. 5,399,346, 5,580,859, 5,589,466, which are incorporated herein by reference in their entirety). In another embodiment, the present invention provides gene therapy vectors.

[0327] Nucleic acids can be cloned into several types of vectors. For example, nucleic acids can be cloned into vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0328] Furthermore, the expression vector may be provided to the cell in the form of a viral vector. Viral vector technology is well known in the art and described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0329] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. Recombinant viruses can then be isolated and delivered to target cells in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Several adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0330] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30-110 bp upstream of the start site, but in recent years, some promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, so that promoter function is maintained 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 a spacing of 50 bp, after which activity begins to decrease. Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription.

[0331] 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 promoting high levels of expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters, such as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter, and the creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or turn off expression when such expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionine promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0332] To evaluate the expression of anti-CTHRC1 fusion protein or a portion thereof, the expression vector introduced into the cell may also contain either a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from the cell population to be transfected or infected via the viral vector. In other embodiments, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences that allow expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0333] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. In general, reporter genes are genes that are not present in or expressed by the recipient organism or tissue and that code for a polypeptide whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein genes (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 that shows the highest expression level of the reporter gene with the minimal 5' flanking region is identified as the promoter. Such promoter regions may be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0334] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, the vectors can be easily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, the expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0335] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). One method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0336] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, especially retroviral vectors, have become the most widely used method for gene insertion into mammalian, e.g., human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type I, adenoviruses and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0337] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle). When utilizing a non-viral delivery system, an exemplary delivery vehicle is a liposome. The use of lipid formulations is contemplated for the introduction of nucleic acid into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid may be associated with lipid. The nucleic acid associated with lipid may be encapsulated in the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, bound to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, entrapped in the liposome, complexed with the liposome, dispersed in a solution containing lipid, mixed with lipid, combined with lipid, contained in a suspension in lipid, contained in or complexed with a micelle, or otherwise associated with lipid. The lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they may exist in a bilayer structure, as a micelle, or in a "collapsed" structure. They may also simply be dispersed in the solution, possibly forming aggregates that are not uniform in size or shape.Lipids are fatty substances that can be naturally occurring lipids or synthetic lipids.For example, lipids include the lipid droplets that naturally occur in cytoplasm, as well as the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes.

[0338] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, Mo., dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY), cholesterol ("Choi") can be obtained from Calbiochem-Behring, and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid stock solutions in chloroform or chloroform / methanol can be stored at about -20 degrees Celsius. Chloroform is used as the sole solvent because it evaporates more easily than methanol. "Liposome" is a generic term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, trapping water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions that have structures in solution that differ from normal vesicular structures are also encompassed. For example, lipids may adopt micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0339] Regardless of the method used to introduce exogenous nucleic acid into a host cell or otherwise expose the cell to an inhibitor of the invention, various assays may be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as detecting the presence or absence of specific peptides, for example, by immunological means (ELISA and Western blot) or by the assays described herein to identify agents within the scope of the invention.

[0340] prokaryotic host cells Polynucleotide sequences encoding the polypeptide components of the antibody of the present invention can be obtained using standard recombinant techniques. The desired polynucleotide sequences can be isolated and sequenced from antibody producing cells, such as hybridoma cells. Alternatively, polynucleotides can be synthesized using a nucleotide synthesizer or PCR techniques. Once obtained, the polypeptide encoding sequence is inserted into a recombinant vector capable of replicating and expressing heterologous polynucleotides in a prokaryotic host. Many vectors available and known in the art can be used for the purposes of the present invention. The selection of an appropriate vector depends primarily on the size of the nucleic acid to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the particular host cell in which it resides.

[0341] Generally, plasmid vectors containing replicon and control sequences derived from species compatible with the host cell are used in connection with these hosts. Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells, as well as marking sequences that can provide phenotypic selection in transformed cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322, which contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides a convenient means for identifying transformed cells, is suitable for most Gram-negative bacteria, the origin of the 2μ plasmid is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by the microorganism for expression of endogenous proteins. Examples of pBR322 derivatives used to express particular antibodies are detailed in Carter et al., US Pat. No. 5,648,237.

[0342] In addition, phage vectors containing replicon and control sequences compatible with the host microorganism can be used as transforming vectors in connection with these hosts. For example, bacteriophage such as λGEM™-11 can be utilized to generate recombinant vectors that can be used to transform susceptible host cells such as E. coli LE392.

[0343] The expression vector of the present invention may contain two or more promoter-cistron pairs, each encoding a polypeptide component. A promoter is a non-translated control sequence located upstream (5') of a cistron that regulates its expression. Prokaryotic promoters are typically divided into two classes: inducible promoters and constitutive promoters. An inducible promoter is a promoter that initiates increased levels of transcription of the cistron under its control in response to a change in culture conditions, such as the presence or absence of a nutrient or a change in temperature.

[0344] A large number of promoters recognized by various potential host cells are well known. The selected promoter can be operably linked to the cistron DNA encoding the light or heavy chain by removing the promoter from the source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Both the native promoter sequence and many heterologous promoters can be used to direct the amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they generally allow greater transcription and higher yields of the expressed target gene compared to the native target polypeptide promoter.

[0345] Promoters recognized by a variety of potential host cells are well known. Suitable promoters for use with prokaryotic hosts include the PhoA promoter, β-galactamase and lactose promoter systems (Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281:544 (1979)), alkaline phosphatase, tryptophan (trp) promoter system (Goeddel, Nucleic Acids Res., 8:4057 (1980); EP 36, 776), as well as hybrid promoters such as tac (deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)) or trc promoter. Promoters for use in bacterial systems will also contain a Shine-Dalgarno (SD) sequence operably linked to the DNA encoding the anti-CTHRC1 antibody. However, other promoters functional in bacteria (e.g., other known bacterial or phage promoters) are also suitable, whose nucleotide sequences have been published, enabling one of skill in the art to operably link them to cistrons encoding the target light and heavy chains, using linkers or adapters to provide any required restriction sites (Siebenlist et al. (1980) Cell 20:269).

[0346] In one aspect of the invention, each cistron in the recombinant vector contains a secretory signal sequence component that directs the translocation of the expressed polypeptide across the membrane. In general, the signal sequence may be a component of the vector or it may be part of the target polypeptide DNA that is inserted into the vector. The signal sequence selected for the purposes of the present invention should be one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. In the case of prokaryotic host cells that do not recognize and process the signal sequence native to the heterologous polypeptide, the signal sequence is replaced by a prokaryotic signal sequence selected from the group consisting of, for example, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II (STII) leader, LamB, PhoE, PelB, OmpA, and MBP. In one embodiment of the invention, the signal sequence used in both cistrons of the expression system is the STII signal sequence or a variant thereof.

[0347] In another embodiment, production of immunoglobulins according to the present invention can occur in the cytoplasm of the host cell and therefore does not require the presence of a secretion signal sequence within each cistron. In this regard, immunoglobulin light and heavy chains are expressed, folded and assembled in the cytoplasm to form functional immunoglobulins. Certain host strains (e.g., E. coli trxB-strains) provide cytoplasmic conditions that are favorable for disulfide bond formation, thereby allowing proper folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0348] The present invention provides an expression system in which the quantitative ratio of expressed polypeptide components can be adjusted to maximize the yield of secreted, properly assembled fusion proteins of the invention, such adjustment being accomplished, at least in part, by simultaneously adjusting the translational strength for the polypeptide components.

[0349] One technique for modulating translation strength is disclosed in US Patent No. 5,840,523 to Simmons et al. It utilizes variants of the translation initiation region (TIR) ​​within a cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be created with varying translation strengths, thereby providing a convenient means to adjust this factor for the desired expression level of a particular chain. TIR variants can be generated by conventional mutagenesis techniques that result in codon changes that can alter the amino acid sequence, although silent changes in the nucleotide sequence are preferred. Changes in the TIR can include, for example, changes in the number or spacing of Shine-Dalgarno sequences in addition to changes in the signal sequence. One method for generating variant signal sequences is to generate a "codon bank" at the beginning of the coding sequence that does not 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. In addition, some amino acids, such as leucine, serine, and arginine, have multiple first and second positions, which can complicate the creation of the bank. This mutagenesis method is detailed in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.

[0350] Preferably, a set of vectors is generated with various TIR strengths for each cistron therein. This limited set provides a comparison of the expression levels of each chain as well as the yield of the desired antibody product under various combinations of TIR strengths. TIR strength can be determined by quantifying the expression level of a reporter gene, as detailed in Simmons et al., U.S. Patent No. 5,840,523. Based on the comparison of translation strengths, the desired individual TIRs are selected and combined in the expression vector construct of the present invention.

[0351] Suitable prokaryotes include, but are not limited to, archaebacteria and eubacteria, e.g., gram-negative or gram-positive organisms, e.g., Enterobacteriaceae, such as E. coli. Various strains of E. coli are publicly available, such as the K12 strains MM294 (ATCC 31,446), X1776 (ATCC 31,537), W3110 (ATCC 27,325), and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P, disclosed in DD 266,710, published April 12, 1989), Pseudomonas such as P. aeruginosa, Rhizobia, Vitreoscilla, Paracoccus, and Streptomyces. These examples are illustrative and not limiting. E. coli strain W3110 is one particularly preferred host or parent host, as it is a common host strain for fermentation of recombinant DNA products. Preferably, the host cells secrete minimal amounts of proteolytic enzymes.For example, strain W3110 (Bachmann, Cellular and Molecular Biology, vol. 2 (Washington, DC: American Society for Microbiology, 1987), pp. 1190-1219; ATCC Accession No. 27,325) can be modified to create genetic mutations in genes encoding proteins endogenous to the host, examples of such hosts include E. coli W3110 strain 1A2 with the complete genotype tonA; E. coli W3110 strain 9E4 with the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244) with the complete genotype tonA ptr3 phoA E15(argF-lac) 169 degP ompT kanr; E. coli W3110 strain 27C7 (ATCC 55,244) with the complete genotype tonA ptr3 phoA E15(argF-lac) 169 degP ompT rbs7 ilvG kanr. E. coli W3110 strain 37D6, E. coli W3110 strain 40B4, which is strain 37D6 with a degP deletion mutation that is not kanamycin resistant, E. coli W3110 strain 33D3 with the genotype W3110ΔfhuA(ΔtonA)ptr3 lac Iq lacL8ΔompTΔ(nmpc-fepE)degP41 kanR (U.S. Patent No. 5,639,635), and E. coli strains with mutant periplasmic proteases as disclosed in U.S. Patent No. 4,946,783 issued August 7, 1990. Other strains and derivatives thereof, such as E. coli 294 (ATCC 31,446), E. coli B, E. coli λ 1776 (ATCC 31,537), and E. coli RV308 (ATCC 31,608), are also suitable. These examples are illustrative and not limiting. Methods for constructing derivatives of any of the above bacteria with defined genotypes are known in the art and are described, for example, in Bass et al., Proteins, 8:309-314 (1990). In general, it is necessary to select an appropriate bacterium taking into account the replicability of the replicon in the bacterial cell.For example, E. coli, Serratia, or Salmonella species may be suitably used as hosts when well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410 are used to supply the replicon. Typically, the host cell should secrete minimal amounts of proteolytic enzymes, and additional protease inhibitors may be desirably incorporated into the cell culture. Alternatively, in vitro methods of cloning, for example by PCR or other nucleic acid polymerase reactions, are suitable.

[0352] Antibody fusion proteins can be produced in bacteria, especially when glycosylation and Fc effector functions are not required. Full-length antibodies have a longer half-life in blood circulation. Production in E. coli is faster and more cost-effective. For expression of antibody fragments and polypeptides in bacteria, see, for example, US 5,648,237, US 5,789,199, and US 5,840,523, which describe translation initiation regions (TIRs) and signal sequences for optimizing expression and secretion, and are incorporated herein by reference. After expression, the fusion protein can be isolated from E. coli cell paste and purified, for example, through a protein A or G column depending on the isotype. Final purification can be performed similarly to the process for purifying antibodies expressed, for example, in CHO cells.

[0353] eukaryotic host cell In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for anti-CTHRC1 fusion protein-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Other microorganisms include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 (1981), EP 139,383 published May 2, 1985); e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 (1983)), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg ... spp. (ATCC 15,102), K. spp. (ATCC 15,102), K. spp. (ATCC 15,102), K. spp. (ATCC 15,102), K. spp. (ATCC 15,102), K. spp. (ATCC 15,102), K. spp. (ATCC 15,102), K. spp. (ATCC 1 Kluyveromyces hosts such as K. al., Bio / Technology, 8:135 (1990)), K. thermotolerans, and K. marxianus (US Pat. No. 4,943,529, Fleer et al., Bio / Technology, 9:968-75 (1991)); yarrowia (EP402,226); Pichia pastoris(EP183,070, Sreekrishna et al.,J.Basic Microbiol.,28:265-278(1988));Candida;Trichoderma reesia(EP244,234);Neurospora crassa(Case et al. al.,Proc.Natl.Acad.Sci.USA,76:5259-5263(1979));Schwanniomyces Schwanniomyces, such as A. occidentalis (EP 394,538 published October 31, 1990); as well as, for example, Neurospora, Penicillium, Tolypocladium (WO 91 / 00357 published January 10, 1991), and Aspergillus hosts, such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289 (1983), Tilburn et al., Gene, 26:205-221 (1983), Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 (1984)) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 (1985)). Methylotrophic yeasts are suitable herein, including, but not limited to, yeasts capable of growing on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species representative of this class of yeast can be found in C. Anthony, The Biochemistry of Methylotrophs, 269 (1982). .

[0354] Suitable host cells for the expression of glycosylated fusion proteins are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, and plant cells such as cotton, corn, potato, soybean, petunia, tomato, and tobacco cell cultures. Numerous baculovirus strains and variants have been identified, as well as corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as viruses herein according to the invention, particularly for transfection of Spodoptera frugiperda cells.

[0355] However, vertebrate cells have received the most attention, and propagation of vertebrate cells in culture (tissue culture) has become routine procedure. Examples of useful mammalian host cell lines are monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL1651), human embryonic kidney cell line (293 cells or 293 cells subcloned to grow in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC CCL10), Chinese hamster ovary cells / -DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)), mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1 ATCC CCL70), African green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical carcinoma cells (HELA, ATCC CCL2), canine kidney cells (MDCK, ATCC CCL34), buffalo rat liver cells (BRL 3A, ATCC CRL1442), human lung cells (W138, ATCC CCL75), human liver cells (Hep G2, HB8065), mouse mammary carcinoma (MMT060562, ATCC CCL51), TRI cells (Mather et al., Annals NYAcad. Sci. 383:44-68 (1982)), MRC5 cells, FS4 cells, and a human hepatocellular carcinoma cell line (Hep G2).

[0356] 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.

[0357] Signal sequence components Vectors for use in eukaryotic host cells may also contain a signal sequence or other polypeptide with a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The heterologous signal sequence selected is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For expression in mammalian cells, mammalian signal sequences as well as viral secretory leaders, such as the herpes simplex gD signal, are available.

[0358] The DNA for such precursor region is ligated in reading frame to DNA encoding the fusion protein.

[0359] origin of replication Generally, the origin of replication component is not needed for mammalian expression vectors For example, the SV40 origin may typically be used only because it contains the early promoter.

[0360] Selection gene components Expression and cloning vectors will typically contain a selection gene, also called a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline, (b) complement an auxotrophic deficiency, or (c) supply vital nutrients unavailable from complex media, such as the gene encoding D-alanine racemase for Bacilli.

[0361] One example of a selection scheme utilizes a drug to arrest the growth of a host cell. Cells that are successfully transformed with a heterologous gene produce a protein that confers drug resistance and thus survive the selection regime. Examples of such dominant selection use the drugs neomycin, mycophenolic acid, and hygromycin.

[0362] Examples of suitable selectable markers for mammalian cells are those that allow the identification of cells that are competent to take up anti-CTHRC1 fusion protein-encoding nucleic acid, such as DHFR, or thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc. When wild-type DHFR is used, a suitable host cell is a CHO cell line (e.g., ATCC CRL-9096) that is deficient in DHFR activity, which is prepared and grown as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Alternatively, host cells transformed or co-transformed with an anti-CTHRC1 fusion protein, a wild-type DHFR protein, and a DNA sequence encoding another selectable marker, such as an aminoglycoside 3'-phosphotransferase (APH), (particularly wild-type hosts containing endogenous DHFR), can be selected by growing the cells in medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

[0363] A suitable selection gene for use in yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)). The trp1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow on tryptophan, e.g., ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)).

[0364] Promoter components Expression and cloning vectors usually contain a promoter operably linked to the anti-CTHRC1 fusion protein-encoding nucleic acid sequence to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known.

[0365] Virtually all eukaryotic genes have an AT-rich region located approximately 25-30 bases upstream from the site where transcription begins. Another sequence found 70-80 bases upstream from the transcription start of many genes is a CNCAAT region, where N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of a poly A tail to the 3' end of the coding sequence. All of these sequences are suitable for insertion into eukaryotic expression vectors.

[0366] Examples of promoting sequences suitable for use with yeast hosts include promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0367] Other yeast promoters that are inducible promoters with the added advantage that transcription is controlled by growth conditions are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes involved in maltose and galactose utilization. Vectors and promoters suitable for use in yeast expression are further described in EP 73,657.

[0368] Transcription of the anti-CTHRC1 fusion protein from the vector in mammalian host cells is controlled by promoters derived from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,211,504 published July 5, 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and Simian Virus 40 (SV40), heterologous mammalian promoters such as actin promoters or immunoglobulin promoters, and heat shock promoters, provided that such promoters are compatible with the host cell system.

[0369] The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982), on the expression of human β-interferon cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus. Alternatively, the long terminal repeat of Rous sarcoma virus can be used as a promoter.

[0370] Enhancer element components Transcription of DNA encoding an anti-CTHRC1 fusion protein by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about 10-300 bp, that act on a promoter to increase transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, enhancers from eukaryotic viruses will be used. Examples include the SV40 enhancer on the late side of the replication origin (base pairs 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) for enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced ​​into the vector at a position 5' or 3' to the anti-CTHRC1 fusion protein coding sequence, but is preferably located at a site 5' from the promoter.

[0371] Transcription termination components Expression vectors used in eukaryotic host cells (yeast, fungi, insects, plants, animals, humans, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and stabilization of mRNA. Such sequences are commonly available from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the anti-CTHRC1 fusion protein. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94 / 11026 and the expression vectors disclosed therein.

[0372] Still other methods, vectors, and host cells suitable for adapting the synthesis of anti-CTHRC1 fusion proteins 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.

[0373] Host cells are transformed with the above-described expression or cloning vectors for producing anti-CTHRC1 fusion proteins and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

[0374] Host cell culture The host cells used to produce the anti-CTHRC1 fusion protein of this invention may be cultured in a variety of media.

[0375] prokaryotic host cells The prokaryotic cells used to produce the polypeptide of the present invention are grown in a medium known in the art and suitable for culturing the selected host cell. An example of a suitable medium is Luria Broth (LB) with necessary nutritional supplements. In some embodiments, the medium also contains a selection agent selected based on the structure of the expression vector to selectively allow the growth of prokaryotic cells containing the expression vector. For example, ampicillin is added to the medium for the growth of cells expressing an ampicillin resistance gene.

[0376] In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary supplements may also be included at appropriate concentrations, introduced alone or in mixtures with other supplements or media, such as complex nitrogen sources. Optionally, the media may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolate, dithioerythritol, and dithiothreitol.

[0377] Prokaryotic host cells are cultured at a suitable temperature. For growth of E. coli, for example, preferred temperatures range from about 20° C. to about 39° C., more preferably from about 25° C. to about 37° C., and even more preferably about 30° C. The pH of the medium may be any pH in the range of about 5 to about 9, depending primarily on the host organism. For E. coli, the pH is preferably about 6.8 to about 7.4, more preferably about 7.0.

[0378] When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for activating the promoter.In one aspect of the present invention, the PhoA promoter is used to control the transcription of polypeptide.Therefore, transformed host cells are cultured in phosphate-limited medium for induction.In some embodiments, the phosphate-limited medium is CRAP medium (see, for example, Simmons et al., J.Immunol.Methods (2002), 263:133-47).As known in the art, various other inducers can be used according to the vector construction employed.

[0379] In one embodiment, the expressed polypeptide of the present invention is secreted into the periplasm of the host cell and recovered therefrom. Protein recovery typically involves disruption of the microorganism, generally by means such as osmotic shock, sonication, or lysis. Once the cells are disrupted, cell debris or whole cells can be removed by centrifugation or filtration. The protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported to the medium and isolated therein. The cells can be removed from the culture and the culture supernatant filtered and concentrated for further purification of the produced protein. The expressed polypeptide can be further isolated and identified using commonly known methods such as polyacrylamide gel electrophoresis (PAGE) and Western blot assays.

[0380] In one aspect of the invention, the production of fusion proteins is carried out in large quantities by fermentation processes. A variety of large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentations have a volume of at least 1000 liters, preferably about 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose (the preferred carbon / energy source). Small-scale fermentation generally refers to fermentation in fermenters with a volume of approximately 100 liters or less, which may range from about 1 liter to about 100 liters.

[0381] In the fermentation process, induction of protein expression is typically initiated after the cells have been grown under suitable conditions until they reach a desired density, e.g., an OD550 of about 180-220, at which stage the cells are in early stationary phase. As known in the art and described above, various inducers may be used according to the vector construct employed. The cells may be grown for a shorter period before induction. The cells are usually induced for about 12-50 hours, although longer or shorter induction times may also be used.

[0382] Various fermentation conditions can be modified to improve the production yield and quality of the polypeptide of the present invention. For example, to improve the proper assembly and folding of the secreted fusion protein polypeptide, the host prokaryotic cell can be co-transformed with an additional vector overexpressing chaperone proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD, and / or DsbG) or FkpA (peptidyl prolyl cis-trans isomerase with chaperone activity). Chaperone proteins have been demonstrated to facilitate the proper folding and solubility of heterologous proteins produced in bacterial host cells. Chen et al. (1999) J Bio Chem 274, 19601-5, U.S. Patent No. 6,083,715, U.S. Patent No. 6,027,888, Bothmann and Pluckthun (2000) J. Biol. Chem. 275:17100-5, Ramm and Pluckthun (2000) J. Biol. Chem. 275:17106-13, Arie et al. (2001) Mol. Microbiol. 39: 199-210.

[0383] Certain host strains deficient in proteolytic enzymes may be used in the present invention to minimize proteolysis of expressed heterologous proteins, especially those that are sensitive to proteolysis. For example, the host cell strain may be modified to introduce genetic mutation(s) in genes encoding known bacterial proteases, such as protease III, OmpT, DegP, Tsp, protease I, protease Mi, protease V, protease VI, and combinations thereof. Several E. coli protease-deficient strains are available and are described, for example, in Joly et al. (1998) (supra), U.S. Patent No. 5,264,365, U.S. Patent No. 5,508,192, and Hara et al., Microbial Drug Resistance, 2:63-72 (1996).

[0384] In one embodiment, an E. coli strain transformed with a plasmid deficient in a proteolytic enzyme and overexpressing one or more chaperone proteins is used as a host cell in the expression system of the present invention.

[0385] eukaryotic host cell Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium (MEM, Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium (DMEM, Sigma) are suitable for culturing the host cells. In addition, the methods described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al. Any of the media described in U.S. Pat. Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO 90 / 03430, WO 87 / 00195, or U.S. Pat. Reissue No. 30,985 may be used as a medium for the host cells. Any of these media may optionally contain hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride), or the like. The medium may be supplemented with nutrients such as phosphate buffer, thionium, calcium, magnesium, and phosphate, buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., 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 one of skill in the art. 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 one of skill in the art.

[0386] Gene Amplification / Expression Detection Gene amplification and / or expression may be measured directly in the sample, for example, by conventional Southern blotting to quantitate mRNA transcription, Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5 (1980)), dot blotting (DNA analysis), or in situ hybridization using appropriately labeled probes based on the sequences provided herein. Alternatively, antibodies may be used that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes, or DNA-protein duplexes. The antibodies may then be labeled and the duplexes bound to a surface, whereby an assay may be performed in which the presence of the antibody bound to the duplex can be detected upon formation of the duplex on the surface.

[0387] Alternatively, gene expression may be measured by immunological methods such as immunohistochemical staining of cells or tissue sections and assay of cell cultures or body fluids to directly quantitate expression of the gene product. Antibodies useful for immunohistochemical staining and / or assay of sample fluids may be either monoclonal or polyclonal and may be prepared in any mammal. Conveniently, antibodies may be prepared against native sequence CTHRC1 polypeptides or against synthetic peptides based on the DNA sequences provided herein, or against exogenous sequences fused to CTHRC1 DNA and encoding specific antibody epitopes.

[0388] Purification of anti-CTHRC1 fusion protein The form of anti-CTHRC1 fusion protein may be recovered from culture medium or from host cell lysate.If it is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton®-X 100) or by enzymatic cleavage.The cells used for expressing anti-CTHRC1 fusion protein can be disrupted by various physical or chemical means, such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysing agents.

[0389] It may be desirable to purify the anti-CTHRC1 fusion protein from recombinant cell proteins or polypeptides. The following procedures are representative of suitable purification procedures: fractionation on an ion exchange column; ethanol precipitation; reversed-phase HPLC; chromatography on a cation exchange resin such as silica or DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration, for example, using Sephadex G-75; protein A sepharose column to remove contaminants such as IgG; and metal chelate column to bind the epitope tag form of the anti-CTHRC1 fusion protein. A variety of protein purification methods may be used, which 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-CTHRC1 fusion protein produced.

[0390] When using recombinant techniques, the fusion protein can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the fusion protein is produced intracellularly, as a first step, particulate debris (either host cells or lysed fragments) is removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-7 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If the fusion protein is secreted into the medium, the supernatant of such expression systems is 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.

[0391] Fusion protein compositions prepared from cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, of which affinity chromatography is the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the fusion protein. Protein A can be used to purify antibodies based on human gamma 1, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human gamma 3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, although other matrices are 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. When the CTHRC1 binding moiety contains a CH3 domain, Bakerbond ABX™ resin (JT Baker, Phillipsburg, NJ) is useful for purification. Depending on the fusion protein to be recovered, other techniques for protein purification are also available, such as fractionation on an ion exchange column, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE™, chromatography on anion or cation exchange resins (e.g., polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0392] After any preliminary purification step(s), the mixture containing the fusion protein of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH of about 2.5-4.5 and generally with a low salt concentration (e.g., about 0-0.25 M salt).

[0393] Assay Activity assay In one embodiment, an assay is provided for identifying the anti-CTHRC1 antibody having biological activity.Biological activity can include, for example, the ability to inhibit cell growth or proliferation (e.g., "cell killing" activity) or the ability to induce cell death, including programmed cell death (apoptosis).Antibodies having such biological activity in vivo and / or in vitro are also provided.

[0394] In certain embodiments, anti-CTHRC1 antibodies are tested for their 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" assay described herein, measure cell viability. One such assay is the CellTiter-Glo™ Luminescent Cell Viability Assay, available from Promega (Madison, WI). The assay determines the number of viable cells in culture based on the quantification of ATP present, which is an indicator of metabolically active cells. See Crouch et al. (1993) J.Immunol.Meth.160:81-8, US Patent No. 6,602,677. This assay may be performed in 96-well or 384-well format, making it suitable for automated high-throughput screening (HTS) (see Cree et al. (1995) AntiCancer Drugs 6:398-404). The assay procedure involves adding a single reagent (CellTiter-Glo® Reagent) directly to cultured cells. This results in cell lysis and the generation of a luminescent signal produced by a luciferase reaction. The luminescent signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells present in the culture. Data can be recorded by a luminometer or a CCD camera imaging device. Luminescent output is expressed as relative light units (RLU).

[0395] Another assay for cell proliferation is the "MTT" assay, a colorimetric assay that measures the oxidation of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan by mitochondrial reductase. Like the CellTiter-Glo™ assay, this assay indicates the number of metabolically active cells present in a cell culture (see, e.g., Mosmann (1983) J. Immunol. Meth. 65:55-63, and Zhang et al. (2005) Cancer Res. 65:3877-82).

[0396] In one aspect, anti-CTHRC1 antibodies are tested for their 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 the loss of membrane integrity as indicated by, for example, 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 (Hy Clone) and 2 mM L-glutamine. Thus, the assay is performed in the absence of complement and immune effector cells. Cells are seeded in 100×20 mm dishes at a density of 3×106 per dish and allowed to attach overnight. The medium is removed and replaced with fresh medium alone or medium containing various concentrations of antibody. The cells are incubated for a period of 3 days. Following treatment, the monolayer is washed with PBS and detached by trypsinization. The cells are then centrifuged at 1200 rpm for 5 minutes at 4° C., and the pellet is resuspended in 3 mL of cold Ca2+ binding buffer (10 mM Hepes (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) and aliquoted into 12×75 mm tubes with 35 mm strainer caps (1 mL per tube, 3 tubes per treatment group) for removal of cell clumps. The tubes are then loaded with PI (10 μg / mL). The samples are analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (Becton Dickinson). Antibodies that induce statistically significant levels of cell death as determined by PI uptake are thus identified.

[0397] In one embodiment, an anti-CTHRC1 antibody is tested for its ability to induce apoptosis (programmed cell death) in vitro. An exemplary assay for antibodies to induce apoptosis is an annexin binding assay. In an exemplary annexin binding assay, cells are cultured and plated in dishes as discussed in the previous paragraph. The medium is removed and replaced with fresh medium alone or medium containing 0.001-10 μg / mL of antibody. Following a 3-day incubation period, the monolayer is washed with PBS and detached by trypsinization. The cells are then centrifuged as discussed in the previous paragraph, resuspended in Ca2+ binding buffer, and aliquoted into tubes. The tubes are then loaded with labeled annexin (e.g., annexin V-FITC) (1 μg / mL). Samples are analyzed using a FACSCAN™ flow cytometer and FACSCONVERT™ CellQuest software (BD Biosciences). The antibody that induces statistically significant annexin binding level compared to control is thus identified.Another exemplary assay for antibody that induces apoptosis is the histone DNA ELISA colorimetric assay for detecting internucleosomal degradation of genomic DNA.Such an assay can be carried out, for example, using Cell Death Detection ELISA Kit (Roche, Palo Alto, CA).

[0398] 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 compared to normal cells from the same tissue. Such cells also include cell lines that express CTHRC1 (including tumor cell lines), and cell lines that do not normally express CTHRC1 but have been transfected with a nucleic acid encoding CTHRC1.

[0399] In one aspect, the anti-CTHRC1 antibody is tested for its ability to inhibit cell growth or proliferation in vivo. In certain embodiments, the anti-CTHRC1 antibody is tested for its ability to inhibit tumor growth in vivo. An in vivo model system, such as a xenograft model, can be used for such testing. In an exemplary xenograft system, human tumor cells are introduced into a suitable immunodeficient non-human animal, such as a SCID mouse. The antibody of the present invention is administered to the animal. The ability of the antibody to inhibit or reduce tumor growth is measured. In certain embodiments of the above xenograft system, the human tumor cells are tumor cells from a human patient. In certain embodiments, the human tumor cells are introduced into a suitable immunodeficient non-human animal by subcutaneous injection or by implantation into a suitable site, such as the mammary fat pad.

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

[0401] In one aspect, a competitive assay may be used to identify monoclonal antibodies that compete for binding to CTHRC1 with a monoclonal antibody comprising a variable domain of any one of SEQ ID NOs: 75-84, or a chimeric antibody comprising a variable domain of a monoclonal antibody comprising a sequence of Tables 2 and 3 and a constant domain from IgG1 or IgG4. In certain embodiments, such a competing antibody binds to the same epitope (e.g., a linear or conformational epitope) as a monoclonal antibody comprising a variable domain of any one of SEQ ID NOs: 75-84, or a chimeric antibody comprising a variable domain of a monoclonal antibody comprising a sequence of Tables 2 and 3 and a constant domain from IgG1 or IgG4. Exemplary competitive 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 epitopes bound by antibodies are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). Two antibodies are said to bind to the same epitope if each blocks binding of the other by 50% or more.

[0402] In one aspect, the purified anti-CTHRC1 antibody can be further characterized by a range of assays including, but not limited to, N-terminal sequencing, amino acid analysis, non-denaturing size-exclusion high pressure liquid chromatography (HPLC), mass spectrometry, ion exchange chromatography, and papain digestion.

[0403] Treatment The anti-CTHRC1 fusion protein of the present invention may be used, for example, in in vitro, ex vivo, and in vivo therapeutic methods. In one aspect, the present invention provides a method for inhibiting cell growth or proliferation, either in vivo or in vitro, comprising exposing a cell to an anti-CTHRC1 fusion protein of the present disclosure or a composition thereof under conditions that allow the binding of the fusion protein to CTHRC1. "Inhibiting cell growth or proliferation" means reducing cell growth or proliferation by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, including inducing cell death. In certain embodiments, the cell is a tumor cell. The anti-CTHRC1 fusion protein or composition thereof may additionally or alternatively (i) inhibit tumor metastasis in vivo; (ii) inhibit tumor growth in vivo; (iii) reduce tumor size in vivo; (iv) inhibit tumor angiogenesis in vivo; (v) exhibit cytotoxic activity against tumor cells and cancer-associated fibroblasts expressing and / or presenting CTHRC1 in vivo; (vi) exhibit cytostatic activity against tumor cells or cancer-associated fibroblasts expressing and / or presenting CTHRC1 in vivo; (vii) enhance the infiltration of anti-tumor immune cells in vivo; or (viii) prevent the suppression of immune cells in the tumor microenvironment in vivo.

[0404] Cancers that can be treated include tumors that are not or have not yet been substantially vascularized, as well as tumors that have been vascularized. Cancers can include non-solid tumors (such as hematological tumors, e.g., leukemias and lymphomas) or solid tumors. Types of cancers that can be treated with the fusion proteins of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, 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 with non-hematologic tumors, such as solid tumors arising from breast, CNS, and skin malignancies.

[0405] Hematological cancer is cancer of blood or bone marrow. Examples of hematological (or hematopoietic) cancer include leukemia, including acute leukemia (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (low-grade and high-grade types), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0406] A solid tumor is an abnormal mass of tissue that does not usually 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 sarcoma, carcinoma, and lymphoma. Examples of solid tumors, such as sarcoma and carcinoma, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Wilms' tumor. , cervical cancer, testicular cancer, seminoma, bladder cancer, melanoma, and CNS tumors, such as gliomas (such as brain stem gliomas and mixed gliomas), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germinomas, medulloblastomas, schwannomas, craniopharyogiomas, ependymomas, pinealomas, hemangioblastomas, acoustic neuromas, oligodendroglioma, meningiomas, neuroblastomas, retinoblastomas, and metastatic brain tumors.

[0407] In some embodiments, a method for treating a cell proliferation disorder may include administering to a subject a therapeutically effective amount of any of the above-mentioned embodiments of an anti-CTHRC1 fusion protein or pharmaceutical composition thereof. In certain embodiments, the cell proliferation disorder is associated with increased expression, presentation, and / or activity of CTHRC1. For example, in certain embodiments, the cell proliferation disorder is associated with increased expression and / or presentation of CTHRC1 on the surface of a cell, either directly or as a complex. In certain embodiments, the cell proliferation disorder is a tumor or cancer. In certain embodiments, the fusion protein may be administered at a dose of 0.001 mg / kg to about 100 mg / kg based on the patient's body weight.

[0408] In some embodiments, the method of treating cancer may include administering to a subject a therapeutically effective amount of any of the above-mentioned embodiments of an anti-CTHRC1 fusion protein or pharmaceutical composition thereof. By way of example and not limitation, the cancer may be selected from the group consisting of breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, renal cancer, kidney cancer, liver cancer, head and neck cancer, colorectal cancer, melanoma, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, and myeloma. In certain embodiments, the cell proliferative disorder is a tumor or cancer. In certain embodiments, the fusion protein may be administered at a dose of 0.001 mg / kg to about 100 mg / kg based on the patient's body weight.

[0409] The pharmaceutical composition of the present invention may be administered in a manner appropriate to the disease to be treated (or prevented). The amount and frequency of administration will be determined by factors such 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.

[0410] When an "immunologically effective amount," "antitumor effective amount," "tumor-inhibiting effective amount," or "therapeutic amount" is indicated, the exact amount of the composition of the present invention to be administered can be determined by a physician taking into account individual differences in age, weight, tumor size, extent of infection or metastasis, and the condition of the patient (subject).

[0411] In any of the above-mentioned embodiments, the step of administering the anti-CTHRC1 fusion protein or pharmaceutical composition thereof (and any additional therapeutic agent or adjuvant) can be performed by any suitable means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, as well as intralesional administration if desired for localized treatment. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. By way of further example, but not limited to, administration can be performed by known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In addition, the anti-CTHRC1 fusion protein is suitably administered by pulse infusion, particularly with tapering doses of the anti-CTHRC1 fusion protein. Dosing can be by any suitable route, e.g., by injection, such as intravenous or subcutaneous injection, depending in part on whether administration is brief or chronic. In some embodiments, intravenous or subcutaneous administration of an anti-CTHRC1 fusion protein is preferred.

[0412] Administration of the subject compositions may be performed in any convenient manner, including aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, by intravenous (iv) injection, or intraperitoneally.

[0413] The anti-CTHRC1 fusion proteins of the present invention will be formulated, dosed and administered in a manner consistent with the principles of good medical practice. Factors to be considered 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 drug, the method of administration, the schedule of administration, and other factors known to medical practitioners.

[0414] As discussed, the anti-CTHRC1 fusion protein is administered to a human patient according to known methods, such as intravenously, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, intrathecal, oral, topical, or inhalation routes. In some embodiments, intravenous or subcutaneous administration of the anti-CTHRC1 fusion protein is preferred.

[0415] The anti-CTHRC1 fusion protein compositions of the present invention will be formulated, dosed and administered in a manner consistent with the principles of good medical practice. Factors to be considered 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 drug, the method of administration, the schedule of administration, and other factors known to medical practitioners.

[0416] The CTHRC1 binding moieties of the present invention can be in different forms, which are encompassed by the definition of "antibody" herein. Thus, antibodies include full-length or intact antibodies, antibody fragments, native sequence antibodies or amino acid variants, humanized, chimeric or fusion antibodies, and functional fragments thereof. In fusion antibodies, the antibody sequence is fused to a heterologous polypeptide sequence. Antibodies can be modified in the Fc region to provide desired effector functions. As discussed in more detail in sections herein, with the appropriate Fc region, naked antibodies bound on the cell surface can induce cytotoxicity, for example, via antibody-dependent cellular cytotoxicity (ADCC), or by recruitment of complement in complement-dependent cytotoxicity, or some other mechanism. Alternatively, certain other Fc regions may be used when it is desirable to eliminate or reduce effector functions to minimize side effects or complications of the treatment.

[0417] In one embodiment, the antibody (i) competes for binding to the same epitope and / or (ii) substantially binds to the same epitope as an antibody of the invention.

[0418] Methods for producing the above fusion proteins are detailed herein.

[0419] The anti-CTHRC1 fusion protein is useful for treating CTHRC1-presenting cancer or alleviating one or more symptoms of cancer in mammals.Cancer includes metastatic cancer of any of the cancers described herein.The fusion protein can bind to at least a portion of cancer cells that present CTHRC1 directly or as a complex in mammals.In a preferred embodiment, the fusion protein is effective in destroying or killing CTHRC1-presenting tumor cells or inhibiting the growth of such tumor cells in vitro or in vivo when it binds to the CTHRC1 epitope on cells. In other preferred embodiments, the fusion protein is effective to (i) inhibit tumor metastasis in vivo, (ii) inhibit tumor growth in vivo, (iii) reduce tumor size in vivo, (iv) inhibit tumor angiogenesis in vivo, (v) exhibit cytotoxic activity against tumor cells and cancer-associated fibroblasts expressing and / or presenting CTHRC1 in vivo, (vi) exhibit cytostatic activity against tumor cells or cancer-associated fibroblasts expressing and / or presenting CTHRC1 in vivo, (vii) enhance infiltration of anti-tumor immune cells in vivo, or (viii) prevent suppression of immune cells in the tumor microenvironment in vivo.

[0420] The present invention provides a composition comprising the anti-CTHRC1 fusion protein of the present invention and a carrier.The present invention also provides a formulation comprising the anti-CTHRC1 fusion protein of the present invention and a carrier.In one embodiment, the formulation is a therapeutic formulation comprising a medicament acceptable carrier.

[0421] Another aspect of the invention is isolated nucleic acids encoding anti-CTHRC1 fusion proteins, including both heavy and light chains and in particular hypervariable region residues, chains encoding native sequence CTHRC1 binding moieties, as well as variants, modifications, and humanized versions of the antibodies.

[0422] The present invention also provides a method useful for treating CTHRC1 polypeptide-presenting cancer in a mammal or alleviating one or more symptoms of cancer, comprising administering a therapeutically effective amount of anti-CTHRC1 fusion protein to the mammal. The therapeutic antibody composition can be administered short-term (acute) or chronically, or intermittently, as directed by a physician. Also provided is a method for inhibiting the growth of CTHRC1 polypeptide-presenting cells and a method for killing them.

[0423] For the prevention or treatment of a disease, the dosage and mode of administration will be selected by the physician according to known criteria. The appropriate dosage of the anti-CTHRC1 fusion protein will depend on the type of disease to be treated, the severity and course of the disease, whether the anti-CTHRC1 fusion protein is administered for prophylactic or therapeutic purposes, previous therapies, the patient's medical history and response to antibodies, and the discretion of the attending physician. The anti-CTHRC1 fusion protein is suitably administered to the patient at one time or over a series of treatments. Preferably, the anti-CTHRC1 fusion protein or a pharmaceutical composition thereof is administered by intravenous infusion or by subcutaneous injection. Depending on the type and severity of the disease, for example, about 1 μg / kg to about 100 mg / kg body weight (e.g., about 0.1 to 30 mg / kg / dose) of the anti-CTHRC1 fusion protein may be the initial candidate dosage for administration to the patient, whether by one or more separate administrations or by continuous infusion. The dosing regimen may include administering an initial loading dose of about 4 mg / kg of anti-CTHRC1 fusion protein, followed by weekly maintenance doses of about 2 mg / kg. However, other dosage regimens may be useful. A typical daily dosage may range from about 1 μg / kg to 1000 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days or more, depending on the condition, treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy can be easily monitored by conventional methods and assays, and based on criteria known to the physician or other skilled in the art.

[0424] In any of the above-described embodiments, the method of treatment may further include administering allogeneic or autologous CAR-T or NK cell therapy to the subject.

[0425] In any of the above embodiments, the subject may be a human or non-human mammal.

[0426] In certain aspects, the pharmaceutical compositions of the present disclosure can be used in the preparation of a medicament for the treatment of a cell proliferative disorder, preferably cancer.

[0427] Manufactured Products and Kits Another embodiment of the present invention is an article of manufacture containing materials useful for treating, preventing, and / or diagnosing CTHRC1-presenting cancer. The article of manufacture includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The containers can be formed from a variety of materials, such as glass or plastic. The container holds a composition effective for treating, preventing, and / or diagnosing a cancer condition and may have a sterile access port (e.g., the container can be an intravenous fluid bag or a vial with a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-CTHRC1 antibody of the present invention, or a CAR-modified immune cell of the present invention, or a nucleic acid of the present invention. Optionally, the composition further comprises a carrier, for example a pharma- ceutically acceptable carrier. The label or package insert indicates that the composition is used to treat cancer. The label or package insert further comprises instructions for administering the antibody composition to a cancer patient. Additionally, the article of manufacture may further comprise a second container containing a pharma- ceutically 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.

[0428] Kits are also provided that are useful for various purposes, such as CTHRC1-presenting cell killing assays, purification of CTHRC1 polypeptide from cells, or immunoprecipitation. For isolation and purification of CTHRC1 polypeptide, the kit may include anti-CTHRC1 antibodies bound to beads (e.g., sepharose beads). Kits may be provided that include antibodies for detection and quantification of CTHRC1 polypeptide in vitro, for example, in ELISA or Western blot. As with articles of manufacture, kits include a container and a label or insert on or associated with the container. The container holds a composition that includes at least one anti-CTHRC1 antibody of the present invention. Additional containers may be included that include, for example, diluents and buffers, control antibodies. The label or insert may provide a description of the composition and instructions for the intended in vitro or detection use.

[0429] For example, the kit may include a first container containing a composition comprising one or more CTHRC1 antibodies of the invention, or CAR-T or CAR-NK cells, or CAR-modified immune cells, such as CAR macrophages, and a second container containing a buffer. The buffer may be pharma- ceutically acceptable.

[0430] The following examples are offered for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0431] All patents, patent applications, and references cited herein are hereby incorporated by reference in their entirety. EXAMPLES

[0432] Example 1: CTHRC1 expression on tumor and stroma in human colorectal cancer samples by IHC Tissue samples from colorectal cancer patients were stained with FAP as shown in Figure 1A. Separate tissue samples from colorectal cancer patients were evaluated by IHC using anti-CTHRC1 rabbit polyclonal antibody (Abcam, ab85739) as shown in Figure 1B. Tissue slides from subjects with stage IV colorectal cancer were obtained from commercial sources and stained with the indicated antibodies at the recommended dilutions for immunohistochemistry. After incubation with the primary antibody, slides were washed and bound mAb was detected by common chromogenic methods. As shown in Figure 1A, tumor stroma is stained with FAP, while tumor nests (blue) are devoid of FAP staining. However, as shown in Figure 1B, CTHRC1 is localized to both tumor stroma and tumor nests, which is compatible with the scRNA data (not shown).

[0433] Example 2: Membrane Binding of CTHRC1 Antibody Staining of tumor cell lines with anti-CTHRC1 mAb was evaluated by flow cytometry. Human SKOv3 ovarian cancer, KP4 PDAC, and HCT115 colorectal cancer lines as well as mouse EMT6 breast cancer line were selected. Cell lines were incubated with recombinant CTHRC1 at 0.25ug / mL to allow complex formation on the cell surface. Following incubation, cells were washed and anti-CTHRC1 mAb was added at 10ug / mL and incubated for 30 minutes. Cells were then washed and incubated with anti-mouse secondary antibody diluted 1:250 in FACS buffer for 20 minutes. Cells were washed again and resuspended in FACS buffer for analysis on a Sony cell analyzer. The results are shown in Figures 2A-2D, which confirm that anti-CTHRC1 antibodies can bind to the surface of tumor cells.

[0434] Example 3: Induction of T cell proliferation by CTHRC1 scFv / 4-1BBL construct Jurkat T cells were labeled with CFSE, washed, and stimulated with 0.1ug / mL anti-CD3 + 5ug / mL anti-CD28 alone or 0.5ug / mL recombinant 4-1BBL or M23-4-1BBL (3-fold molar excess of ligand vs fusion protein) for 72 hours. Stimuli were removed and cells remained in culture for an additional 24 hours. CFSE dilution was assessed by flow cytometry on a BD LSR Fortessa X20. Proliferation index was assessed as a CFSE dilution series, and the degree of proliferation was deemed significant by two-way ANOVA. Results are shown in Figures 3A-3B.

[0435] Example 4: In vivo efficacy of CTHRC1 scFv / 4-1BBL construct Female BALB / c mice aged 8–10 weeks were injected with 1 × 10 5 EMT6 cells were inoculated subcutaneously. Tumor growth was measured and the average tumor volume was 120 mm 3 Mice were randomized to treatments when

[0436] M23-41BBL (SEQ ID NO: 101), a fusion protein of anti-CTHRC1 scFv and 4-1BBL, recombinantly produced in HEK cells and purified via binding of the C-terminal His tag using nickel-NTA resin, was administered to each mouse at a dose of 1 mg / kg, 2.5 mg / kg, or 5 mg / kg. Tumor growth and survival were evaluated three times a week. Dengue-4-1BBL was used as a control.

[0437] As shown in FIG. 4, tumor volume did not increase significantly to the same extent following administration of M23-41BBL versus Dengue-41BBL.

[0438] As shown in Figure 5, mice treated with M23-41BBL had improved survival compared to Dengue-41BBL treated mice. The M23-41BBL group was dosed at 2.5 mg / kg, while M5-41BBL was dosed at 1 mg / kg.

[0439] This data is consistent with the tumor volume over time data shown in FIG.

[0440] As shown in FIG. 7, low dose (1 mg / kg) treatment with M23-41BBL significantly reduced tumor growth in a subset of mice.

[0441] Example 5: In vitro T cell stimulation - normal PBMC (targeting 4-1BBL, OX40L, or GITRL) CD4 and CD8 T cells are isolated separately from normal healthy donor PBMCs by negative selection using a commercial enrichment kit. Isolated cells are labeled with CFSE and transferred to tissue culture plates coated with suboptimal concentrations of anti-CD3 antibodies + / - CTHRC1-targeting fusions. Following incubation, cell proliferation is assessed by measuring CFSE dilution by flow cytometry. OX40L and GITRL fusions should induce proliferation of both CD4 and CD8 T cells. 4-1BBL fusions should predominantly stimulate proliferation of CD8 T cells and a subset of activated CD4 cells.

[0442] Example 6: In vitro T cell and NK cell stimulation - tumor TIL / isolated tumor (targeted 4-1BBL, OX40L, or GITRL) Isolated human tumor samples are procured from commercial suppliers. Cells are incubated in tissue culture plates + / - CTHRC1 targeting fusions or controls. Following incubation, cell proliferation is assessed by measuring Ki-67 expression on individual cell populations by flow cytometry. OX40L and GITRL fusions should induce proliferation of both CD4 and CD8 T cells. 4-1BBL fusions should predominantly stimulate proliferation of NK cells and CD8 T cells and a subset of activated CD4 cells.

[0443] Example 7: In vitro NK cell stimulation - normal PBMC (targeted 4-1BBL) CD16+ NK cells are isolated from normal healthy donor PBMCs by negative selection using a commercial enrichment kit. Isolated cells are labeled with CFSE and transferred to tissue culture plates + / - CTHRC1 targeting fusions. Following incubation, cell proliferation is assessed by measuring CFSE dilution and upregulation of NK activation markers by flow cytometry. The 4-1BBL fusion should induce proliferation and upregulation of NK activating receptors.

[0444] Example 8: ADCC Enhancement - Engineered Cell Line (Targeted 4-1BBL) CD16+ NK cells from normal healthy donors are isolated from PBMCs by negative selection using a commercial enrichment kit. The NK cells are then plated with target (Her2, CD20, EGFR, etc.) expressing tumor cells at various E:T ratios (50:1, 20:1, 10:1, and 5:1) and incubated with a dilution series of target-specific monoclonal antibodies (trastuzumab, rituximab, cetuximab, etc.) capable of inducing ADCC + / - targeting 4-1BBL. ADCC should be enhanced by the addition of 4-1BBL-mediated co-stimulation.

[0445] Example 9: ADCC Enhancement - Primary Tumor and Effector Cells (DTC and Matched PBMC) (Targeting 4-1BBL) Autologous CD16+ NK cells from cancer patients are isolated from PBMCs by negative selection using a commercial enrichment kit. The NK cells are then plated with autologous isolated tumor cells at various E:T ratios (50:1, 20:1, 10:1, and 5:1) and incubated with a dilution series of target-specific monoclonal antibodies (trastuzumab, rituximab, cetuximab, etc.) capable of eliciting ADCC + / - targeting 4-1BBL. ADCC should be enhanced by the addition of 4-1BBL-mediated co-stimulation.

[0446] Example 10: In vitro B cell activation and proliferation - cell lines / normal B cells (targeting CD40L) B cells are isolated from healthy donor PBMCs by negative selection using a commercially available enrichment kit. Isolated B cells are stimulated with CD40L fusion protein or a negative control, and proliferation, activation, and antibody class switching are assessed by flow cytometry. CD40L has been demonstrated to enhance B cell activation, proliferation, and induce antibody class switching.

[0447] Example 11: Stimulation and activation / differentiation of DCs in vitro - primary cells Monocytes from normal human PBMCs are differentiated in vitro using standard procedures + / - targeting CD40L or control proteins in the presence of CEFT peptides. After 7 days, flow profiling of DC maturation, activation, and HLA expression is assessed, which should be enhanced under CD40L treatment conditions.

[0448] Example 12: In vivo generation of antigen-specific T cells (DC and T co-cultures) - tumor lysate pulsing and ELISPOT readout after 2 weeks Monocytes from normal human PBMCs are differentiated in vitro using standard techniques + / - targeting CD40L or control proteins in the presence of CEFT peptide. After 7 days, autologous T cells are added and cultured for another 7 days. At 2 weeks, T cells are restimulated with CEFT peptide and cytokine responses are assessed by ELISPOT. CD40L fusion protein should enhance the development of antigen-specific memory T cells upon restimulation as well as result in higher IFNg secretion.

[0449] Example 13: B cell activation and proliferation - tumor TIL (targeting CD40L) B cells are isolated from human tumor-infiltrating lymphocyte samples by negative selection using a commercially available enrichment kit. Isolated B cells are stimulated with CD40L fusion protein or a negative control, and proliferation, activation, and antibody class switching are assessed by flow cytometry. CD40L has been demonstrated to enhance B cell activation, proliferation, and induce antibody class switching.

[0450] Example 14: In vivo efficacy: dose response and schedule as monotherapy and in combination with checkpoints (aPD-1, aPD-L1, a-CTLA-4, and aLAG3) Models for EMT6, B16-OVA, MC38, etc.

[0451] In vivo efficacy is evaluated in common syngenic tumor-bearing mice. Naive mice are inoculated subcutaneously with tumor cells and tumor growth is monitored. When tumors reach approximately 150 mm 3 When the tumor reaches a tumor size of 1000, the mice are randomized into treatment groups. The mice are treated with the targeted fusion protein as monotherapy or in combination with a checkpoint inhibitor. Tumor growth inhibition and survival rates are evaluated over the duration of the study. The fusion protein as monotherapy should result in tumor growth inhibition in the model tested, and the combination with a checkpoint inhibitor should result in enhanced efficacy.

[0452] Example 15: In vivo efficacy: non-checkpoint combinations (cytotoxic agents) A similar combination to experiment 4, but performed in mice

[0453] Performing with cell line-derived xenografts

[0454] In vivo efficacy is evaluated in mice (SCID / nude) bearing common cell line derived xenograft tumors. Naive mice are inoculated subcutaneously with tumor cells and tumor growth is monitored. When tumors reach approximately 150 mm 3 When the tumor reaches a tumor size of 1000, the mice are randomized into treatment groups. The mice are treated with the targeted fusion protein as monotherapy or in combination with mAbs with ADCC capabilities (Trastuzumab for Her2+ xenografts; Cetuximab for EGFR+ xenografts, Rituximab for CD20+ xenografts, etc.). Tumor growth inhibition and survival rates are evaluated over the duration of the study. The fusion protein as monotherapy should result in tumor growth inhibition in the model tested, and combination with a cytotoxic antibody should result in enhanced efficacy.

[0455] Example 16: In vivo efficacy: Combination with cell therapy NK cells and CD8 T cells

[0456] Performing with cell line-derived xenografts

[0457] In vivo efficacy is evaluated in mice (SCID / nude) bearing common cell line derived xenograft tumors. Naive mice are inoculated subcutaneously with tumor cells and tumor growth is monitored. When tumors reach approximately 150 mm 3 When the tumor cell count reaches 100%, mice are randomized into treatment groups. Mice are injected with CAR-T or CAR-NK cells and treated with the targeted fusion protein or a relevant control protein. Tumor growth inhibition and survival rates are assessed for the duration of the study. The fusion protein should enhance the anti-tumor activity of CAR-T and CAR-NK cells.

[0458] Example 17: In vivo enhancement of antigen-specific immune responses OTI model, evaluation of SIINFEKL peptide-reactive T cells upon treatment with targeted constructs

[0459] The in vivo activity and development of antigen-specific responses are evaluated in OTI mice bearing B16-OVA tumors. Naive mice are inoculated subcutaneously with tumor cells and tumor growth is monitored. When tumors reach approximately 150 mm 3 When the tumor reaches 100% OVA-specific T cell response, mice are randomized into treatment groups. Mice are treated with the targeted fusion protein as monotherapy. Two to three weeks after the start of dosing, tumors, dLNs, and spleens are harvested and assessed for frequency and phenotype of SIINFEKL (OVA peptide sequence) tetramer-specific T cells. The fusion protein should result in enhanced induction of OVA-specific T cell responses in treated mice.

[0460] Example 18: PD evaluation demonstrating in vivo activity Assessment of CD4, CD8, B, NK, and DC activity and phenotype in tumors and dLNs following short-term (single dose or weekly dosing) targeted ligand treatment

[0461] Models for EMT6, B16-OVA, MC38, etc.

[0462] In vivo activity and immune cell activation and polarization are evaluated in syngenic tumor-bearing mice. Naive mice are inoculated subcutaneously with tumor cells and tumor growth is monitored. When tumors reach approximately 150 mm 3 When the mice reach 100% NK cell count, they are randomized into treatment groups. Mice are treated with targeted fusion proteins as monotherapy. Following one week of dosing, tumors, dLNs, and spleens are harvested and evaluated for frequency and phenotype of immune cells present in tumors, dLNs, and spleens. OX40L, GITRL, and 4-1BBL should stimulate T cell proliferation and activation. Additional NK activity should be observed for 4-1BBL, and CD40L should stimulate proliferation of macrophages / DCs, B cells, and CD8 T cells.

[0463] Example 19: Assessment of durability of response in cured mice Immunocompetent mice that have been cured of syngenic tumors by treatment with targeted fusion proteins are restimulated with the same tumors after a 10-week washout period following the termination of the initial study. Tumor growth is measured. Mice that were previously cured of tumors following fusion protein treatment should spontaneously reject the re-inoculated tumors, demonstrating the induction of adaptive immune responses during the initial treatment and persistent anti-tumor immunity.

[0464] Example 20: CTHRC1 is highly selectively localized in cancer, associated with poor outcome, and is most upregulated on cancer-associated fibroblasts (CAFs) in the immune-cold tumor microenvironment Without being bound by theory, it is expected that in the context of cancer, inhibition of CTHRC1 may provide therapeutic benefit by blocking CAF and autocrine pro-survival signaling to cancer cells, together with interfering with CTHRC1-mediated immunosuppression.Targeting CTHRC1 with antibodies that are conjugated to toxins or that bind to immune cells may further drive anti-tumor activity.Data has been collected that support the idea that CTHRC1 is selectively upregulated in cance...

Claims

1. 1. An anti-collagen triple helix repeat containing 1 (anti-CTHRC1) fusion protein, (a) a first domain comprising a CTHRC1-binding portion; (b) a second domain comprising a T cell engager, preferably wherein the T cell engager comprises a ligand for a receptor expressed on a T cell. The anti-collagen triple helix repeat containing 1 (anti-CTHRC1) fusion protein.

2. 2. The anti-CTHRC1 fusion protein of claim 1, wherein the T cell engager comprises a ligand for a costimulatory receptor expressed on a T cell, or a functional fragment, variant, or mutant thereof, and preferably the costimulatory receptor is a member of the tumor necrosis factor superfamily (TNFSF).

3. 3. The anti-CTHRC1 fusion protein of claim 2, wherein the costimulatory receptor is selected from the group consisting of 4-1BB (CD137), OX40 (CD134), CD40, CD27, CD28, herpes virus entry mediator (HVEM), ICOS, CD2, and GITR.

4. 4. The anti-CTHRC1 fusion protein of claim 3, wherein the costimulatory receptor is 4-1BB (CD137), and preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 1-10, or a functional fragment thereof.

5. 4. The anti-CTHRC1 fusion protein of claim 3, wherein the costimulatory receptor is OX40 (CD134), and preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 11-13, or a functional fragment thereof.

6. The anti-CTHRC1 fusion protein of claim 3, wherein the costimulatory receptor is CD40, and preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 14-16, or a functional fragment thereof.

7. The anti-CTHRC1 fusion protein of claim 3, wherein the costimulatory receptor is GITR, and preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 17-18, or a functional fragment thereof.

8. 4. The anti-CTHRC1 fusion protein of claim 3, wherein the costimulatory receptor is CD27, and preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 19-20, or a functional fragment thereof.

9. 4. The anti-CTHRC1 fusion protein of claim 3, wherein the costimulatory receptor is HVEM, and preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 21-23, or a functional fragment thereof.

10. 4. The anti-CTHRC1 fusion protein of claim 3, wherein the costimulatory receptor is ICOS, and preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 30-31, or a functional fragment thereof.

11. 4. The anti-CTHRC1 fusion protein of claim 3, wherein the costimulatory receptor is CD28, and preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 32-35, or a functional fragment thereof.

12. The anti-CTHRC1 fusion protein of claim 3, wherein the costimulatory receptor is CD2 and preferably the ligand comprises an amino acid sequence having at least 80% identity to any one of SEQ ID NOs: 39-40, or a functional fragment thereof.

13. and further comprising a linker positioned between the CTHRC1 binding moiety and the ligand, preferably the linker is ４ S) ｎ or (GSG) ｎ 4. The anti-CTHRC1 fusion protein of any one of the preceding claims, selected from the group consisting of: (wherein n is 1-4).

14. An anti-CTHRC1 fusion protein according to any one of the preceding claims, wherein the CTHRC1 binding portion comprises an anti-CTHRC1 antibody that binds to CTHRC1, preferably wherein the CTHRC1 antibody binds to CTHRC1 with a binding affinity of less than 10 nM.

15. The anti-CTHRC1 antibody of claim 14 comprises a heavy chain variable region comprising a CDR1 sequence comprising the sequence of SEQ ID NO: 46, a CDR2 sequence comprising the sequence of SEQ ID NO: 51, and a CDR3 sequence comprising the sequence of SEQ ID NO: 56, and a light chain variable region comprising a CDR1 sequence comprising the sequence of SEQ ID NO: 61, a CDR2 sequence comprising the sequence of SEQ ID NO: 66, and a CDR3 sequence comprising the sequence of SEQ ID NO:

71.

16. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 77 and a light chain variable region comprising SEQ ID NO:

78.

17. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence comprising the sequence of SEQ ID NO: 45, a CDR2 sequence comprising the sequence of SEQ ID NO: 50, and a CDR3 sequence comprising the sequence of SEQ ID NO: 55, and a light chain variable region comprising a CDR1 sequence comprising the sequence of SEQ ID NO: 60, a CDR2 sequence comprising the sequence of SEQ ID NO: 65, and a CDR3 sequence comprising the sequence of SEQ ID NO:

70.

18. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 75 and a light chain variable region comprising SEQ ID NO:

76.

19. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 75, 77, 79, 81, and 83.

20. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a heavy chain variable region comprising an amino acid sequence selected from Table 4.

21. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 76, 78, 80, 82, and 84.

22. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a light chain variable region comprising an amino acid sequence selected from Table 5.

23. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a heavy chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 45-49, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 50-54, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 55-59.

24. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a light chain variable region comprising a CDR1 sequence selected from the group consisting of SEQ ID NOs: 60-64, a CDR2 sequence selected from the group consisting of SEQ ID NOs: 65-69, and a CDR3 sequence selected from the group consisting of SEQ ID NOs: 70-74.

25. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 75 and a light chain variable region comprising SEQ ID NO:

76.

26. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 79 and a light chain variable region comprising SEQ ID NO:

80.

27. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 81 and a light chain variable region comprising SEQ ID NO:

82.

28. The anti-CTHRC1 fusion protein of claim 14, wherein the anti-CTHRC1 antibody comprises a heavy chain variable region comprising SEQ ID NO: 83 and a light chain variable region comprising SEQ ID NO:

84.

29. The anti-CTHRC1 fusion protein of any one of the preceding claims, wherein the anti-CTHRC1 antibody is a chimeric, humanized, or human antibody.

30. The anti-CTHRC1 fusion protein of any one of claims 1 to 29, wherein the anti-CTHRC1 antibody is a monoclonal antibody.

31. The anti-CTHRC1 fusion protein of any one of claims 1 to 29, wherein the anti-CTHRC1 antibody is an antibody fragment.

32. The anti-CTHRC1 fusion protein of any one of claims 1 to 29, wherein the anti-CTHRC1 antibody comprises a single chain antibody.

33. The anti-CTHRC1 fusion protein of any one of claims 1 to 29, wherein the anti-CTHRC1 antibody is a heavy chain only antibody (single domain antibody).

34. A method for activating T cells in a tumor microenvironment, comprising contacting the tumor with an anti-CTHRC1 fusion protein according to any one of claims 1 to 33.

35. A method for inhibiting the growth of a cell presenting a CTHRC1 tumor epitope, comprising contacting said cell with an anti-CTHRC1 fusion protein according to any one of claims 1 to 33.

36. 34. A method of treating a subject having cancer, comprising contacting said cells with an anti-CTHRC1 fusion protein according to any one of claims 1 to 33.

37. 37. The method of claim 36, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma cancer, bladder cancer, renal cancer, kidney cancer, liver cancer, head and neck cancer, colorectal cancer, melanoma, pancreatic cancer, gastric cancer, esophageal cancer, mesothelioma, prostate cancer, leukemia, lymphoma, and myeloma.

38. The method of any one of claims 34 to 37, further comprising administering an allogeneic or autologous T cell therapy in combination with the anti-CTHRC1 fusion protein of any one of claims 1 to 33.

39. A pharmaceutical composition comprising an anti-CTHRC1 fusion protein according to any one of claims 1 to 33 and a pharma- ceutically acceptable carrier.

40. 40. Use of the pharmaceutical composition according to claim 39 in the preparation of a medicament for the treatment of a cell proliferative disorder, preferably cancer.

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