Anti-CTHRC1 T cell engager and method of use thereof

Abstract

The present invention relates to anti-CTHRC1 T cell activators, compositions comprising the same, and methods of using such anti-CTHRC1 T cell activators and compositions for the prevention and / or treatment of cancer.

Description

[Background technology]

[0001] Cross-reference of related applications This application claims the benefits of U.S. Provisional Application No. 63 / 526,444, filed on 12 July 2023, which is incorporated herein by reference in its entirety.

[0002] Collagen triple helix repeat-containing protein 1 (CTHRC1) is a highly conserved protein found in both normal and cancerous tissues and is thought to be involved in metabolism, arterial remodeling, osteogenesis, and myelination of the peripheral nervous system (Liu, et al. 2023). Although CTHRC1 is associated with cancer, the precise mechanism of its function in cancer, as well as its morphology and location, remains difficult to ascertain. Several studies have found that CTHRC1 is present only in the tumor stroma, which constitutes a major barrier to anti-tumor immunity and therapeutic targeting, and the stroma is considered to be the main immunosuppressive component that promotes resistance to immune checkpoint inhibitors. However, CTHRC1 has been found to exist in multiple different molecular weight forms with potentially different epitope mappings, and its association with certain cytoplasmic proteins may block the exposure of certain epitopes, and anti-CTHRC1 antibodies have been found to exhibit varying specificity (Liu, et al. 2023).

[0003] The development of therapeutically effective T-cell engagers also faces numerous challenges. One such problem is antibody internalization, which results in a reduced therapeutic area due to the antibody's short residence time on the cell surface. In the case of bispecific antibodies, upon internalization, the T-cell engager arm becomes unable to function in recruiting T cells to cells containing tumor antigens. This invention addresses this and other unmet needs. [Overview of the project]

[0004] As demonstrated herein, certain antibodies against the interstitial target CTHRC1 may have varying properties regarding internalization into cells. In relation to anti-CTHRC1 T cell engagers, this lack of internalization may enhance therapeutic efficacy by avoiding internalization that would otherwise reduce the T cell engager's ability to recruit T cells into those cells. Given that certain antibodies may bind to the CTHRC1 epitope on cells without being internalized, this may offer an effective solution to the problems in the prior art.

[0005] For this purpose, the Disclosure provides an anti-CTHRC1 T cell engager and a composition thereof, the anti-CTHRC1 T cell engager comprising at least one collagen triple helix repeat-containing 1(CTHRC) binding moiety that selectively binds to human CTHRC1 and cells presenting the CTHRC1 epitope without being internalized, and at least one T cell engager moiety, preferably the T cell engager moiety comprising an anti-CD3 antibody or a fragment thereof. The Disclosure further provides a method for producing the same and its use in the treatment of cancer.

[0006] In one embodiment, the anti-CTHRC1 T cell engager comprises at least one first domain including a CTHRC1 binding moiety that selectively binds to human CTHRC1 and cells presenting the CTHRC1 epitope without being internalized, and at least one second domain including a T cell engager moiety. In several embodiments, the T cell engager moiety comprises an anti-CD3 antibody or a fragment thereof.

[0007] In some embodiments, the CTHRC1 binding moiety includes an anti-CTHRC1 antibody that binds to CTHRC1, preferably with a binding affinity of less than 10 nM. In some embodiments, the anti-CTHRC1 T cell engager binds to CTHRC1 with a binding affinity of less than 10 nM.

[0008] In several embodiments, the CTHRC1 binding moiety comprises an anti-CTHRC1 antibody comprising heavy chain complementarity-determining regions (HCDR) 1, HCDR2, and HCDR3, respectively, including SEQ ID NOs. 48, 53, and 58, and light chain complementarity-determining regions (LCDR) 1, LCDR2, and LCDR3, respectively, including SEQ ID NOs. 63, 68, and 73.

[0009] In several embodiments, the anti-CTHRC1 antibody includes a heavy chain variable region (HCVR) containing SEQ ID NO: 81 and a light chain variable region (LCVR) containing SEQ ID NO: 82.

[0010] In several embodiments, the anti-CTHRC1 antibody is a chimeric, humanized, or human antibody.

[0011] In several embodiments, the anti-CTHRC1 antibody is a monoclonal antibody.

[0012] In several embodiments, the anti-CTHRC1 antibody is an antibody fragment.

[0013] In several embodiments, the anti-CTHRC1 antibody comprises a single-chain antibody.

[0014] In several embodiments, the anti-CTHRC1 antibody is a heavy-chain-only antibody (single-domain antibody).

[0015] In some embodiments, the anti-CTHRC1 antibody fragment is selected from the group consisting of Fab, F(ab')2, Fv, scFv, dsFv, and single-domain antibodies.

[0016] In several embodiments, the anti-CD3 antibody is a chimeric, humanized, or human antibody.

[0017] In several embodiments, the anti-CD3 antibody is a monoclonal antibody.

[0018] In several embodiments, the anti-CD3 antibody is an antibody fragment.

[0019] In multiple embodiments, the anti-CD3 antibody includes a single-chain antibody.

[0020] In multiple embodiments, the anti-CD3 antibody is a heavy-chain only antibody (single-domain antibody).

[0021] In multiple embodiments, the anti-CD3 antibody fragment is selected from the group consisting of Fab, F(ab’)2, Fv, scFv, dsFv, and single-domain antibodies.

[0022] In multiple embodiments, the anti-CTHRC1 T cell engager includes two first domains and / or two second domains. In multiple embodiments, the anti-CTHRC1 T cell engager has a first domain and a second domain in a ratio of 2:1, 1:2, or 2:2. In multiple embodiments, the anti-CTHRC1 T cell engager has a 2:1 ratio in a cis configuration.

[0023] In multiple embodiments, a method of activating T cells in the tumor microenvironment includes contacting a tumor with an anti-CTHRC1 T cell engager of any of the above embodiments.

[0024] In multiple embodiments, a method of inhibiting the growth of cells presenting a CTHRC1 tumor epitope includes contacting the cells with an anti-CTHRC1 T cell engager of any of the above embodiments.

[0025] In multiple embodiments, a method of treating a subject having cancer includes administering an anti-CTHRC1 T cell engager of any of the above embodiments.

[0026] In multiple embodiments, the cancer is selected from the group consisting of breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, 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.

[0027] In some embodiments, the above-described method may further include administering allogeneic or autologous immunotherapy, including T cell therapy (e.g., CAR-T and TiL), NK cell therapy (e.g., CAR-NK and LAK), and macrophage therapy (e.g., CAR-macrophage), in combination with an anti-CTHRC1 T cell engager from any of the embodiments described above.

[0028] In some embodiments, the pharmaceutical composition comprises an anti-CTHRC1 T cell engager from any of the embodiments described above and a pharmaceutically acceptable carrier.

[0029] In some embodiments, the use of the pharmaceutical compositions of the embodiments described above may be in the preparation of pharmaceuticals for the treatment of cell proliferation disorders, preferably cancer. [Brief explanation of the drawing]

[0030] [Figure 1A] The image shows a sample from a colorectal cancer patient stained with FAP, indicating tumor and stroma. [Figure 1B] The image shows samples from colorectal cancer patients stained with anti-CTHRC1 antibody, which indicates tumor and stroma. [Figure 2A] FACS data for staining cancer cell lines with anti-CTHRC1 antibody are shown. [Figure 2B] FACS data for staining cancer cell lines with anti-CTHRC1 antibody are shown. [Figure 2C] FACS data for staining SKOV3 cells with PAI-0323(M23), PAI-0314(M14), PAI-0305(M5), and PAI-0303(M3) against palivizumab control and secondary antibody-only control are shown in the figure. [Figure 2D] The MFI (Multiple Factor Indication) of fluorescent dye experimental data for PAI-0303(M3) and PAI-0323(M23), as well as for necitumumab controls and isotype controls, is shown in the figure, illustrating the lack of internalization of M3. [Figure 2E]The results of the bridging ELISA between CTHRC1 and CD3 are shown in the figure. [Figure 3A] This diagram illustrates that CTHRC1 mRNA is a leading marker for cancer-associated fibroblasts (CAFs) in cancer-rich, immunocold tumor samples. The collected tumor samples were profiled across cancer scRNA studies to create a large scRNA atlas. The samples were then divided into two groups: T-cell rich, cancer-poor (immune-hot) and cancer-rich, T-cell poor (immunocold). [Figure 3B] This diagram illustrates that CTHRC1 mRNA is a top marker for cancer-associated fibroblasts (CAFs) in cancer-rich, immunocold tumor samples. Genes expressed by CAFs were compared between the two groups to determine which were most associated with cancer-rich samples (Wilcoxon ranking). These genes were then narrowed down to those specifically expressed by CAFs in all samples (top 500; Wilcoxon ranking). CTHRC1 was the 11th most expressed gene in this analysis. [Figure 4] This diagram illustrates that CTHRC1 is highly upregulated in cancer compared to normal adjacent tissue, as determined by bulk RNA measurements analyzed from cancer genome atlases. Across the profiled set of indications, the highest levels of CTHRC1 expression were observed in solid tumors, particularly breast cancer, lung cancer, ovarian cancer, pancreatic cancer, sarcoma, melanoma, and uterine carcinosarcoma (P<0.001 in all cases tested). In summary, this suggests that CTHRC1 expression is highest in cancers with more fibrous stroma, aligning with the idea that it is primarily a target secreted by CAFs. [Figure 5] A series of survival plots illustrating CTHRC1 survival curves for several solid tumors associated with low survival rates is shown. Values ​​were obtained from the Cancer Genome Atlas. Survival curves were calculated and plotted using the online tool GEPIA. [Figure 6]The diagram 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 stage I and stage IV. Stages II and III show intermediate CTHRC1 expression levels. [Figure 7A] This histogram shows the bulk RNA expression levels of CTHRC1 (Figure 7C) and known stromal targets FAP (Figure 7A) and LRRC15 (Figure 7B) in pancreatic cancer samples (cancer genome atlas) compared to all normal tissue samples (GTEX). The histogram highlights the existence of a significant therapeutic region for targeting CTHRC1 in pancreatic cancer based on bulk RNA measurements. This region is similar to, though not larger than, that of the known / developed stromal targets FAP and LRRC15. [Figure 7B] This histogram shows the bulk RNA expression levels of CTHRC1 (Figure 7C) and known stromal targets FAP (Figure 7A) and LRRC15 (Figure 7B) in pancreatic cancer samples (cancer genome atlas) compared to all normal tissue samples (GTEX). The histogram highlights the existence of a significant therapeutic region for targeting CTHRC1 in pancreatic cancer based on bulk RNA measurements. This region is similar to, though not larger than, that of the known / developed stromal targets FAP and LRRC15. [Figure 7C] This histogram shows the bulk RNA expression levels of CTHRC1 (Figure 7C) and known stromal targets FAP (Figure 7A) and LRRC15 (Figure 7B) in pancreatic cancer samples (cancer genome atlas) compared to all normal tissue samples (GTEX). The histogram highlights the existence of a significant therapeutic region for targeting CTHRC1 in pancreatic cancer based on bulk RNA measurements. This region is similar to, though not larger than, that of the known / developed stromal targets FAP and LRRC15. [Figure 8]This dataset illustrates very high levels of CTHRC1 expression in cancer epithelial expression across many solid tumors (CAFs), as well as in breast, pancreatic, lung, ovarian, and skin cancers. In contrast, minimal CTHRC1 expression is observed in normal tissues. The data were obtained based on a previously generated large-scale integrated single-cell RNA sequencing atlas to enable probing of gene expression at the single-cell level across toxic cancer and normal tissue samples (Swechha, 2021). The data highlight the potential large therapeutic spectrum for blocking CTHRC1 and the value of using mAbs against CTHRC1 to target payloads, e.g., ADCs to the tumor microenvironment. [Figure 9] This dataset, using the same atlas discussed in Figure 8, was applied to LRRC15, a known non-toxic stromal target. Here, the antibody ADC was engineered and shown to be safe in clinical settings. Low levels of LRRC15 were observed in normal tissue compared to CTHRC1. In cancer single-cell RNA (scRNA) datasets, LRRC15 expression was observed to be selectively localized to specific CAFs in certain breast cancers, unlike CTHRC1, and low levels of expression were also observed on sarcoma cancer cells. [Figure 10] This graph illustrates the quantitative ELISA results for CTHRC1 in single-cell and co-cell cultures. Supernatants from different sets of single-cell cultures (fibroblasts or cancer cells) and co-cell cultures (fibroblasts and cancer cells) were profiled for CTHRC1 levels. CTHRC1 was expressed at low levels in single-cell fibroblast cultures (BJ,CCD18-Co) and upregulated in co-cell cultures, indicating that the interaction between fibroblasts and cancer cells drives CTHRC1 expression. [Figure 11] A series of tissue images from three mouse models probed with a CTHRC1-specific mAb are shown. Extensive staining is observed within the tumor region, indicating that the CTHRC1 protein localizes to cancerous areas in vivo. [Figure 12] This series of images shows variations in CTHRC1 expression patterns at the protein level, illustrating different expression dynamics, from tissue samples from three types of human cancer. In head and neck cancer and melanoma cancer samples, CTHRC1 is localized at the boundary between the cancer and stromal tissue. In pancreatic cancer, CTHRC1 is observed to be widely expressed in areas with high CAF / stromal tissue density. [Figure 13] Various schematic diagrams of exemplary anti-CTHRC1 T cell engagers, including M3 Fab (shown in yellow) and SP34-based scFv (shown in purple), are illustrated. [Figure 14A] The efficacy of CTHRC1 tested in the syngenic mouse mammary tumor model EMT6 is illustrated. Results for the anti-CTHRC1 antibody, specifically M5, are shown. [Figure 14B] The efficacy of CTHRC1 tested in the syngeneic mouse mammary tumor model EMT6 is illustrated. The results for the anti-CTHRC1 antibody, specifically M23, are shown. [Figure 14C] The efficacy of CTHRC1 tested in the syngeneic mouse mammary tumor model EMT6 is illustrated. The results for the anti-CTHRC1 antibody, specifically M14, are shown. [Figure 15A] The efficacy of anti-CTHRC1 (clone M5) in a PD-1 resistant Pan02 pancreatic cancer model is illustrated. [Figure 15B] The efficacy of anti-CTHRC1 (clone M5) in a PD-1 resistant Pan02 pancreatic cancer model is illustrated. [Figure 16] The effect of cell pretreatment with an anti-CTHRC1 antibody on CD8 T cell infiltration is illustrated. [Figure 17A] A schematic diagram of the anti-CTHRC1 T cell engager tested in Example 11 is shown. [Figure 17B]Figure 17A illustrates the results of a cell-killing assay using the anti-CTHRC1 T cell engager, in contrast to a control bispecific molecule. It visualizes caspase activation following treatment with the anti-CTHRC1 T cell engager (1:1 at the top, 2:1 cis at the bottom) or the control bispecific molecule for matched KP4 pancreatic tumors and CAFs. [Figure 17C] Figure 17A illustrates the results of a cell-killing assay using the anti-CTHRC1 T cell engager, contrasting it with a control bispecific molecule. Quantitative analysis of caspase activation across different treatment groups is also shown. [Figure 18A] The relative tumor cell viability of samples treated with either an anti-CTHRC1 T cell engager (cis-2:1 configuration) or a control dengue bispecific molecule from two donors is illustrated. [Figure 18B] The number of CD8 T cells in samples treated with either an anti-CTHRC1 T cell engager (cis-2:1 configuration) or a control dengue bispecific molecule from two donors is illustrated. [Figure 18C] The expression levels of 4-1BB and CD8 per 1 mg of tumor in donor 2 of Example 12 are shown in the figure. [Figure 18D] The characteristics of the supernatant for perforin in the sample from donor 2 are illustrated. [Figure 18E] The characteristics of the supernatant for granzyme B in the donor 2 sample are illustrated. [Figure 18F] The characteristics of the supernatant for IFN-γ in the donor 2 sample are illustrated. [Figure 19A] The test design for Example 13 is shown in the diagram. [Figure 19B] The mean growth curves after medication in both treatment groups of Example 13 are shown, with the small dots on the x-axis indicating the medication time. The data are presented as median + / - median absolute deviation. [Figure 19C] The individual growth curves after drug administration in both treatment groups of Example 13 are shown, with the small dots on the x-axis indicating the drug administration time. [Modes for carrying out the invention]

[0031] This disclosure provides an anti-CTHRC1 T cell engager and a composition thereof, the anti-CTHRC1 T cell engager comprising a first domain containing a collagen triple helix repeat-containing 1(CTHRC) binding moiety that selectively binds to human CTHRC1 and cells presenting the CTHRC1 epitope without internalization, and a second domain containing a T cell engager, e.g., an anti-CD3 antibody or a fragment thereof. In some embodiments, the T cell engager comprises an anti-CD3 antibody or a fragment thereof. This disclosure further provides a method for producing the same and its use in the treatment of cancer.

[0032] The present invention will be carried out using conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, within the scope of the skills of those skilled in the art, unless otherwise specified. Such techniques are well explained in literature such 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” (FMAusubel et al., eds., 1987, and regularly updated editions), “PCR: The Polymerase Chain Reaction” (Mullis et al., ed., 1994), “A Practical Guide to Molecular Cloning” (Perbal Bernard V., 1988), and “Phage Display: A Laboratory Manual” (Barbas et al., 2001).

[0033] Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein that can be used in carrying out the present invention. In fact, the present invention is by no means limited to the methods and materials described herein. In the present invention, the following terms are defined as follows:

[0034] definition For the purposes of interpreting this specification, the following definitions apply, and wherever appropriate, a term used in the singular also includes its plural form, and vice versa. In the event of any conflict between any definition set forth herein and any reference incorporated herein by reference, the definition set forth below shall prevail.

[0035] When used herein, unless explicitly indicated otherwise by the context, the singular forms "a," "an," and "the" include plural referents.

[0036] The use of the term “or” in the claims and this disclosure is intended to mean “and / or” unless it is expressly directed to refer only to one of the options or the options are mutually exclusive.

[0037] The use of the term "approximately" when used with a number is intended to include a + / - 10% range. While not limiting, for example, if some amino acids are identified as "approximately 100," this would include a range of 90-110 (plus or minus 10%).

[0038] When used herein, "comprising," "including," "containing," "having," etc., are intended to be open and inclusive, unless otherwise noted, so as not to exclude additional unlisted elements.

[0039] As used herein, the term “collagen triple helix repeat-containing 1 (CTHRC1)” refers to any natural CTHRC1 from any vertebrate source, including mammals such as primates (e.g., humans, primates, and rodents (e.g., mice and rats)), unless otherwise indicated. This term encompasses several isoforms (see, for example, SEQ ID NOs. 85–87). Human CTHRC1 is encoded by the nucleotide sequence corresponding to GenBank accession number NG031985.

[0040] The term “collagen triple helix repeat-containing 1” encompasses unprocessed “full-length” CTHRC1 and any form of CTHRC1 resulting from cellular processing. This term also encompasses naturally occurring variants of CTHRC1, such as splice variants, allele variants, and isoforms. The CTHRC1 polypeptides described herein may be isolated from various sources, such as from human tissue species or other sources, or prepared by recombinant or synthetic methods. “Natural sequence CTHRC1 polypeptide” includes polypeptides having the same amino acid sequence as the corresponding CTHRC1 polypeptide derived in nature. Such natural sequence CTHRC1 polypeptides may be isolated from nature or produced by recombinant or synthetic means. The term “natural sequence CTHRC1 polypeptide” specifically encompasses naturally occurring truncated or secreted forms (e.g., extracellular domain sequences), naturally occurring variant forms (e.g., alternative splicing forms), and naturally occurring allele variants of a particular CTHRC1 polypeptide. In certain embodiments of the present invention, the natural sequence CTHRC1 polypeptide disclosed herein is a mature or full-length natural sequence polypeptide comprising the full-length amino acid sequence shown in the appendix disclosure.

[0041] The "identity" percentage between an amino acid sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to amino acid residues in the reference sequence, after the sequences have been aligned and gaps introduced, if necessary, to obtain the maximum possible sequence identity percentage. Alignment for the purpose of determining the amino acid sequence identity percentage can be achieved in various ways within the scope of the skill of a 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. A person skilled in the art can determine appropriate parameters for aligning the sequences, including any algorithm required to obtain the maximum possible alignment over the entire length of the sequences being compared. In certain embodiments, default parameters are used.

[0042] 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 containing the amino acid residue / position. Typical modifications include, for example, substitution of a residue (or at that position) with another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more (usually five or fewer than three) amino acids adjacent to the residue / position, and deletion of the residue / position. “Amino acid substitution” or its variations refers to the replacement of an existing amino acid residue in the default (starting) amino acid sequence with a different amino acid residue. Generally, a modification results in a change in at least one physicobiochemical activity of the variant polypeptide compared to the polypeptide containing the starting (or “wild-type”) amino acid sequence. For example, in the case of an antibody, the altered physicobiochemical activity may be binding affinity, binding ability, and / or binding effect to the target molecule.

[0043] The term "antibody" is used in its broadest sense and specifically includes, for example, single anti-CTHRC1 monoclonal antibodies (including agonists, antagonists, neutralizing antibodies, full-length or intact monoclonal antibodies), polyepitope-specific anti-CTHRC1 antibody compositions, polyclonal antibodies, multivalent antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies (as 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, and single-domain antibodies (sdAb) (as long as they exhibit the desired biological or immunological activity). Furthermore, some anti-CTHRC1 antibodies, particularly fragments, include portions of anti-CTHRC1 antibodies (and combinations of portions of anti-CTHRC1 antibodies, e.g., scFv) that can be used as targeted arms, directed to, for example, CTHRC1 tumor epitopes, in chimeric antigen receptors of CAR-T cells, CAR-NK cells, or CAR-macrophages, and in anti-CTHRC1 T cell engagers provided herein. Such fragments are not necessarily proteolytic fragments, but rather portions of polypeptide sequences that can confer affinity to a target. The term "immunoglobulin" (Ig) is used herein interchangeably with antibody. Antibodies may be, for example, human antibodies, humanized antibodies, and / or affinity-mature antibodies.

[0044] The terms “anti-CTHRC1 antibody,” “CTHRC1 antibody,” and “antibody that binds to CTHRC1” are used interchangeably. The anti-CTHRC1 antibody is preferably able to bind with sufficient affinity, whether isolated or as part of a T cell engager, cell, or cell composition, so that the antibody is useful as a diagnostic and / or therapeutic agent.

[0045] In one embodiment, the term CTHRC1 antibody is used herein to specifically refer to an anti-CTHRC1 monoclonal antibody comprising (i) the heavy chain variable domain of SEQ ID NO: 81 and / or the light chain variable domain of SEQ ID NO: 82, or (ii) one, two, three, four, five, or six CDRs from SEQ ID NOs: 48, 53, 58, 63, 68, and 73.

[0046] "Isolated antibodies" are antibodies identified, isolated, and / or recovered from components of their natural environment. Contaminants in that natural environment are materials that interfere with the therapeutic use of the antibody and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes.

[0047] An "intact" antibody is one that contains the antigen-binding site, along with CL and at least the heavy chain constant domains CH1, CH2, and CH3. The constant domains may be the constant domains of the natural sequence (e.g., the constant domains of the natural human sequence) or amino acid sequence variants thereof. Preferably, the intact antibody has one or more effector functions.

[0048] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies; that is, the individual antibodies constituting the population are identical except for the possibility of naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific and directed to a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations, which contain different antibodies directed to different determinants (epitopes), each monoclonal antibody is directed to a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized without contamination by other antibodies. The modifier “monoclonal” should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies useful in this invention may be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or they may be produced using recombinant DNA methods in bacterial cells, eukaryotic cells, or plant cells (e.g., 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using techniques such as those described in Clackson et al., Nature, 352:624-8 (1991) and Marks et al., J. Mol. Biol., 222:581-97 (1991).

[0049] A basic four-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. In the case of IgG, a four-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has interchain disulfide bridges spaced at regular intervals. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for the α and γ chains respectively, and four CH domains for the μ and ε isotypes. Each L chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at the other end. The VL aligns with the VH, and the CL aligns with the first constant domain (CH1) of the heavy chain. It is thought that specific amino acid residues 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 structures 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.

[0050] The light chains (L chains) of any vertebrate species can be assigned to one of two 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, each having a heavy chain denoted as α, δ, ε, γ, and μ, respectively. The γ and α classes are further classified 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.

[0051] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain is "VH" or "V H It is sometimes referred to as "VL". The variable domain of the light chain is called "VL" or "V L These domains are sometimes referred to as "antigen-binding sites." These domains are generally the most variable parts of an antibody and include the antigen-binding site.

[0052] The term "variable" refers to the fact that certain segments of the variable domain have significantly different sequences among antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody to that particular antigen. However, variability is not evenly distributed across the entire 110-amino acid length of the variable domain. Instead, the V region consists of relatively invariant segments called framework regions (FRs) of 15-30 amino acids, separated by shorter, highly variable regions called "hypervariable regions," each 9-12 amino acids long. The variable domains of the natural heavy and light chains each contain four FRs, primarily in a β-sheet configuration, and these FRs are connected by three hypervariable regions that form loops connecting, and sometimes forming part of, the β-sheet structure. The hypervariable regions in each chain are held together in close proximity by fiber layers (FRs), and together with the hypervariable region from the other chain, they contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)).

[0053] An "antibody fragment" is a portion of an intact antibody, preferably comprising the antigen-binding region or one or more variable regions of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (see U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-62 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. In one embodiment, the antibody fragment contains the antigen-binding site of an intact antibody and therefore retains the ability to bind to an antigen. Furthermore, anti-CTHRC1 antibody fragments also include portions of anti-CTHRC1 antibodies (and combinations of anti-CTHRC1 antibody portions, e.g., scFv) that can be used as targeted arms directed to, for example, CTHRC1 tumor epitopes in chimeric antigen receptors of CAR-T cells or CAR-NK cells or CAR macrophages, and in anti-CTHRC1 T cell engagers provided herein. Such fragments are not necessarily proteolytic fragments, but rather portions of polypeptide sequences that can confer affinity to a target.

[0054] Papain digestion of the antibody yields two identical antigen-binding fragments called "Fab" fragments, and the remaining "Fc" fragment, a designation reflecting its ability to readily crystallize. The Fab fragment consists of the entire L chain, plus the variable region domain (VH) of the H chain and the first constant domain (CH1) of one of the heavy chains. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen-binding site. Pepsin treatment of the antibody yields a single large F(ab')2 fragment, which roughly corresponds to the two disulfide-bonded Fab fragments with divalent antigen-binding activity and is still capable of crosslinking antigens. The Fab' fragment differs from the Fab fragment by having a few additional residues, including one or more cysteines from the hinge region of the antibody at the carboxyl terminus of the CH1 domain. Fab'-SH is the designation herein for Fab' in which the cysteine ​​residue(s) of the constant domain have a free thiol group. The F(ab')2 antibody fragment was originally produced as a pair with the Fab' fragment, which has a hinged cysteine ​​in between. Other chemical couplings of antibody fragments are also known.

[0055] The Fc fragment contains the carboxyl-terminal portions of both H chains held together by a disulfide. The effector function of the antibody is determined by the sequence in the Fc region, which is also the region recognized by the Fc receptor (FcR) found on certain types of cells.

[0056] "Fv" is the smallest antibody fragment containing a complete antigen recognition site and an antigen binding site. This fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain that are tightly associated by non-covalent bonds. In single-stranded Fv (scFv) species, the one heavy chain variable domain and the 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 "dimer" structure similar to that in double-stranded Fv species. The folding of these two domains results in six hypervariable loops (three from the H chain and three from the L chain) that provide the antibody with amino acid residues for antigen binding and confer antigen-binding specificity. However, even a single variable domain (or half of Fv containing only three antigen-specific CDRs) has the ability to recognize and bind to an antigen, although with lower affinity than a complete binding site.

[0057] A "single-stranded Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. In some embodiments, the sFv polypeptide further includes a polypeptide linker between the VH and VL domains, thereby enabling the scFv to form a structure desirable for antigen binding. For an overview 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), and Borrebaeck 1995 (see below). In one embodiment, an scFv derived from an anti-CTHRC1 antibody is used as a targeting arm for CAR-T cells, CAR-NK cells, or CAR-macrophages as disclosed herein.

[0058] As used herein, the terms “hypervariable region,” “HVR,” or “HV” refer to regions of antibody variable domains whose sequences are highly variable and / or form structurally defined loops. Generally, antibodies contain six hypervariable regions, three of which are located in the VH (H1, H2, H3) and three in the VL (L1, L2, L3). Several hypervariable region delimitations are used and are incorporated herein. The Kabat complementarity-determining region (CDR) is 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, on the other hand, refers to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). When numbered using Kabat numbering rules, the end of the Chothia CDR-H1 loop differs between H32 and H34 depending on the loop length (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B is 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 region corresponds to a compromise between Kabat's CDR and Chothia's structural loop and is used by Oxford Molecular's AbM antibody modeling software. The "contact" hypervariable region is based on the analysis of available complex crystal structures. The residues from each of these hypervariable regions are listed below. [Table 1]

[0059] The hypervariable region may include the following "extended hypervariable regions": 24–36 or 24–34 (L1), 46–56 or 50–56 (L2), and 89–97 (L3) in the VL, and 26–35B (H1), 50–65, 47–65, or 49–65 (H2), and 93–102, 94–102, or 95–102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al. (above) for each of these definitions.

[0060] A “framework” or “FR” residue is a variable domain residue other than a hypervariable region residue as defined herein.

[0061] The terms “Kabat-like variable domain residue numbering” or “Kabat-like amino acid position numbering,” and their variations, refer to the numbering system used in Kabat et al. (above) for the heavy-chain or light-chain variable domains of antibody recording. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of FR or CDR residues in the variable domain. For example, a heavy-chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a by Kabat) and a residue inserted after heavy-chain FR residue 82 (e.g., residues 82a, 82b, and 82c by Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with a homologous region of the “standard” Kabat-numbered sequence.

[0062] The Kabat numbering system is generally used to refer to residues in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al. (above)). The "EU numbering system" or "EU index" is generally used to refer to residues in the constant region of the immunoglobulin heavy chain (e.g., the EU index reported in Kabat et al. (above)). "EU index as found in Kabat" refers to the residue numbering of human IgG1 EU antibodies. Unless otherwise specified herein, references to residue numbers in the variable domain of an antibody mean residue numbering according to the Kabat numbering system.

[0063] An antibody that "binds" to a target antigen or epitope is one that binds to the antigen or epitope with sufficient affinity to be measurably different from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule compared to the binding of a control molecule (which is generally a molecule with a similar structure that does not possess binding activity).

[0064] Antibodies that inhibit tumor cell growth are antibodies that result in measurable inhibition of cancer cell growth. In one embodiment, an anti-CTHRC1 antibody can inhibit the growth of cancer cells presenting a CTHRC1 tumor epitope. When referred to herein, a CTHRC1 tumor epitope includes a CTHRC1 epitope to which an anti-CTHRC1 antibody, or a fragment thereof, as disclosed herein, can be bound, or to which an antibody or other molecule that competes with an anti-CTHRC1 antibody, as disclosed herein, for binding to the epitope can be at least partially bound. A preferred growth-inhibiting anti-CTHRC1 antibody inhibits the growth of CTHRC1-presenting tumor cells by more than 20%, preferably about 20% to about 50%, and more preferably more than 50% (e.g., about 50% to about 100%) compared to a suitable control, where the control is typically tumor cells not treated with the antibody being tested.

[0065] Anti-CTHRC1 antibodies may (i) inhibit tumor metastasis in vivo, (ii) inhibit tumor growth in vivo, (iii) reduce tumor size in vivo, (iv) inhibit tumor angiogenesis in vivo, (v) exhibit cytotoxic activity against CTHRC1-presenting tumor cells and cancer-associated fibroblasts in vivo, (vi) exhibit cell division inhibitory activity against CTHRC1-presenting tumor cells or cancer-associated fibroblasts in vivo, (vii) enhance the infiltration of anti-tumor immune cells in vivo, or (viii) inhibit the suppression of immune cells in the tumor microenvironment in vivo.

[0066] The terms “cancer” and “malignant” refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell growth. A “tumor” includes one or more cancerous cells. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas, sarcomas, and leukemias or lymphoid malignancies. More specific examples of such cancers include squamous cell carcinoma (e.g., squamous cell carcinoma of the epithelium), skin cancer, melanoma, small cell lung cancer, non-small cell lung cancer ("NSCLC"), lung cancer including adenocarcinoma and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, gastric cancer or stomach 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. Examples of cancers include (for example, renal cell carcinoma, nephroblastoma, or Wilms' tumor), prostate cancer, vulvar cancer, thyroid cancer, liver cancer, anal cancer, penile cancer, and head and neck cancer. Additional examples of cancers, but not limited to, include adrenocortical carcinoma, cholangiocarcinoma, colon adenocarcinoma, B-cell lymphoma, esophageal cancer, glioblastoma multiforme, clear cell carcinoma of the kidney, papillary renal cell carcinoma, myeloid leukemia, lung adenocarcinoma, lung squamous cell carcinoma, prostate cancer, rectal adenocarcinoma, sarcoma, gastric adenocarcinoma, thymoma, uterine and uterine carcinosarcoma.

[0067] The term "metastatic cancer" refers to a cancerous condition in which cancer cells from a primary site in the body spread from the primary site to one or more other sites via blood vessels or lymphatic vessels, forming one or more secondary tumors in one or more organs in addition to the primary site. A prominent example is metastatic breast cancer.

[0068] As used herein, “CTHRC1-related cancer” refers to cancer associated with the overexpression of the CTHRC1 gene or its gene product and / or the presentation of the CTHRC1 epitope. Suitable control cells may be, for example, cells from an individual without cancer, or non-cancerous cells from a subject with cancer.

[0069] Anti-CTHRC1 T cell engagers may (i) inhibit tumor metastasis in vivo, (ii) inhibit tumor growth in vivo, (iii) reduce tumor size in vivo, (iv) inhibit tumor angiogenesis in vivo, (v) exhibit cytotoxic activity against CTHRC1-presenting tumor cells and cancer-associated fibroblasts in vivo, (vi) exhibit cell division inhibitory activity against CTHRC1-presenting tumor cells or cancer-associated fibroblasts in vivo, (vii) enhance the infiltration of anti-tumor immune cells in vivo, or (viii) inhibit the suppression of immune cells in the tumor microenvironment in vivo.

[0070] The terms "proliferative disorder" and "proliferative disorder" refer to disorders associated with a certain degree of abnormal cell proliferation. In one embodiment, the proliferative disorder is cancer.

[0071] As used herein, “tumor” refers to any neoplastic cell growth and proliferation, whether malignant or benign, as well as any precancerous and cancerous cells and tissues.

[0072] The terms “patient,” “subject,” and “individual” are used interchangeably herein and refer to any animal conforming to the methods described herein. In certain non-limiting embodiments, the patient, subject, or individual is a human.

[0073] The terms “therapeutically effective dose” or simply “effective dose” refer to the amount of a drug or composition (e.g., a drug-containing composition) that elicits a biological or medical response in a tissue, system, or subject as sought by a researcher, veterinarian, physician, or other clinician. The term “therapeutically effective dose” includes the amount of a drug or drug-containing composition that, when administered, is sufficient to prevent or, to some extent, alleviate the manifestation of one or more signs or symptoms of the disorder or disease being treated (e.g., a hematological malignancy or a solid tumor). The therapeutically effective dose may vary depending on the composition, the disease and its severity, and the age, weight, etc., of the subject being treated.

[0074] As used herein, the term “administration” means providing or giving to a target one or more agents, such as agents, that treat one or more signs or symptoms associated with a condition / disorder or disease, including but not limited to cancer (e.g., lymphoma), viral infections, bacterial infections, etc. Exemplary routes of administration include, but are not limited to, injection (subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), oral, sublingual, rectal, transdermal, intranasal, vaginal, and inhalation routes. Administration “in combination” with one or more further therapeutic agents includes simultaneous (parallel) administration and sequential administration in any order.

[0075] As used herein, the term “pharmaceutically acceptable” refers to materials, including but not limited to salts, carriers, or diluents, that do not inhibit the biological activity or properties of a compound and are relatively non-toxic; that is, materials that can be administered to an individual without causing undesirable biological effects or interacting in a harmful manner with any of the components of the composition containing them. The pharmaceutically acceptable carriers (vehicles) useful in this disclosure are conventional. Remington's Pharmaceutical Sciences, 19th Edition (1995) by EW Martin, Mack Publishing Co., Easton, Pa. describes compositions and formulations suitable for the pharmaceutically acceptable delivery of one or more drugs, such as one or more modifiers. Generally, the properties of the carrier will depend on the specific mode of administration used. For example, parenteral formulations may include injection solutions containing pharmaceutically and physiologically acceptable fluids as vehicles, such as water, physiological saline, equilibrium salt solutions, aqueous dextrose, glycerol, etc. In addition to a biologically neutral carrier, the administered pharmaceutical may contain small amounts of non-toxic auxiliary substances such as humectants or emulsifiers, preservatives, and pH buffers, for example, sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate.

[0076] "Code" refers to the inherent nature of a particular nucleotide sequence in polynucleotides such as genes, cDNA, or mRNA, and the biological properties derived therefrom, that 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. Thus, a gene codes for a protein if the transcription and translation of the mRNA corresponding to that gene produces a 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 the sequence listing, and the non-coding strand, which is used as a template for the transcription of the gene or cDNA, can be said to code for a protein or other product of that gene or cDNA.

[0077] Unless otherwise specified, "nucleotide sequences encoding amino acid sequences" include all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may contain introns.

[0078] In one aspect of the present invention, an anti-CTHRC1 T cell engager provides activation signals to T cells and NK cells present in the tumor microenvironment.

[0079] In another aspect of the present invention, T cell and NK cell activation signals induce T cell and / or NK cell-dependent cytotoxicity directed towards cancer cells.

[0080] In another embodiment, anti-CTHRC1 T cell engagers bring immune cells, including T cells and / or NK cells, to proximity to CAFs or cancer cells in a CTHRC1-dependent manner.

[0081] In another embodiment, this enhances CAF and T cell and NK cell-mediated killing of cancer cells.

[0082] In one aspect of the present invention, the anti-CTHRC1 T cell engager provides a pro-inflammatory signal to a broader range of receptor-expressing immune cells, which may include macrophages, dendritic cells, monocytes, B cells, plasma cells, neutrophils, mast cells, and other blood-derived immune cells, as well as antigen-presenting cells.

[0083] In another aspect of the present invention, an immune activation signal induces an inflammatory immune response in the tumor microenvironment that would result in the killing of tumor cells.

[0084] In another aspect of the present invention, anti-CTHRC1 T cell engagers bring inflammatory macrophages close to CAFs or cancer cells, inducing direct macrophage-mediated cytotoxicity, for example, through complement system and complement receptor-mediated phagocytosis.

[0085] In another aspect of the present invention, anti-CTHRC1 T cell engagers bring inflammatory macrophages, dendritic cells, or other antigen-presenting cells into close proximity to CAFs and cancer cells to induce enhanced presentation of antigens associated with CAFs or cancer cells and associated indirect adaptive immune responses.

[0086] In another aspect of the present invention, proximity of immune cells triggers a broad immune response, including the release of inflammatory cytokines.

[0087] In several embodiments, an anti-CTHRC1 T-cell engager is provided, which may comprise at least one first domain including a CTHRC1 binding moiety that selectively binds to human CTHRC1 and cells presenting the CTHRC1 epitope without being internalized, and at least one second domain including a T-cell engager. In several embodiments, the T-cell engager comprises an anti-CD3 antibody or a fragment thereof. In several embodiments, the anti-CTHRC1 T-cell engager comprises two first domains and / or two second domains. In several embodiments, the anti-CTHRC1 T-cell engager has first and second domains in a ratio of 2:1, 1:2, or 2:2. In embodiments having more than one first and / or second domains, the CTHRC1 binding moiety and / or T-cell engager moiety may be the same or different.

[0088] The subject matter of this disclosure includes multispecific antibodies, such as bispecific antibodies. For example, proteins can be linked together by either chemical or genetic manipulation using methods known in the art.

[0089] In one example, this disclosure encompasses anti-CTHRC1 T cell engagers. In principle, anti-CTHRC1 antibodies, such as those disclosed herein, can be fused to any T cell engager moiety using recombinant molecular biology techniques. In some embodiments, the anti-CTHRC1 T cell engager may comprise an anti-CTHRC1 antibody or a fragment thereof and an anti-CD3 antibody or a fragment thereof.

[0090] multispecific antibodies In any aspect of this disclosure, the anti-CTHRC1 T cell engagers provided herein are multispecific antibodies, for example, bispecific antibodies. A bispecific antibody is an antibody that has binding specificity to at least two different epitopes. Exemplary bispecific antibodies may bind to the epitope of the CTHRC1 protein described herein and to an epitope on T cells, for example, CD3. Other such antibodies may combine the CTHRC1 binding site with a binding site to another protein present on T cells. In some examples, the anti-CTHRC1 arm may be combined with an arm that binds to a trigger molecule on leukocytes, for example, a T cell receptor molecule (e.g., CD3), or to an Fc receptor for IgG (FcγR), for example, FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), to concentrate and localize the cellular defense mechanism to CTHRC1-expressing cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies), as well as in various forms including, for example, BiTE, HLE-Bite, Duobody, TDB, common light chain, DART, and DART-HLE.

[0091] The anti-CTHRC1 T cell engagers of this disclosure in the form of bispecific antibodies may be prepared in either cis or trans configuration. The cis configuration refers to an engager in which the first and second domains are located on the same side of the bispecific antibody, for example, at the end of each binding arm, while the trans configuration refers to an engager in which the first and second domains are located at different ends of the engager, for example, the N-terminus and the C-terminus. A description of the cis and trans configurations is provided in Santich, et al. (Sci Transl Med. 12(534):eaax1315(2020)), which is incorporated herein by reference in its entirety.

[0092] The ratio of the first domain to the second domain, or vice versa, can also be used to describe an engager by reference to the number of first and second domains. For example, a 2:1 configuration can refer to an engager having two first domains and one second domain, where, if the configuration is cis, then for an antibody, there is one first binding domain on the first binding arm, one first domain and one second domain on the second binding arm. Exemplary configurations of such engagers are provided in Figures 13 and 17A.

[0093] In addition, the anti-CTHRC1 T cell engagers of this disclosure may be designed in a symmetric or asymmetric form, with the first and second domains located on the same binding arm or on different binding arms. Symmetric and asymmetric dual-specific designs are disclosed in Madsen, et al. (Front. Bioeng. Biotechnol. 12 (2024)), which is incorporated herein by reference in its entirety.

[0094] Bispecific constructs in cis or trans configurations can be synthesized by methods known in the art, including recombinant expression techniques, or by methods described herein.

[0095] In several embodiments, the anti-CTHRC1 T cell engager is a bispecific antibody in a cis configuration. In several embodiments, the anti-CTHRC1 T cell engager is a bispecific antibody in a trans configuration.

[0096] In several embodiments, the anti-CTHRC1 T cell engager has a first domain and a second domain in a ratio of 1:1, 2:1, 1:2, or 2:2. In several embodiments, the anti-CTHRC1 T cell engager has a 2:1 ratio in cis configuration.

[0097] In several embodiments, the anti-CTHRC1 T cell engager is asymmetric. In several embodiments, the anti-CTHRC1 T cell engager is symmetric.

[0098] Methods for producing bispecific antibodies are known in the art. Conventional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where these two chains have different specificities (Millstein et al., Nature 305:537-9 (1983)). Due to the random combination of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a possible mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually performed by affinity chromatography steps, is somewhat cumbersome and results in low product yields. Similar procedures are disclosed in WO93 / 08829 and Traunecker et al., EMBO J.10:3655-3659 (1991).

[0099] Other approaches are known for producing bispecific antibodies. One approach is the “knob-into-hole” or “protuberance-into-cavity” approach (see, for example, U.S. Patent No. 5,731,168). In this approach, two immunoglobulin polypeptides (e.g., heavy-chain polypeptides) each contain an interface. The interface of one immunoglobulin polypeptide interacts with the corresponding interface of the other immunoglobulin polypeptide, thereby enabling the association of the two immunoglobulin polypeptides. These interfaces can be manipulated so that a “knob” or “protuberance” (these terms may be used interchangeably herein) located at the interface of one immunoglobulin polypeptide corresponds to a “hole” or “cavity” (these terms may be used interchangeably herein) located at the interface of the other immunoglobulin polypeptide. In some embodiments, the hole is the same size as or similar to the knob and is preferably positioned so that when the two interfaces interact, the knob of one interface can be positioned within the corresponding hole of the other interface. While we do not wish to be bound by theory, this approach is thought to stabilize heteromultimers and be more favorable to heteromultimer formation than other species, such as homomultimers. In some embodiments, this approach may be used to promote heteromultimerization of two different immunoglobulin polypeptides and to produce bispecific antibodies containing two immunoglobulin polypeptides with binding specificity to different epitopes.

[0100] A schematic diagram of an exemplary anti-CTHRC1 T cell engager is shown in Figure 13, in which M3 Fab (shown in yellow) and SP34 scFv are bound to the Fc scaffold in different ratios (1:1 and 2:1 (two alternative configurations)). The exemplary Fc scaffold may include knob-into-hole designs, which may include, for example, knob mutations S354C and T366W and hole mutations Y349C, T366S, L368A and Y407V. These include the addition of disulfide bonds to stabilize the heterodimer Fc. In certain embodiments, the hole side may be designed to always contain the anti-CTHRC1 moiety to avoid homodimerization of the “hole” chain. As will be discussed in more detail elsewhere in this disclosure, the construct is (G4S) n This may include various linkers, including (where n is 2, 3, or 4).

[0101] According to a different approach, an antibody variable domain (antibody-antigen binding site) with desired binding specificity is fused to an immunoglobulin constant domain sequence. This fusion is preferably with an immunoglobulin heavy chain constant domain that includes at least a portion of the hinge, CH2, and CH3 regions. Typically, a first heavy chain constant region (CH1) containing the site required for light chain binding is present in at least one of the fusions. The immunoglobulin heavy chain fusions, and optionally the DNA encoding the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into a suitable host organism. This provides excellent flexibility in adjusting the relative proportions of the three polypeptide fragments in embodiments where an unequal ratio of the three polypeptide chains used in construction provides optimal yield. However, if high yield is obtained by expressing at least two polypeptide chains in equal ratios, or if their ratios are not particularly important, it is possible to insert the coding sequences of two or all three polypeptide chains into a single expression vector.

[0102] In one embodiment of this approach, a bispecific antibody comprises a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate the separation of desired bispecific compounds from undesirable immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one arm of the bispecific molecule provides an easy separation method. This approach is disclosed in WO94 / 04690. For further details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986).

[0103] According to another approach described in WO96 / 27011, the interface between a pair of antibody molecules can be manipulated to maximize the proportion of heterodimers recovered from recombinant cell cultures. One interface is the C of the antibody constant domain. H This method involves at least a portion of three domains. In this method, one or more smaller amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of the same or similar size as the larger side chains are created on the interface of the second antibody molecule by replacing the larger amino acid side chains with smaller side chains (e.g., alanine or threonine). This provides a mechanism for increasing the yield of heterodimers over other undesirable end products such as homodimers.

[0104] Bispecific antibodies include crosslinked antibodies or “heteroconjugate” antibodies. For example, in a heteroconjugate, one antibody may be coupled to avidin and the other to biotin. Such antibodies have been proposed, for example, to target unwanted cells with immune system cells (U.S. Patent No. 4,676,980) and to treat HIV infection (WO91 / 00360, WO92 / 200373, and EP03089). Heteroconjugate antibodies can be produced using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 along with several crosslinking techniques.

[0105] Techniques for generating bispecific antibodies from antibody fragments are also described in this literature. For example, bispecific antibodies can be prepared using chemical bonding. Brennan et al., Science, 229:81 (1985) describe a procedure in which intact antibodies are cleaved by proteolysis to generate F(ab')2 fragments. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to thionitrobenzoic acid (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to a Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form a bispecific antibody. The produced bispecific antibody can be used as an agent for selective enzyme immobilization. Shalaby et al., J.Exp.Med., 175:217-225 (1992) describes the production of a fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment was secreted separately from E. coli and subjected to directed chemical coupling in vitro to form a bispecific antibody.

[0106] Various techniques for directly producing and isolating bispecific antibody fragments from recombinant cell cultures are also described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553(1992). Leucine zipper peptides derived from Fos and Jun proteins were linked to the Fab’ portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers, and then re-oxidized to form antibody heterodimers. This method can also be utilized for the production of antibody homodimers. An alternative mechanism for producing bispecific antibody fragments is provided by the "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448(1993). This fragment contains heavy chain variable domains (V L ) connected by a linker that is too short to allow pairing between two domains on the same chain to the light chain variable domains (V H ). Thus, the V H and V L domains of one fragment are forced to pair with the complementary V L and V H domains of another fragment, thereby forming two antigen-binding sites. Another strategy for producing bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al, J. Immunol, 152:5368(1994).

[0107] Another technique for producing bispecific antibody fragments is the "bispecific T cell engager" or BiTE® approach (see, for example, WO2004 / 106381, WO2005 / 061547, WO2007 / 042261, and WO2008 / 119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain contains a variable heavy chain (V H ) and a variable light chain (V LThe scFv comprises two single-stranded Fv (scFv) fragments having domains, these domains separated by a polypeptide linker long enough to allow intramolecular association between the two domains. This single polypeptide further contains a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific to different cell types, thereby bringing cells of two different cell types into proximity or linking them together when each scFv binds to its homologous epitope. One particular embodiment of this approach comprises an scFv that recognizes a cell surface antigen expressed on immune cells, e.g., the CD3 polypeptide on T cells, which is linked to another scFv that recognizes a cell surface antigen expressed on target cells such as malignant or tumor cells.

[0108] Because it is a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic or eukaryotic cell expression system known in the art, e.g., CHO cell lines. However, specific purification techniques (see, e.g., EP1691833) may be required to separate the monomeric bispecific T cell engager from other multimeric species that may have biological activities other than the intended monomeric activity. In one exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted using an imidazole concentration gradient. This eluate is further purified using anion exchange chromatography, and the polypeptide is eluted using a sodium chloride concentration gradient. Finally, this eluate is subjected to size exclusion chromatography to separate the monomer from the multimeric species.

[0109] Other relevant bispecific antibody fragment forms include, but are not limited to, dual-affinity re-targeting proteins (DARTs) and tandem diabolic proteins (TandAbs). DARTs consist of two Fv fragments, and two unique antigen-binding sites are formed when the two Fv fragments heterodimerize (Holliger et al.). al., Proc. Natl. Acad. Sci. USA. 90:6444-6448 (1993). Specifically, Fv1 consists of VH derived from antibody "A" and VL derived from antibody "B", while Fv2 is constructed from VH derived from antibody "B" and VL derived from antibody "A". Unlike BiTE antibodies linked by polypeptide linkers, this combination allows DART to mimic innate interactions within the IgG molecule. Stability is improved by the formation of C-terminal disulfide crosslinks by adding another cysteine ​​residue to the end of each heavy chain. TandAb is a tetravalent bispecific antibody, providing two binding sites for each antigen to maintain the binding activity of the innate bivalent antibody. Furthermore, TandAb has a molecular weight (approximately 105 kDa) that exceeds the first-pass renal clearance threshold, and therefore provides a longer half-life compared to smaller antibody constructs (Reusch et al., Clin. Cancer). Res.Off.J.Am.Assoc.Cancer Res.22:5829-5838(2016), Reusch et al., MAbs.6:728-739(2014); Compte et al., Oncoimmunology.3:e28810(2014).For a recent overview of common forms of bispecific antibodies (including their production methods), including BiTE, scFv-based antibodies and full-length IgG-like asymmetric antibodies, see Madsen et al., Design and engineering of bispecific antibodies: insights and practical considerations; Front. Bioeng. Biotechnol. 12 (2024), Santich, et al. (Sci Transl Med. 12 (534):eaax1315 (2020)), and Wang et al., Antibodies (Basel), 8 (3):43 (2019).

[0110] For additional instructions on exemplary bispecific forms and their preparation methods, please refer to Bacac et al., Clin Cancer Res; 24(19) October 1, 2018; Bacac et al., Clin Cancer Res; 22(13) July 1, 2016; Ravandi et al., Blood advances; 7(21) November 14, 2023; Engelberts et al., EBioMedicine 52, 2020; Giffin et al., Clin Cancer Res 2021; 27: 1526-37; and Chichili et al., Science Transl Med 27 May 2015 Vol 7 Issue. 289, as well as US_9914776_B2, US11124577, US10155815, WO_2022_262959_A1, US_11672858_B2, US9850320, US9856327, US10544220, US10294300, US9657102, and US20160200827, the disclosures of which are expressly incorporated herein by reference in their entirety.

[0111] T cell engager portion, anti-CD3 antibody and its fragments. As described herein, the second domain of the anti-CTHRC1 T cell engager includes a T cell engager moiety. In some embodiments, the anti-CTHRC1 T cell engager of this disclosure includes one or more second domains including a T cell engager moiety. In some embodiments, the anti-CTHRC1 T cell engager has a first domain and a second domain in a ratio of 1:1, 2:1, 1:2, or 2:2.

[0112] In any of the embodiments described above, the second domain comprises an anti-CD3 antibody or a fragment thereof. In a particular embodiment, the anti-CD3 antibody is SP34 or a fragment thereof. In a particular embodiment, the anti-CD3 antibody is UCHT-1 or a fragment thereof. In a particular embodiment, the anti-CD3 antibody is BC-3 or a fragment thereof.

[0113] In any of the embodiments described above, the anti-CD3 antibody or a fragment thereof may include heavy chain complementarity-determining regions (HCDRs) 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, and light chain complementarity-determining regions (LCDRs) 1, LCDR2, and LCDR3 of SEQ ID NOs: 4, 5, and 6, respectively.

[0114] In any of the embodiments described above, the anti-CD3 antibody or a fragment thereof may include the variable heavy chain region (HCVR) of SEQ ID NO: 7 and the light chain variable chain region of SEQ ID NO: 8.

[0115] In several embodiments, the T cell engager, for example, an anti-CD3 antibody or a fragment thereof, may include a heavy chain variable region (HCVR) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 7. In several embodiments, the T cell engager, for example, an anti-CD3 antibody or a fragment thereof, may include a light chain variable region (LCVR) having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to SEQ ID NO: 8.

[0116] In some embodiments, the anti-CD3 antibody or fragment thereof may include HCDR1, HCDR2, and HCDR3 of HCVR having any one of the following HCVR sequences. [Table 2]

[0117] In several embodiments, the anti-CD3 antibody or a fragment thereof may include LCDR1, LCDR2, and LCDR3 of LCVRs having any one of the following LCVR sequences. [Table 3]

[0118] In multiple embodiments, the anti-CD3 antibody or fragment thereof may include HCDR1, HCDR2, and HCDR3 of any one of the following HCVR / LCVR pairs, as well as LCDR1, LCDR2, and LCDR3 of LCVR, including any combination of HCVR / LCVR for epcolitamab and any combination of HCVR / LC for talketamab. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] [Table 4-6]

[0119] In some embodiments, the anti-CD3 antibody or fragment thereof may include any combination of eccolitamab HCVR / LCVR pairs, or any one of the following HCVR / LCVR pairs: [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] [Table 5-5] [Table 5-6]

[0120] In several embodiments, the anti-CD3 antibody or fragment thereof may include any combination of epcolitamab HCVR / LCVRs, and may comprise HCVR / LCVR pairs having sequences with at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of the following HCVR / LCVR pairs: [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

[0121] In several embodiments, if the T cell engager, e.g., anti-CD3, is an antibody, it may be a chimeric, humanized, or human antibody. In several embodiments, if the T cell engager is an antibody, it may be a fragment of a full-length antibody, 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.

[0122] In several embodiments, the T cell engager may be a T cell engager, for example, an antibody or a fragment thereof, which may be described in or derived from any of U.S. Patent Publication No. 2017 / 0355767, U.S. Patent No. 10,407,501, U.S. Patent No. 10,562,968, U.S. Patent Publication No. 2021 / 0253701, U.S. Patent No. 9,657,102, WO2000 / 041474, and U.S. Patent Publication No. 2023 / 0002506 (each of which is incorporated herein by reference in its entirety). Additional T-cell activators, including anti-CD3 antibodies and their fragments, include alnuctamab, grofitamab, gresonitamab, akapatamab, pasotuxizumab, pacanalotamab, elbixtamab, emerfetamab, solitomab, cebostamab, erranatamab, odronextamab, muromonab, huCLB-T3 / 4, blinatumomab, PAI-SP34, bicilizumab, otelixizumab, epcolitamab, talketamab, flotetuzumab, tecristamab, and pabul. Tamab, Cibistamab, Vibecotamab, Pabultamab, MGD007, Tepositamab, GBR1302, M802, Paramotamab, GEM333, PF-06671008, AMV564, JNJ-63709178, ERY974, Obrindatamab, Chidutamab, IGM-2323, JNJ-63898081, Duvoltuxizumab, Etebritamab, Tarulatamab, OKT3, Teplizumab (trademark) (MGA031, Eli This includes Lilly), UCHT1, BC-3, NI0401, X35-3, VIT3, BMA030 (BW264 / 56), CLB-T3 / 3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, WT31, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, OKT3D, M-T301, SP34, SMC2 and F101.01.An anti-CD3 antibody or fragment thereof may comprise six CDRs (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) of any of these disclosures or the antibody or fragment thereof, and / or heavy chain variable regions (HCVRs) and / or light chain variable regions (LCVRs) having 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with respect to the sequence of such antibody or fragment thereof.

[0123] In some embodiments, the second domain includes means for coupling to CD3 according to any of the embodiments described above.

[0124] CTHRC1 binding part In some embodiments of the present disclosure, the anti-CTHRC1 T cell engager of the present disclosure comprises one or more first domains including a CTHRC1 binding moiety. In some embodiments, the anti-CTHRC1 T cell engager has a first domain and a second domain in a ratio of 1:1, 2:1, 1:2, or 2:2.

[0125] In several embodiments, the CTHRC1 binding moiety can (i) selectively bind to CTHRC1 and / or (ii) block cell adhesion to CTHRC1. In several embodiments, the CTHRC1 binding moiety can selectively bind to human CTHRC1 and cells presenting the CTHRC1 epitope without being internalized.

[0126] In any of the embodiments described above, the CTHRC1 binding moiety may be an antibody as defined herein that can bind to CTHRC1. In some embodiments, the anti-CTHRC1 antibody of the present invention further comprises 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. In one embodiment, the anti-CTHRC1 antibody inhibits or neutralizes one or more functions of human CTHRC1.

[0127] It should be understood that the CTHRC1 binding portion may further include peptide linkers as described herein. For example, if the CTHRC1 binding portion includes scFv, the VH and VL portions may be linked by any suitable linker, such as those described herein.

[0128] Exemplary CDRs and antibody sequences are provided in the table below. [Table 7] [Table 8] [Table 9] [Table 10]

[0129] AB990(M3) exhibits a unique characteristic in that it is not internalized upon binding to the CTHRC1 epitope on cells. This characteristic is surprising and unexpected, as it exhibits variability in internalization characteristics among antibodies against CTHRC1. Therefore, in any of the embodiments described above, the CTHRC1 binding moiety may include an antibody moiety containing HCDR1, HCDR2, and HCDR3 of AB990 (or, alternatively, any antibody from Tables 2 and 3, if antibody internalization is permitted) and / or LCDR1, LCDR2, and LCDR3 of AB990. In any of the embodiments described above, the CTHRC1 binding moiety may include an antibody moiety containing VH and / or VL of any of the antibodies from Tables 4 and 5 (or, alternatively, any antibody from Tables 4 and 5, if antibody internalization is permitted).

[0130] Preferably, the CTHRC1 binding portion of the present invention is scFv or scFab, where the nucleic acid sequence of scFv comprises nucleic acid sequences (or more) encoding one or more light chain CDRs and one or more heavy chain CDRs disclosed herein with respect to an anti-CTHRC1 antibody, and the nucleic acid sequence of scFab comprises nucleic acid sequences (or more) encoding one or more light chain CDRs and one or more heavy chain CDRs disclosed herein with respect to an anti-CTHRC1 antibody.

[0131] 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: 81 to 82.

[0132] Preferably, the CTHRC1 binding portion of the present invention is scFv or scFab containing the amino acid sequence of SEQ ID NO: 81.

[0133] Preferably, the CTHRC1 binding portion of the present invention is scFv or scFab containing the amino acid sequence of SEQ ID NO: 82.

[0134] Preferably, the CTHRC1 binding portion of the present invention is an scFv or scFab containing the amino acid sequence of SEQ ID NO: 81 and the amino acid sequence of SEQ ID NO: 82.

[0135] In a preferred embodiment, the CTHRC1 binding portion includes HCDR1 of SEQ ID NO: 48, HCDR2 of SEQ ID NO: 53, HCDR3 of SEQ ID NO: 58, LCDR1 of SEQ ID NO: 63, LCDR2 of SEQ ID NO: 68, and LCDR3 of SEQ ID NO: 73.

[0136] In a preferred embodiment, the CTHRC1 binding moiety includes a heavy chain variable domain containing the sequence of SEQ ID NO: 81 and a light chain variable domain containing the sequence of SEQ ID NO: 82.

[0137] In any of the embodiments described above, the anti-CTHRC1 binding moiety and / or anti-CTHRC1 T cell engager may have a binding affinity of less than 10 nM, preferably less than 5 nM, and more preferably less than 1 nM to CTHRC1. The binding affinity of the anti-CTHRC1 T cell engager can be determined, for example, by scatchard analysis as described in Munson et al., Anal. Biochem. 107:220 (1980).

[0138] In some embodiments, if the CTHRC1 binding portion is an antibody, it may be a chimeric, humanized, or human antibody. In some embodiments, if the CTHRC1 binding portion is an antibody, it may be a fragment of a full-length antibody, a single-chain antibody, a single-domain antibody (e.g., an antibody consisting only of the heavy chain), a single-chain variable fragment (scFv), or any other type of “antibody” as defined herein. Exemplary antibodies include monoclonal, chimeric, humanized, and human antibodies.

[0139] In several embodiments, the CTHRC1 coupling portion is WO2010 / 047448, U.S. Patent Application Publication 2005 / 0147602, U.S. Patent Application Publication 2016 / 0000866, WO2014 / 200134, U.S. Patent No. 9,050,296, U.S. Patent Application Publication 2013 / 0190357, U.S. Patent Application Publication 2018 / 0313846, U.S. Patent No. 9,718, This could be a CTHRC1 binding portion as described in any of the following: U.S. Patent Application Publication No. 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 herein by reference in its entirety).Additional CTHRC1 binding moieties include, but are not limited to, 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). Biotechnology, Inc., Dallas, Texas), anti-CTHRC1 antibodies: SAB1102667, HPA059806, SAB2107469, and SAB1402656 (each of which is incorporated herein by reference) (Sigma-Aldrich®, St. Louis, Mo.), and anti-CTHRC1 antibody PA5-38054 (incorporated herein by reference) (Thermo Antibodies or fragments thereof containing HCDR1, HCDR2, and HCDR3 of SEQ ID NOs. 9-11 and 13-15 respectively, and LCDR1, LCDR2, and LCDR3 of US Patent Application Publication No. 2022 / 0204599, respectively, and HCDR1, HCDR2 of SEQ ID NOs. 1-3 or 11-13 (HCDR) and 4-6 or 14-16 (LCDR), The antibodies or fragments thereof (including clones cCMAb45, cCMAb96, hCMAb45, and hCMab96, 6C1, and 1D1) (each of which is incorporated herein by reference in whole) may include HCDR2 and any of the heavy chain variable regions of LCDR1, LCDR2, and LCDR3 and / or SEQ ID NOs. 7, 17, 21, or 25 and / or any of the light chain variable regions of SEQ ID NOs. 9, 19, 23, or 27.

[0140] In some embodiments, the first domain includes means for coupling to CTHRC1 according to any of the embodiments described above.

[0141] Monoclonal antibodies Monoclonal antibodies (mAbs) against the target antigen can be prepared using any technique known in the art. These include, but are not limited to, the hybridoma technique originally described by Kohler and Milstein (1975, Nature 256, 495-497), the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). 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 may be of any immunoglobulin class, including IgG, IgM, IgE, IgA, and IgD, and any subclass thereof. Hybridomas for producing mAbs useful in this invention can be cultured in vitro or in vivo.

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

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

[0144] The hybridoma cells thus prepared are seeded and grown in a suitable medium that may contain one or more substances that inhibit the proliferation or survival of non-fused parent myeloma cells (also called fusion partners). For example, if the parent myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the selective medium for hybridomas will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibit the proliferation of HGPRT-deficient cells.

[0145] Preferred fusion partner myeloma cells are those that efficiently fuse, support stable high levels of antibody production by selected antibody-producing cells, and are sensitive to selective media that are selective for non-fused parent cells. Preferred myeloma cell lines include mouse myeloma lines, such as MOPC-21 and MPC-11 mouse tumor-derived cells available from Salk Institute Cell Distribution Center, San Diego, Calif., USA, as well as SP-2 and its derivatives, such as X63-Ag8-653 cells, available from American Type Culture Collection, Manassas, Va., USA. Human myeloma and mouse-human heterozygous myeloma cell lines have also been described for human monoclonal antibody production (Kozbor, J. Immunol., 133:3001 (1984), and Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

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

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

[0148] Once hybridoma cells producing antibodies with desired specificity, affinity, and / or activity are identified, the clones may be subcloned using 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 in animals as ascites carcinoma, for example, by intraperitoneal injection of these cells into mice.

[0149] Monoclonal antibodies secreted by subclones can be suitably separated from culture media, ascites fluid, or serum by conventional antibody purification procedures such as affinity chromatography (e.g., using protein A or protein G-Sepharose), ion exchange chromatography, hydroxyl apatite chromatography, gel electrophoresis, or dialysis.

[0150] DNA encoding monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of mouse antibodies). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA may be placed in 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 would otherwise not produce antibody proteins, resulting in the synthesis of monoclonal antibodies in recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol. 5:256-62 (1993) and Pluckthun, Immunol. Rev. 130:151-88 (1992).

[0151] In further embodiments, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the technique 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 using phage libraries, respectively. Subsequent publications describe the production of high-affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology, 10:779-783 (1992)), and combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nuc.Acids.Res. 21:2265-6 (1993)). Therefore, these techniques are viable alternatives to conventional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0152] The DNA encoding the antibody may be modified to produce a chimeric or fusion antibody polypeptide, for example, by substituting homologous mouse sequences in the human heavy and light chain constant domains (CH and CO sequences) (U.S. Patent No. 4,816,567, and Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence of a non-immunoglobulin polypeptide (heterogeneous polypeptide). The non-immunoglobulin polypeptide sequence can substitute for the constant domain of the antibody, or substitute for the variable domain of one antigen-binding site of the antibody, thereby creating a chimeric bivalent antibody having one antigen-binding site specific to a certain antigen and another antigen-binding site specific to a different antigen.

[0153] Chimeras, humanization, and human antibodies In some embodiments, the CTHRC1 binding moiety is a chimeric antibody. In some embodiments, the T cell engager moiety, e.g., an anti-CD3 antibody or a fragment thereof, is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-5 (1984). In one example, a chimeric antibody includes 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 further examples, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include their antigen-binding fragments.

[0154] In some embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce its immunogenicity to humans while retaining the specificity and affinity of the non-human parent antibody. Generally, a humanized antibody contains one or more variable domains, such as an HVR, where a CDR (or a portion thereof) is derived from the non-human antibody and an FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody also optionally contains at least a portion of the human constant region. In some embodiments, several FR residues in the humanized antibody are replaced with corresponding residues derived from the non-human antibody (e.g., the antibody from which the CDR residue is derived) to restore or improve antibody specificity or affinity, for example.

[0155] The anti-CTHRC1 antibody and T-cell engager moiety of the present invention may include a humanized antibody or a human antibody. The humanized form of a non-human (e.g., mouse or rabbit) antibody is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of the antibody) containing a minimal sequence derived from a non-human immunoglobulin. The humanized antibody includes a human immunoglobulin (recipient antibody) in which residues derived from the recipient's complementarity-determining region (CDR) are replaced with residues derived from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and ability. In some cases, Fv framework residues of the human immunoglobulin are replaced with corresponding non-human residues. The humanized antibody may also include residues not found in the recipient antibody or in the transferred CDR or framework sequence. Generally, humanized antibodies contain substantially all of at least one, typically two, variable domains, where all or substantially all of the CDR region corresponds to that of a non-human immunoglobulin, and all or substantially all of the FR region corresponds to that of the human immunoglobulin consensus sequence. Humanized antibodies also optimally contain at least a portion of the 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)).

[0156] The humanized antibody of the present invention, as the CTHRC1 binding moiety and / or T cell engager moiety, may contain one or more human and / or human consensus non-hypervariable region (e.g., framework) sequences in its heavy chain and / or light chain variable domains. In some embodiments, one or more additional modifications exist within the human and / or human consensus non-hypervariable region sequence. In one embodiment, the heavy chain variable domain of the antibody of the present invention contains 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 contains a variant subgroup III consensus framework sequence modified at at least one amino acid position.

[0157] As is known in the art and as will be described in more detail herein, the amino acid positions / boundaries that define the hypervariable region of an antibody can vary depending on the context and various definitions known in the art (described below). Some positions within the variable domain can be considered hybrid hypervariable positions in that these positions may be considered within the hypervariable region under one set of criteria, while being considered outside the hypervariable region under a different set of criteria. One or more of these positions may also be found in an extended hypervariable region (as further defined below). The present invention provides antibodies with modifications to these hybrid hypervariable positions. In one embodiment, these hypervariable positions include one or more positions 26-30, 33-35B, 47-49, 57-65, 93, 94, and 101-102 in the heavy chain variable domain. In one embodiment, these hybrid hypervariable positions include one or more positions 24-29, 35-36, 46-49, 56, and 97 in the light chain variable domain. In one embodiment, the antibody of the present invention comprises a human variant human subgroup consensus framework sequence modified at one or more hybrid hypervariable positions.

[0158] The CTHRC1 binding moiety of the present invention may contain any suitable human or human consensus light chain framework sequence, insofar as the antibody exhibits the desired biological properties (e.g., desired binding affinity). In one embodiment, the antibody of the present invention contains at least a portion (or all) of a human κ light chain framework sequence. In one embodiment, the CTHRC1 binding moiety of the present invention contains at least a portion (or all) of a human κ subgroup I framework consensus sequence.

[0159] Methods for humanizing non-human antibodies are well known in the art. Generally, humanized antibodies have one or more amino acid residues introduced into them from a non-human source. These non-human amino acid residues are often referred to as “implant” residues, and these residues are typically obtained from the “implant” variable domain. Humanization is essentially carried out by substituting the corresponding sequence of a human antibody with a rodent CDR or CDR sequence, according to the method of Winter and collaborators (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)). Thus, such “humanized” antibodies are essentially chimeric antibodies (U.S. Patent No. 4,816,567) in which less than a intact human variable domain is substituted with the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which several CDR residues and possibly several FR residues are substituted with residues derived from similar sites in rodent antibodies.

[0160] When antibodies are intended for human therapeutic use, the selection of both light and heavy chain human variable domains used in the production of humanized antibodies is crucial for reducing antigenicity and HAMA response (human anti-mouse antibody). Reducing or eliminating the HAMA response is an important aspect of the clinical development of suitable therapeutic agents (see, for example, Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et al., Transplantation (1986), 41:572; Shawler et al., J. Immunol. (1985), 135:1530; Sears et al., J. Biol. Response Mod. (1984), 3:138; Miller et al., Blood (1983), 62:988; Hakimi et al., J. Immunol. (1991), 147:1352; Reichmann et al., Nature (1988), 332:323; Junghans et al., Cancer Res. (1990), 50:1495). As described herein, the present invention provides antibodies that have been humanized such that the HAMA response is reduced or eliminated. Variants of these antibodies can be further obtained using methods commonly known in the art, some of which are further described below. The variable domain sequence of a rodent antibody is screened against an entire library of known human variable domain sequences according to a so-called "best-fit" method. The human V-domain sequence most closely resembling the rodent V-domain sequence is identified, and the human framework region (FR) within it 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 particular subgroup of the light or heavy chain.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)).

[0161] For example, an amino acid sequence from an antibody, such as those described herein, may serve as a starting (parent) sequence for diversifying the framework and / or hypervariable sequence(s). The selected framework sequence to which the starting hypervariable sequence is ligated is referred herein to as the acceptor human framework. The acceptor human framework may be from or derived from human immunoglobulin (its VL and / or VH regions), but preferably, the acceptor human framework is from or derived from a human consensus framework sequence, such that immunogenicity has been demonstrated to be minimal or absent in human patients.

[0162] If the acceptor is derived from human immunoglobulin, the donor framework sequence may be optionally aligned with various human framework sequences in a population of human framework sequences, and the framework sequence most homologous to the acceptor may be selected, thereby selecting a human framework sequence based on its homology to the donor framework sequence.

[0163] In one embodiment, the human consensus framework described herein is derived from or is derived from the VH subgroup III and / or VL kappa subgroup I consensus framework sequence.

[0164] The acceptor may be identical in sequence to the selected human framework sequence, regardless of whether it originates from human immunoglobulin or the human consensus framework; however, the present invention intends that the acceptor sequence may contain existing amino acid substitutions compared to the human immunoglobulin sequence or the human consensus framework sequence. These existing substitutions are preferably minimal, typically consisting of only four, three, two, or one amino acid difference compared to the human immunoglobulin sequence or the consensus framework sequence.

[0165] The hypervariable region residues of non-human antibodies are incorporated into the VL and / or VH acceptor human framework. For example, residues corresponding to the CDR residues of Kabat, the hypervariable loop residues of Chothia, the residues of Abm, and / or contact residues may be introduced. Optionally, the following extended hypervariable region residues are incorporated: 24-34 (L1), 50-56 (L2), and 89-97 (L3), 26-35B (H1), 50-65, 47-65, or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3).

[0166] The "incorporation" of hypervariable region residues is discussed herein, and it will be understood that this can be achieved in various ways. For example, nucleic acids encoding a desired amino acid sequence can be generated by mutating a nucleic acid encoding a mouse 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 hypervariable domain residues are changed to non-human residues, or by synthesizing a nucleic acid encoding the desired sequence.

[0167] As described herein, a hypervariable region-implanted variant may be generated by the Kunkel mutagenesis method of nucleic acids encoding a human acceptor sequence, using separate oligonucleotides for each hypervariable region. (Kunkel et al., Methods Enzymol. 154:367-382 (1987)). To modify the hypervariable region-antigen interaction and reconstruct it appropriately, appropriate changes can be introduced into the framework and / or hypervariable region using conventional techniques.

[0168] 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 a library produced by sequence randomization. However, other methods for producing and screening variant antibodies are available to those skilled in the art.

[0169] Phage(mid) display technology provides a powerful tool for generating and selecting novel proteins that bind to ligands such as antigens. Using phage(mid) display techniques, it is possible to generate large libraries of protein variants that can be rapidly sorted for sequences that bind to target molecules with high affinity. The nucleic acid encoding the variant polypeptide is typically fused to a nucleic acid sequence encoding a viral coat protein, such as gene III protein or gene VIII protein. Monovalent phagemide 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 gene III protein (Bass, S., Proteins, 8:309 (1990), Lowman and Wells, Methods: A Companion to Methods in Enzymology, 3:205 (1991)). In monovalent phagemide display systems, the gene fusion is expressed at low levels, while the wild-type gene III protein is also expressed to maintain particle infectivity. Methods for generating peptide libraries and screening those libraries are disclosed in numerous patents (e.g., U.S. Patents 5,723,286, 5,432,018, 5,580,717, 5,427,908, and 5,498,530).

[0170] Libraries of antibodies or antigen-binding polypeptides are prepared in several ways, including by altering a single gene, inserting random DNA sequences, or cloning a family of related genes. Methods for displaying antibodies or antigen-binding fragments using phage (mid) displays are described in U.S. Patents 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 expression of antibodies or antigen-binding proteins with desired properties.

[0171] Methods for substituting selected amino acids into template nucleic acids are well-established in the art, and some of these are described herein. For example, hypervariable region residues can be substituted using the Kunkel method (e.g., Kunkel et al., Methods Enzymol. 154:367-382 (1987)).

[0172] The sequence of an oligonucleotide contains one or more codon sets designed for the hypervariable region residues to be altered. A codon set is a set of different nucleotide triplet sequences used to encode a desired variant amino acid. Codon sets can be represented using symbols that specify a particular nucleotide or an equimolar mixture of nucleotides, as shown below in IUB code. IUB code G Guanine Adenine T Chimin 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, C, or G) D(A or G or T)H N (A or C or G or T)

[0173] For example, in the codon set DVK, D can be nucleotide A, G, or T, V can be A, G, or C, and K can be G or T. This codon set can present 18 different codons and can encode the amino acids Ala, Trp, Tyr, Lys, Thr, Asn, Lys, Ser, Arg, Asp, Glu, Gly, and Cys.

[0174] Oligonucleotides or primer sets can be synthesized using standard methods. A set of oligonucleotides containing sequences that represent all possible combinations of nucleotide triplets provided by a codon set and that will encode a desired group of amino acids can be synthesized, for example, by solid-phase synthesis. The synthesis of oligonucleotides having nucleotide "degeneracy" selected at a particular position is well known in the art. Oligonucleotides of such a set having a particular codon set can be synthesized using commercial nucleic acid synthesizers (e.g., available from Applied Biosystems, Foster City, Calif.) or are commercially available (e.g., from Life Technologies, Rockville, Md.). Thus, a set of synthesized oligonucleotides having a particular codon set typically includes multiple oligonucleotides with different sequences, the difference being established by the codon set within the overall sequence. Oligonucleotides used in this invention have sequences that enable hybridization into a variable domain nucleic acid template and may also include restriction enzyme sites for cloning purposes.

[0175] One method involves oligonucleotide-mediated mutagenesis to generate nucleic acid sequences encoding variant amino acids. This technique is well-known in the art, as described by Zoller et al. Nucleic Acids Res. 10:6487-6504 (1987). Briefly, nucleic acid sequences encoding variant amino acids are generated by hybridizing a set of oligonucleotides encoding a desired codon set to a DNA template, where the template is a single-stranded plasmid containing a nucleic acid template sequence in a variable region. After hybridization, a complete second complementary strand of the template is synthesized using DNA polymerase, so that it incorporates oligonucleotide primers and contains the codon set provided by the oligonucleotide set.

[0176] Generally, oligonucleotides of at least 25 nucleotides in length are used. An optimal oligonucleotide should have 12 to 15 nucleotides that are perfectly complementary to the template on each side of the nucleotide(s) encoding the mutation(s). This ensures that the oligonucleotide hybridizes properly to the single-stranded DNA template molecule. Oligonucleotides are readily synthesized using techniques known in the art, such as the technique described by Crea et al., Proc. Nat'l. Acad. Sci. USA, 75:5765 (1978).

[0177] The DNA template is generated by either a vector derived from the bacteriophage M13 vector (commercially available M13 mp 18 and M13 mp 19 vectors are preferred) or a vector containing a single-stranded phage replication origin, as described by Viera et al., Meth. Enzymol., 153:3 (1987). Therefore, to generate a single-stranded template, the DNA to be mutated can be inserted into one of these vectors. The production of the single-stranded template is described in sections 4.21-4.41 of Sambrook et al. mentioned above.

[0178] To alter the native DNA sequence, oligonucleotides are hybridized to a single-strand template under suitable hybridization conditions. Then, a DNA polymerase, typically T7 DNA polymerase or a Klenow fragment of DNA polymerase I, is 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 codes for the mutant gene 1, and the other strand (the original template) codes for the unchanged native sequence of gene 1. This heteroduplex molecule is then introduced into suitable host cells, typically prokaryotes such as E. coli JM101, to induce transformation. After cell proliferation, the cells are plated on agarose plates and screened using oligonucleotide primers radiolabeled with 32-phosphate to identify bacterial colonies containing the mutant DNA.

[0179] The method described above may be modified to produce a homo-double-stranded molecule in which both strands of the plasmid contain mutations (multiple mutations). The modification is as follows: A single-stranded oligonucleotide is annealed to a single-stranded template as described above. A mixture of three deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP), and deoxyribothymidine (dTT), is combined with a modified thiodeoxyribocytosine called dCTP-(aS) (which can be obtained from Amersham). This mixture is added to the template-oligonucleotide complex. When DNA polymerase is added to this mixture, a DNA strand identical to the template is produced, except for the mutated base. In addition, this new DNA strand will contain dCTP-(aS) instead of dCTP, which serves to protect it from restriction endonuclease digestion. After the template strand of a double-stranded heteroduplex is cleaved with an appropriate restriction enzyme, the template strand can be digested with an ExoIII nuclease or another suitable nuclease beyond the region containing the mutagenesis site(s) to be mutagenesized. The reaction is then stopped, leaving a partially single-stranded molecule. A complete double-stranded 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 introduced into a suitable host cell for transformation.

[0180] As previously shown, the sequence of the oligonucleotide set is long enough to hybridize to the template nucleic acid and may, though not necessarily, contain restriction sites. The DNA template can be generated by either a vector derived from the bacteriophage M13 vector or a vector containing a single-stranded phage replication origin, as described by Viera et al., Meth. Enzymol., 153:3 (1987). Therefore, in order to generate a single-stranded template, the DNA to be mutated must be inserted into one of these vectors. The production of the single-stranded template is described in sections 4.21-4.41 of Sambrook et al. (above).

[0181] Alternatively, antigen binding can be restored during antibody humanization by selecting repaired hypervariable regions (see, for example, U.S. Patent Application No. 11 / 061,841, filed February 18, 2005). This method involves incorporating non-human hypervariable regions onto an acceptor framework and further introducing one or more amino acid substitutions into one or more hypervariable regions without modifying the acceptor framework sequence. Alternatively, the introduction of one or more amino acid substitutions may be carried out by modification of the acceptor framework sequence.

[0182] Alternatively, a library can be generated by providing upstream and downstream oligonucleotide sets, each set having multiple oligonucleotides with different sequences, these different sequences established by a set of codons provided within the oligonucleotide sequences. The upstream and downstream oligonucleotide sets can be used in a polymerase chain reaction along 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 with other related or unrelated nucleic acid sequences, such as viral coat proteins and dimerization domains, using established molecular biology techniques.

[0183] PCR primer sequences contain one or more codon sets designed for solvent-accessible and highly diverse positions in the hypervariable region. As mentioned above, a codon set is a set of different nucleotide triplet sequences used to encode a desired variant amino acid.

[0184] Antibody selections that meet the desired criteria, such as those selected through appropriate screening / selection steps, can be isolated and cloned using standard recombinant techniques.

[0185] It is even more important that antibodies are humanized while retaining high binding affinity to the antigen and other desirable biological properties. To achieve this goal, according to preferred methods, humanized antibodies are prepared by an analytical process of the parental sequence and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the estimated three-dimensional structure of selected candidate immunoglobulin sequences. Examination of these displays allows for the analysis of the possible roles of residues in the function of the candidate immunoglobulin sequence, i.e., the analysis of residues that affect 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 transfer sequences so that desired antibody properties, such as increased affinity to the target antigen(s), are achieved. Generally, hypervariable region residues are directly and most substantially involved in the effect on antigen binding.

[0186] Various forms of humanized anti-CTHRC1 antibodies are intended. For example, the humanized antibody may be an antibody fragment such as Fab. Alternatively, the humanized antibody may be an intact antibody such as an intact IgG1 antibody.

[0187] As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that, upon immunization, can produce a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For instance, homozygous deletion of the antibody heavy chain binding region (JH) gene in chimeric and germline mutant mice has been shown to result in complete inhibition of endogenous antibody production. Transplantation of a human germline immunoglobulin gene array into such germline mutant mice results in the production of human antibodies upon antigen stimulation (see, for example, 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. Patents No. 5,545,806, 5,569,825, 5,591,669, 5,545,807, and WO97 / 17852).

[0188] Alternatively, phage display technology (McCafferty et al., Nature 348:552-53 (1990)) can be used to in vitro produce human antibodies and antibody fragments from an immunoglobulin variable (V) domain gene repertoire from non-immunized donors. According to this technique, the antibody V domain gene is cloned in-frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and presented as a functional antibody fragment on the surface of a phage particle. Since the filamentous particle contains a single-stranded DNA copy of the phage genome, selection based on the functional properties of the antibody also leads to the selection of genes encoding antibodies exhibiting those properties. Thus, the phage mimics some of the properties of B cells. Phage display can be carried out in various forms, as outlined, for example, in Johnson, Kevin S, and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several sources of V-gene segments can be used for phage display. Clackson et al., Nature, 352:624-628 (1991) isolated a wide variety of anti-oxazolone antibodies from a small, random combinatorial library of V genes derived from the pancreas of immunized mice. A repertoire of V genes can be constructed from non-immunized human donors, 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. Patents No. 5,565,332 and 5,573,905). Antibodies against a wide variety of antigens (including autoantigens) can be isolated.

[0189] As discussed above, human antibodies may also be produced by B cells activated in vitro (see, for example, U.S. Patent Nos. 5,567,610 and 5,229,275).

[0190] In another embodiment, the anti-CTHRC1 T cell engager of this 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 possess a portion of the human immune system rather than a mouse lineage. These transgenic and transchromosomic mice include mice referred to herein as HuMAb mice® and KM mice®, respectively, and are collectively referred herein as “human Ig mice.”

[0191] HuMAb mice (trademark) (Medarex, Inc.) contain human immunoglobulin gene miniloci encoding unreorganized human heavy chain (μ and γ) and κ light chain immunoglobulin sequences, along with targeted mutations that inactivate endogenous μ and κ chain loci (see, for example, Lonberg, et al. (1994) Nature 368(6474):856-9). Therefore, mice exhibit reduced expression of mouse IgM or κ, and in response to immunization, the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgGκ monoclonal antibodies (Lonberg, N. et al. (1994) (above), Lonberg, N. (1994) Handbook of Experimental Pharmacology 113:49-101 (overviewed), Lonberg, N. and Huszar, D. (1995) Intern. Rev. Immunol. 13:65-93, and Harding, F. and Lonberg, N. (1995) Ann. NYAcad. Sci. 764:536-46). The preparation and use of HuMAb mice (trademark), and the genomic modifications possessed by such mice, have been reported 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, and Taylor, L. et al. (1994) International Immunology. Further details are provided in 6:579-91 and Fishwild, D. et al. (1996) Nature Biotechnology 14:845-51, all of which are explicitly incorporated herein by reference.Furthermore, 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 See U.S. Patent No. 5,545,807, PCT Publications WO92 / 03918, WO93 / 12227, WO94 / 25585, WO97 / 13852, WO98 / 24884, and WO99 / 45962, as well as PCT Publication WO01 / 14424.

[0192] In another embodiment, the human antibodies of this disclosure can be produced using mice that possess a human immunoglobulin sequence on a transgene and a human light chain transchromosome, such as mice that possess a human heavy chain transgene and a human light chain transchromosome. These mice are referred to herein as “KM mice (trademark)” and are described in detail in PCT Publication WO02 / 43478.

[0193] Furthermore, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the anti-CTHRC1 antibodies of this disclosure. For example, an alternative transgenic system called Xenomouse (Abgenix, Inc.) can be used, such mice are described, for example, in U.S. Patents 5,939,598, 6,075,181, 6,114,598, 6,150,584, and 6,162,963.

[0194] Furthermore, alternative transchromosomal animal lines expressing human immunoglobulin genes are available in the art and can be used to produce the anti-CTHRC1 antibody of this disclosure. For example, mice possessing both human heavy chain and human light chain transchromosomes, referred to as "TC mice," can be used, as described in Tomizuka et al. (2000) Proc. Natl. Acad. Sci. USA 97:722-7. As another example, cattle possessing 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 antibody of this 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, and European Patent No. 2336329B1). Yet another example is the use of VELOCIMMUNE® technology (see, for example, U.S. Patent No. 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®). In short, the VELOCIMMUNE® technology involves generating transgenic mice having a genome containing human heavy and light chain variable regions operably ligated to the locus of the endogenous mouse constant region, so that the mouse produces antigen-binding proteins, such as antibodies, containing human variable regions and mouse constant regions in response to antigen stimulation. The DNA encoding the variable regions of the antibody's heavy and light chains is isolated and operably ligated to the DNA encoding the human heavy and light chain constant regions.Next, this DNA is expressed in cells capable of expressing fully human antibodies.

[0195] antibody fragment In certain situations, there are advantages to using antibody fragments rather than the whole antibody. The smaller fragment size allows for faster removal, which can lead to improved access to solid tumors.

[0196] Various techniques have been developed for the production of antibody fragments. Conventionally, these fragments were obtained via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-7 (1992), and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Since Fab, Fv, and scFv antibody fragments are all expressed in and secreted from E. coli, easy mass production of these fragments is possible. Antibody fragments can be isolated from the antibody phage library described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-7 (1992)). By another method, the F(ab')2 fragment can be isolated directly from recombinant host cell cultures. Fab and F(ab')2 fragments with increased in vivo half-life containing salvage receptor-binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single-stranded Fv fragment (scFv) (see WO93 / 16185, U.S. Patents No. 5,571,894, and U.S. Patents No. 5,587,458). Fv and sFv are the only species with intact binding sites lacking a constant region; therefore, they are suitable for reducing nonspecific binding during in vivo use. sFv T cell engagers can be constructed to produce effector protein fusions at either the amino or carboxyl terminus of sFv (see Antibody Engineering, ed. Borrebaeck (above)). Antibody fragments can also be "linear antibodies," such as those described in U.S. Patent No. 5,641,870.

[0197] Antibody variants and modifications Substitution, insertion, and deletion variants In addition to the anti-CTHRC1 and anti-CD3 antibodies described herein, it is intended that anti-CTHRC1 and / or anti-CD3 antibody variants may be prepared. Anti-CTHRC1 and / or anti-CD3 antibody variants can be prepared by introducing appropriate nucleotide changes into coding DNA and / or by synthesizing the desired antibody or polypeptide. Those skilled in the art will understand that amino acid changes can alter the post-translational processes of anti-CTHRC1 and / or anti-CD3 antibodies, such as by changing the number or position of glycosylation sites or altering membrane anchoring properties.

[0198] The alterations in the anti-CTHRC1 antibody and / or anti-CD3 antibody described herein can be produced using any of the techniques and guidelines for conserved and non-conserved mutations, for example, as described in U.S. Patent No. 5,364,934. The alteration may be a substitution, deletion, or insertion of one or more codons encoding the antibody or polypeptide, resulting in a change in the amino acid sequence compared to the native sequence antibody or polypeptide. Optionally, the alteration may be a substitution of at least one amino acid with any other amino acid in one or more domains of the anti-CTHRC1 antibody and / or anti-CD3 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 sequences of the anti-CTHRC1 antibody and / or anti-CD3 antibody with the sequences of known homologous protein molecules and by minimizing the number of amino acid sequence changes in highly homologous regions. Amino acid substitutions may be the result of conserved amino acid substitutions, such as replacing one amino acid with another amino acid having similar structural and / or chemical properties, for example, the substitution of leucine with serine. Insertions or deletions may be optional and may range from approximately 1 to 5 amino acids. Acceptable changes may be determined by systematically performing amino acid insertions, deletions, or substitutions in the sequence and testing the resulting variants for activity indicated by the full-length or mature native sequence.

[0199] Anti-CTHRC1 antibody fragments and anti-CD3 antibody fragments are provided herein. Such fragments may have shortened N-terminuses or C-terminuses, or lack internal residues, compared to, for example, full-length natural antibodies or proteins. Certain fragments lack amino acid residues that are not essential for the desired biological activity of the anti-CTHRC1 antibody and / or anti-CD3 antibody.

[0200] Anti-CTHRC1 antibody fragments and anti-CD3 antibody fragments may be prepared by any of several conventional techniques. The desired peptide fragment may be chemically synthesized. Alternative methods involve generating antibodies or polypeptide fragments by enzymatic digestion, for example, by treating a protein with an enzyme known to cleave the protein at a site defined by a specific amino acid residue, or by digesting DNA with a suitable restriction enzyme and isolating the desired fragment. Yet another preferred technique involves isolating a DNA fragment encoding the desired antibody or polypeptide fragment and amplifying it by polymerase chain reaction (PCR). Oligonucleotides defining the desired ends of the DNA fragment are used as 5' and 3' primers for PCR. Preferably, the anti-CTHRC1 antibody fragment shares at least one biological and / or immunological activity with the innate anti-CTHRC1 antibody disclosed herein, and the same applies to the anti-CD3 antibody fragment and at least one biological and / or immunological activity with the innate anti-CD3 antibody.

[0201] In certain embodiments, the desired conservative substitutions are shown in Table 6 under the heading "Preferred Substitutions." If such substitutions result in changes to biological activity, more substantial changes are introduced, as referred to as exemplary substitutions in Table 6 or as further described below with reference to amino acid classes, and the product is screened. [Table 11]

[0202] Substantial modifications to the function or immunological properties of anti-CTHRC1 and anti-CD3 antibodies are achieved by selecting substitutions that have significantly different effects on maintaining (a) the structure of the polypeptide backbone in the substitution region, for example, as a sheet or helical structure, (b) the molecular charge or hydrophobicity at the target site, or (c) the bulk of the side chains. Naturally occurring residues are classified into the following groups based on the common properties of their side chains. (1) Hydrophobic: norleucine, met, ala, val, leu, ile, (2) Neutral hydrophilicity: cys, ser, thr, (3) Acidic: asp, glu, (4) Basicity: asn, gln, his, lys, arg, (5) Residues that affect chain orientation: gly, pro, and (6) Aromatic: trp, tyr, phe

[0203] Non-conservative substitutions involve exchanging one member of one of these classes for another. The residue thus substituted may also be introduced into a conserved substitution site, or more preferably, into the remaining (non-conserved) site.

[0204] It should be understood that the anti-CTHRC1 T cell engager or a conservatively substituted version of the first or second domain of the anti-CTHRC1 T cell engager described herein is included within the scope of this disclosure.

[0205] The mutations can be produced 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-selective mutagenesis (Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)), or other known techniques can be applied to cloned DNA to produce anti-CTHRC1 antibody variant DNA.

[0206] Scanning amino acid analysis can also be used to identify one or more amino acids along a continuous sequence. Among the preferred scanning amino acids are relatively small neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a preferred scanning amino acid in this group because it is unlikely to alter the back chain stereochemistry of the variant, as it eliminates the side chain beyond the beta-carbon (Cunningham and Wells, Science, 244:1081-5 (1989)). Alanine is also typically preferred because it is the most common amino acid. Furthermore, it is frequently found in both buried and exposed positions (Creighton, The Proteins, (WHNY), Chothia, J. Mol. Biol., 150:1 (1976)). If alanine substitution does not produce a sufficient amount of variant, isosteric amino acids may be used.

[0207] Furthermore, any cysteine ​​residue that does not contribute to maintaining the proper three-dimensional structure of anti-CTHRC1 antibodies and anti-CD3 antibodies may be replaced with serine in general to improve the oxidative stability of the molecules and prevent abnormal cross-linking. Conversely, cysteine ​​bonds (multiple may be possible) may be added to anti-CTHRC1 antibodies or anti-CD3 antibodies to improve their stability (especially when the antibody is an antibody fragment such as an Fv fragment).

[0208] Particularly preferred types of substitution variants involve the substitution of one or more hypervariable region residues of the parent antibody (e.g., humanized or human antibody). Generally, the resulting variant(s) selected for further development have improved biological properties compared to the parent antibody from which they were 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 presented in a monovalent manner from filamentous phage particles as fusions to the M13 gene III product 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 significantly contribute to antigen binding. Alternatively, or additionally, it may be beneficial to analyze the crystalline structure of the antigen-antibody complex to identify contact sites between the antibody and the CTHRC1 polypeptide and / or between the anti-CD3 antibody and CD3. Such contact residues 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 exhibiting superior properties in one or more relevant assays may be selected for further development.

[0209] Nucleic acid molecules encoding amino acid sequence variants of anti-CTHRC1 antibodies and / or anti-CD3 antibodies are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from natural sources (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis of pre-prepared variant or non-variant versions of anti-CTHRC1 antibodies and / or anti-CD3 antibodies.

[0210] qualification Covalent modifications of anti-CTHRC1 antibodies and anti-CD3 antibodies are included within the scope of the present invention. One type of covalent modification involves reacting a target amino acid residue of an anti-CTHRC1 antibody with an organic derivatizing agent that can react with a selected side chain or N-terminal or C-terminal residue of an anti-CTHRC1 antibody or anti-CD3 antibody. For example, derivatization with a bifunctional agent is useful for crosslinking an anti-CTHRC1 antibody to a water-insoluble support matrix or surface for use in a method for purifying an anti-CTHRC1 antibody, and vice versa. Commonly used crosslinking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, such as esters with 4-azidosalicylic acid, homodifunctional imide esters including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), difunctional maleimides such as bis-N-maleimide-1,8-octane, and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0211] Other modifications include deamidation of glutaminyl and asparaginyl residues to their corresponding glutamyl and aspartyl residues, respectively; hydroxylation of proline and lysine; phosphorylation of hydroxyl groups of ceryl or threonyl residues; methylation of α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)); acetylation of N-terminal amines; and amidation of any C-terminal carboxyl group.

[0212] Another type of covalent modification of an anti-CTHRC1 antibody or anti-CD antibody that falls within the scope of the present invention includes altering the natural glycosylation pattern of the antibody or polypeptide. “Altering the natural glycosylation pattern” is intended, for the purposes of this specification, to mean deleting one or more carbohydrate moieties found in the natural 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 natural sequence anti-CTHRC1 antibody. In addition, this phrase includes qualitative changes in the glycosylation of the natural protein, involving changes in the properties and proportions of the various carbohydrate moieties present.

[0213] Glycosylation of antibodies and other polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are recognition sequences for the enzymatic attachment of a 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 hydroxy amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0214] The addition of glycosylation sites to an anti-CTHRC1 antibody or an anti-CD3 antibody is conveniently accomplished by changing the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (in the case of N-linked glycosylation sites). The change may also be made by the addition or substitution of one or more serine or threonine residues to the sequence of the original anti-CTHRC1 antibody or anti-CD3 antibody (in the case of O-linked glycosylation sites). The amino acid sequence of the anti-CTHRC1 antibody or anti-CD3 antibody may optionally be changed via a change at the DNA level by mutating the DNA encoding the anti-CTHRC1 antibody or anti-CD3 antibody with a preselected base such that a codon that translates to the desired amino acid is generated.

[0215] Another means of increasing the number of carbohydrate moieties on an anti-CTHRC1 antibody or an anti-CD3 antibody is by chemical or enzymatic coupling of a glycoside to the polypeptide. Such methods are described in the art, for example, in WO87 / 05330, published September 11, 1987, and Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0216] Removal of carbohydrate moieties present on the anti-CTHRC1 antibody or anti-CD3 antibody may be accomplished chemically or enzymatically or by mutagenic 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 a variety of endo-glycosidases and exo-glycosidases, as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).

[0217] Fc region variant For example, it may be desirable to modify the antibody of the present invention in terms of effector function to enhance the 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 ​​residues may be introduced into the Fc region to enable interchain disulfide bond formation in this region. Homodimerated antibodies thus produced may have improved internal migration ability and / or increased complement-mediated cytotoxicity and antibody-dependent cytotoxicity (ADCC) (see Caron et al., J.Exp Med. 176:1191-5 (1992), Shopes, BJImmunol. 148:2918-22 (1992)). Homodimerated 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 manipulated to have enhanced complement lysis and ADCC activity by having a double Fc region. See Stevenson et al., Anti-Cancer Drug Design 3:219-30 (1989). To increase the serum half-life of the antibody, salvage receptor-binding epitopes may be incorporated into the antibody (particularly the antibody fragment), as described, for example, in U.S. Patent 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 involved in increasing the in vivo serum half-life of the IgG molecule.

[0218] In some embodiments, the Fc portion of the anti-CTHRC1 T cell engager may include a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of the following Fc sequences, [Table 12-1] [Table 12-2] Alternatively, it may include sequence pairs having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with respect to any one of the following Fc sequence pairs. [Table 13]

[0219] In some embodiments, the Fc portion of the anti-CTHRC1 T cell engager may include a sequence having one of the following Fc sequences, [Table 14-1] [Table 14-2] Alternatively, each sequence may contain a sequence pair in which one of the following Fc sequence pairs is present. [Table 15]

[0220] Cysteine-modified antibody variant In certain embodiments, it may be desirable to produce a cysteine-modified antibody, e.g., "thioMAb," in which one or more residues of the antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites on the antibody. By substituting these residues with cysteine, a reactive thiol group is thereby positioned at an accessible site on the antibody, which may be used to conjugate the antibody to a drug moiety or other moiety, such as a linker-drug moiety, to produce an immunoconjugate as further described herein. Cysteine-modified antibodies can be produced, for example, as described in U.S. Patent No. 7,521,541.

[0221] A specific method for producing antibodies Screening for anti-CTHRC1 antibodies with desired properties Techniques for generating antibodies that bind to the CTHRC1 polypeptide have been described above. If desired, antibodies with certain biological properties, such as lack of intracellular integration, may be further selected.

[0222] The growth inhibitory effect of the anti-CTHRC1 antibody of the present invention may be evaluated by methods known in the art, for example, using cells expressing the CTHRC1 polypeptide either endogenously or after transfection with the CTHRC1 gene. For example, suitable tumor cell lines and CTHRC1-transfected cells may be treated with various concentrations of the anti-CTHRC1 monoclonal antibody of the present invention for several days (e.g., 2-7 days) and stained with crystal violet or MTT, or analyzed by some other colorimetric assay. Another method of measuring proliferation would be by 3H-thymidine uptake by cells treated in the presence or absence of the anti-CTHRC1 antibody of the present invention. After treatment, cells are harvested and the amount of radioactivity incorporated into the DNA is quantified using a scintillation counter. Suitable positive controls include treatment of selected cell lines with growth-inhibiting antibodies known to inhibit the growth of that cell line. In vivo inhibition of tumor cell growth can be determined by various methods known in the art. Tumor cells may overexpress and / or display the CTHRC1 polypeptide. In one embodiment, anti-CTHRC1 antibodies inhibit the proliferation of CTHRC1-presenting tumor cells in vitro or in vivo by about 25–100%, more preferably about 30–100%, and even more preferably about 50–100% or 70–100% compared to untreated tumor cells, at antibody concentrations of about 0.5–30 μg / mL. Growth inhibition can be measured in cell cultures at antibody concentrations of about 0.5–30 μg / mL or about 0.5 nM–200 nM, where growth inhibition is determined 1–10 days after exposure of tumor cells to the antibody. The antibody is growth inhibitory in vivo if administration of anti-CTHRC1 antibody at about 1 μg / kg–100 mg / kg body weight results in a reduction in tumor size or tumor cell proliferation within about 5 days–3 months, preferably about 5–30 days, from the first administration of the antibody.

[0223] To select anti-CTHRC1 antibodies that induce cell death, loss of membrane integrity, as indicated by the uptake of, for example, propidium iodide (PI), trypan blue, or 7AAD, can be evaluated compared to a control. PI uptake assays can be performed in the absence of complement and immunoeffector cells. CTHRC1 polypeptide-presenting tumor cells are incubated in medium alone or with medium containing a suitable anti-CTHRC1 antibody (e.g., approximately 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 to remove cell clumps (1 mL per tube, 3 tubes per treatment group). PI (10 μg / mL) is then added to the tubes. Samples can be analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). Anti-CTHRC1 antibodies that induce statistically significant levels of cell death when determined by PI uptake can be selected as cell death-inducing anti-CTHRC1 antibodies.

[0224] To screen for antibodies that bind to epitopes on the CTHRC1 polypeptide to which the antibody of interest is bound, conventional cross-blocking assays, such as those described in Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine whether the test antibody binds to the same site or epitope as known anti-CTHRC1 antibodies. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, mutations can be induced in the antibody sequence, for instance, by alanine scanning, to identify contact residues. The mutated antibody is first tested for binding to a polyclonal antibody to ensure proper folding. Alternatively, peptides corresponding to different regions of the CTHRC1 polypeptide may be used in competitive assays with the test antibody or with the test antibody and antibodies having characterized or known epitopes.

[0225] In addition, candidate antibodies may also be screened for function using one or more of the following: in vivo screening for inhibition of metastasis, inhibition of chemotaxis by in vitro method (e.g., US2010 / 0061978 (which is incorporated herein by reference in its entirety)), inhibition of angiogenesis, inhibition of tumor growth, and reduction of tumor size.

[0226] A specific library screening method The anti-CTHRC1 antibodies of the present invention can be produced by screening a combinatorial library for antibodies having the desired activity(ies). For example, various methods are known in the art for generating a phage display library and screening such library for antibodies having the desired binding characteristics. Such methods are generally described in Hoogenboom et al. (2001) in Methods in Molecular Biology 178:1-37 (O’Brien et al., ed., Human Press, Totowa, NJ), and in certain embodiments, in Lee et al. (2004) J. Mol. Biol. 340:1073-93.

[0227] In principle, synthetic antibody clones are selected by screening a phage library containing phages that display various antibody variable region fragments (Fvs) fused to a phage coat protein. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments that can bind to the desired antigen are adsorbed to the antigen and thus separated from non-binding clones within the library. The bound clones can then be eluted from the antigen and further enriched by additional antigen adsorption / elution cycles. Any of the anti-CTHRC1 antibodies of the present invention can be obtained by designing an antigen screening procedure suitable for selecting 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 a suitable constant region (Fc) sequence as described in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 91-3242, Bethesda MD (1991), vols. 1-3.

[0228] In certain embodiments, the antigen-binding domain of an antibody is formed from two variable (V) regions of approximately 110 amino acids each, one from the light (VL) chain and one from the heavy (VH) chain, each presenting three hypervariable loops (HVRs) or complementarity-determining regions (CDRs). The variable domains can be functionally presented on a phage either as a single-stranded Fv (scFv) fragment in which VH and VL are covalently linked via a short, flexible peptide, as described in Winter et al., Ann. Rev. Immunol., 12:433-55 (1994), or as a Fab fragment in which they are fused to constant domains and interact noncovalently. As used herein, phage clones encoding scFv and phage clones encoding Fab are collectively referred to as "Fv phage clones" or "Fv clones."

[0229] The VH and VL gene repertoires can be separately cloned by polymerase chain reaction (PCR), as described in Winter et al., Ann. Rev. Immunol., 12:433-55 (1994), randomly recombined in a phage library, and then searched for antigen-binding clones. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12:725-34 (1993), naive repertoires can be cloned to provide a single source of human antibodies against a wide range of non-self and autoantigens without any immunization. Finally, naive libraries can also be synthetically constructed by cloning an unreorganized V gene segment from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and performing in vitro rearrangement, as described by Hoogenboom and Winter, J. Mol. Biol., 227:381-8 (1992).

[0230] In certain embodiments, filamentous phages are used to present antibody fragments by fusion to the minor coat protein pIII. The antibody fragments may be presented as single-stranded Fv fragments in which the VH and VL domains are linked on the same polypeptide chain by a flexible polypeptide spacer, as described, for example, by Marks et al., J. Mol. Biol., 222:581-97 (1991), or as Fab fragments in which one chain is fused to pIII and the other is secreted into the bacterial host cell periplasm, and an assembly of Fab coat protein structures is presented on the phage surface by replacing a portion of the wild-type coat protein, as described, for example, by Hoogenboom et al., Nucl. Acids Res., 19:4133-7 (1991).

[0231] Generally, nucleic acids encoding antibody gene fragments are obtained from immune cells isolated from humans or animals. When a biased library favoring anti-CTHRC1 clones is desired, subjects are immunized with CTHRC1 to induce an antibody response, and spleen cells and / or circulating B cells, other peripheral blood lymphocytes (PBLs) are recovered for library construction. In some embodiments, a biased human antibody gene fragment library favoring anti-CTHRC1 clones is obtained by inducing an anti-CTHRC1 antibody response in transgenic mice possessing a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system) so that CTHRC1 immunization produces B cells that produce human antibodies against CTHRC1. The generation of human antibody-producing transgenic mice is described later.

[0232] 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 cell adsorption to fluorescently labeled CTHRC1, followed by flow-activated cell sorting (FACS).

[0233] Alternatively, the use of spleen cells and / or B cells or other PBLs from non-immunized donors provides better reproduction of the possible antibody repertoire and also allows for the construction of antibody libraries using any animal (human or non-human) species in which CTHRC1 is not antigenic. In the case of libraries incorporating in vitro antibody gene constructs, stem cells are harvested from the subject to provide nucleic acids encoding unreorganized antibody gene segments. The immune cells of interest can be obtained from a variety of animal species, including humans, mice, rats, rabbits, wolves, dogs, cats, pigs, cattle, horses, and birds.

[0234] Nucleic acids encoding antibody-variable gene segments (including VH and VL segments) are recovered from target cells and amplified. In the case of rearranged VH and VL gene libraries, the desired DNA can be obtained by isolating genomic DNA or mRNA from lymphocytes and then performing 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 creating a diverse V gene repertoire for expression.

[0235] The V gene 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 placed within the constant region, as described in Sastry et al., Proc. Natl. Acad. Sci. (USA), 86:5728-32 (1989). To maximize complementarity, degeneracy may be incorporated into the primers, as described in Orlandi et al. (1989) or Sastry et al. (1989). In certain embodiments, library diversity is maximized by using PCR primers targeted to each V gene family to amplify all available VH and VL configurations present in an immunocellular nucleic acid sample, as described, for example, in the method of Marks et al., J. Mol. Biol., 222:581-97 (1991), or in the method of Orum et al., Nucleic Acids Res., 21:4491-98 (1993). When cloning the amplified DNA into an expression vector, a rare restriction site can be introduced into the PCR primer as a tag at one end, as described in Orlandi et al. (1989), or further PCR amplification can be performed using tagged primers, as described in Clackson et al., Nature, 352:624-628 (1991).

[0236] A repertoire of synthetically rearranged V genes can be obtained in vitro from V gene segments. Most human VH gene segments have been cloned and sequenced (reported in Tomlinson et al., J.Mol.Biol., 227:776-98 (1992)) and mapped (reported in Matsuda et al., Nature Genet., 3:88-94 (1993)). Using these cloned segments (including all major three-dimensional structures of the H1 and H2 loops), a diverse VH gene repertoire can be generated using PCR primers encoding H3 loops of diverse sequences and lengths, as described in Hoogenboom and Winter, J.Mol.Biol., 227:381-388 (1992). The VH repertoire can also be constructed by concentrating all sequence diversity into a single long H3 loop, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 89:4457-61 (1992). Human Vκ and Vλ segments have been cloned and sequenced (reported in Williams and Winter, Eur. J. Immunol., 23:1456-61 (1993)) and can be used to construct a synthetic light chain repertoire. A synthetic V gene repertoire based on various VH and VL foldings, as well as L3 and H3 lengths, will encode antibodies with considerable structural diversity. Following amplification of the 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).

[0237] The antibody fragment repertoire can be constructed by combining the VH and VL gene repertoires in several ways. Each repertoire can be produced in a different vector, which can be recombined in vitro as described in Hogrefe et al., Gene, 128:119-26 (1993), or in vivo by combinatorial infection, such as the loxP system, as described in Waterhouse et al., Nucl. Acids Res., 21:2265-66 (1993). The in vivo recombination method utilizes the double-stranded nature of the Fab fragment to overcome the library size limitations imposed by the transformation efficiency of E. coli. The naive VH and VL repertoires are cloned separately, one into a phagemide and the other into a phage vector. These two libraries are then combined by phage infection of phagemide-containing bacteria, so that each cell contains a different combination, and the library size is limited only by the number of cells present (approximately 10¹² clones). Both vectors contain in vivo recombination signals, so that the VH and VL genes are recombined on a single replicon and co-packaged into phage virions. These massive libraries provide a large number of diverse antibodies with good affinity (approximately 10⁻⁸ M of Kd-1).

[0238] Alternatively, the repertoire may be sequentially cloned into the same vector, for example, as described in Barbas et al., Proc. Natl. Acad. Sci. USA, 88:7978-7982 (1991), or assembled together by PCR and then cloned, for example, as described in Clackson et al., Nature, 352:624-628 (1991). PCR assembly can also be used to ligate the VH and VL DNA with DNA encoding a flexible peptide spacer to form a single-stranded Fv (scFv) repertoire. Yet another technique involves using "intracellular PCR assembly," as described in Embleton et al., Nucl. Acids Res., 20:3831-3837 (1992), to combine the VH and VL genes in lymphocytes by PCR, and then clone the ligated gene repertoire.

[0239] Antibodies produced by a naive library (either natural or synthetic) have a moderate affinity (approximately 10%). 6 ~10 7While it may have M-1 (Kd-1), affinity maturation can also be mimicked in vitro by constructing a secondary library as described in Winter et al. (1994) (above) and re-selecting from it. For example, mutations can be randomly introduced in vitro by using error-prone polymerase (reported in Leung et al., Technique, 1:11-5 (1989)), in the method of Hawkins et al., J. Mol. Biol., 226:889-96 (1992), or in the method of Gram et al., Proc. Natl. Acad. Sci USA, 89:3576-80 (1992). Additionally, affinity maturation can be performed, for example, by randomly mutating one or more CDRs using PCR with primers containing random sequences spanning the target CDRs in individual selected Fv clones, and then screening for higher affinity clones. WO9607754 describes a method for generating a library of light chain genes by inducing mutagenesis in the complementarity-determining region of immunoglobulin light chains. Another effective method, as described in Marks et al., Biotechnol., 10:779-83 (1992), involves recombining VH or VL domains selected by phage display using a repertoire of naturally occurring V-domain variants obtained from non-immunized donors, and screening for higher affinity through several chain reshufflings. This technique enables the production of antibodies and antibody fragments with affinity of approximately 10⁻⁹ M or less.

[0240] Library screening can be carried out by various techniques known in the art. For example, CTHRC1 can be used to coat the wells of an adsorption plate, expressed on host cells attached to an adsorption plate, used in cell sorting, conjugated to biotin for capture with streptavidin-coated beads, or used in any other way for panning a phage display library.

[0241] Phage library samples are brought into contact with immobilized CTHRC1 under conditions suitable for binding at least a portion of the phage particles by an adsorbent. Typically, conditions, including pH, ionic strength, and temperature, are selected to mimic physiological conditions. The phages bound to the solid phase are washed and then eluted with acid, for example, as described in Barbas et al., Proc. Natl. Acad. Sci USA, 88:7978-82 (1991), or with alkali, for example, as described in Marks et al., J. Mol. Biol., 222:581-97 (1991), or by CTHRC1 antigen competition in a procedure similar to the antigen competition method described in Clackson et al., Nature, 352:624-8 (1991). The phages can be enriched 20 to 1,000 times in a single selection round. Furthermore, the concentrated phages can be grown in bacterial cultures and subjected to further selection rounds.

[0242] The efficiency of selection depends on many factors, including the dissociation rate during washing and whether multiple antibody fragments on a single phage can simultaneously bind to the antigen. Antibodies with a fast dissociation rate (and weak binding affinity) can be retained by short washing times, multivalent phage display, and the use of a high coating density of the antigen on the solid phase. High density not only stabilizes the phage through multivalent interactions but also facilitates the rebinding of dissociated phages. The selection of antibodies with a slow dissociation rate (and good binding affinity) can be facilitated by the use of long washing times and monovalent phage display, as described in Bass et al., Proteins, 8:309-314 (1990) and WO92 / 09690, and by the use of a low coating density of the antigen, as described in Marks et al., Biotechnol., 10:779-783 (1992).

[0243] Even if they have slightly different affinities to CTHRC1, selection is possible between phage antibodies with different affinities. However, random mutations in selected antibodies (as performed, for example, in some affinity maturation techniques) can produce many variants, most of which bind to the antigen and only a few with higher affinity. Restricting CTHRC1 may competitively exclude rare high-affinity phages. To retain all higher-affinity variants, phages can be incubated with an excess of 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 enables the isolation of variant clones with only twice the affinity from a large excess of lower-affinity phages. The conditions used when washing the solid-bound phages can also be manipulated to distinguish based on dissociation rate.

[0244] Anti-CTHRC1 clones may be selected based on activity. In certain embodiments, the present invention provides an anti-CTHRC1 antibody that binds to living cells that naturally express CTHRC1. In one embodiment, the present invention provides an anti-CTHRC1 antibody that blocks the binding between the CTHRC1 ligand and CTHRC1, but does not block the binding between the 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 isolated population of phage clones by growing this population in a suitable bacterial host; (2) selecting CTHRC1 and a second protein for which blocking and non-blocking activity are desired, respectively; (3) adsorbing the anti-CTHRC1 phage clones onto immobilized CTHRC1; (4) eluting any unwanted clones that recognize CTHRC1 binding determinants that overlap with or share with the binding determinants of the second protein using excess second protein; and (5) eluting clones that remain adsorbed after step (4). Optionally, clones with the desired blocking / non-blocking properties can be further enriched by repeating the selection procedure described herein once or multiple times.

[0245] The hybridoma-derived monoclonal antibody or phage-display Fv clone encoding DNA of the present invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide primers designed to specifically amplify the desired heavy and light chain coding regions from a hybridoma or phage DNA template). Once isolated, the DNA can be placed in an expression vector, which can then be transfected into host cells such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that would otherwise not produce immunoglobulin proteins, to obtain the synthesis of the desired monoclonal antibody in 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).

[0246] The DNA encoding the Fv clone of the present invention can be combined with a known DNA sequence encoding the heavy chain and / or light chain constant region (for example, a suitable DNA sequence can be obtained from Kabat et al. (above)) to form a clone encoding a full-length or partial-length heavy chain and / or light chain. Any isotype of constant region, including IgG, IgM, IgA, IgD, and IgE constant regions, may be used for this purpose, and such constant regions may be obtained from any human or animal species. Fv clones derived from variable domain DNA of one animal (e.g., human) species and then fused to a constant region DNA of another animal species to form a coding sequence(s) for a “hybrid” full-length heavy chain and / or light chain are included in the definitions of “chimeric” and “hybrid” antibodies as used herein. In certain embodiments, an Fv clone derived from human variable DNA is fused to human constant region DNA to form a coding sequence(s) for a full-length or partial-length human heavy chain and / or light chain.

[0247] The DNA encoding the hybridoma-derived anti-CTHRC1 antibody can also be modified, for example, by substituting the homologous mouse sequence derived from the hybridoma clone with the coding sequence for human heavy and light chain constant domains (as in the method of Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-5 (1984)). The DNA encoding the hybridoma-derived or Fv clone-derived antibody or fragment can be further modified by covalently linking all or part of the coding sequence for non-immunoglobulin polypeptides to the immunoglobulin coding sequence. In this way, a “chimeric” or “hybrid” antibody having the binding specificity of the Fv clone or hybridoma clone-derived antibody of the present invention is prepared.

[0248] Antibody generation using CAR-T cells The anti-CTHRC1 antibodies of the present invention can be produced by using a CAR T cell platform to screen for antibodies having the desired activity(s) or activity(s). Chimeric antigen receptors (CARs) consist of an extracellular antigen recognition domain (usually a single-strand variable fragment (scFv) antibody) bound to a transmembrane domain and a cytoplasmic signaling domain. Alvarez-Vallina, L, Curr Gene Ther 1:385-97 (2001). CAR-mediated recognition converts tumor-associated antigens (TAAs) expressed on the cell surface into mobilization sites for effector function, addressing the target of effector cell activation independent of major histocompatibility complex. First-generation CARs were typically constructed by fusing an scFv-based TAA-binding domain to a cytoplasmic signaling domain derived from either the ζ chain of the T cell receptor (TCR) / CD3 complex or the γ 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 include the TCRζ signaling region in series with a signaling domain derived from the T cell costimulatory receptor 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)).

[0249] Upon encountering an antigen, the interaction of gene-transplanted CARs can induce effector function and mediate cytolysis of tumor cells. The practicality and effectiveness of CAR techniques have been demonstrated in various animal models and in ongoing clinical trials using CAR-based genetically engineered T lymphocytes for the treatment of cancer patients. Lipowska-Bhalla, G. et al., Cancer Immunol Immunother 61:953-62 (2012). CARs enable the targeting of effector cells to any native extracellular antigen for which suitable antibodies exist. Engineered cells can be targeted not only to proteins but also to structures such as carbohydrates 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)).

[0250] Current methods for generating recombinant antibodies are primarily based on the use of purified proteins. Hoogenboom, HR et al., Nat Biotechnol 23:1105-1116 (2005). However, mammalian cell-based antibody display platforms that leverage the functional capabilities of T lymphocytes have recently been described. Alonso-Camino et al, Molecular Therapy Nucleic Acids (2013) 2, e93. Antibody presentation on the surface of T lymphocytes as part of CAR-mediated signaling can, ideally, link antigen-antibody interactions to demonstrable changes in cellular phenotype resulting from the surface expression of activation markers. Alonso-Camino, V. et al., PLoS ONE 4:e7174 (2009). By using scFv-based CARs that recognize TAAs, CAR-mediated activation can be combined with fluorescence-activated cell separation (FACS) of CD69+ T cells to achieve at least 10 after two rounds. 3It has been demonstrated that by using a 2x enrichment factor, it becomes possible to isolate binding factors to surface TAAs, resulting in a homogeneous population of T cells expressing TAA-specific CARs. Alonso-Camino, V, et al., PLoS ONE 4:e7174 (2009).

[0251] Preparation of anti-CTHRC1 antibody The following description primarily relates to the production of anti-CTHRC1 antibodies by culturing cells transformed or transfected with vectors containing anti-CTHRC1 antibody-coding nucleic acids. Naturally, it is intended that anti-CTHRC1 antibodies may be prepared using alternative methods well known in the art. For example, a suitable 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 carried out using manual techniques or automated operations. Automated synthesis may be performed, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, CA) following the manufacturer's instructions. Various parts of an anti-CTHRC1 antibody may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired anti-CTHRC1 antibody.

[0252] Isolation of DNA encoding anti-CTHRC1 antibody DNA encoding anti-CTHRC1 antibodies may be obtained from a cDNA library prepared from tissues that are thought to possess anti-CTHRC1 antibody mRNA and express it at detectable levels. Therefore, human anti-CTHRC1 antibody DNA can be conveniently obtained from cDNA libraries prepared from human tissues. Anti-CTHRC1 antibody-coding genes may also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0253] Libraries can be screened using probes (such as oligonucleotides of at least approximately 20–80 nucleotides) designed to identify the gene of interest or the protein it encodes. Screening of cDNA or genomic libraries with selected probes can be performed using standard procedures, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989). An alternative method for isolating the gene encoding the anti-CTHRC1 antibody is to use the PCR method (Sambrook et al. (above), Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)).

[0254] Techniques for screening cDNA libraries are well known in the art. Oligonucleotide sequences selected as probes should be of sufficient length and clearly descriptive enough to minimize false positives. Oligonucleotides are preferably labeled so that they can be detected when hybridized to DNA in the library being screened. Labeling methods are well known in the art and include the use of radiolabeling such as 32P-labeled ATP, biotinylation, or enzymatic labeling. Hybridization conditions, including moderate and high stringency, are provided in Sambrook et al. (above).

[0255] Sequences identified in such library screening methods can be compared and aligned with other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid level or the nucleotide level) within a defined region of a molecule or across the full-length sequence can be determined using methods known in the art or methods described herein.

[0256] Nucleic acids having protein-coding sequences may be obtained by screening selected cDNA or genomic libraries using putative amino acid sequences disclosed herein for the first time, and, if necessary, using conventional primer extension procedures such as those described in Sambrook et al. (above), to detect mRNA precursors and processing intermediates that may not be reverse transcribed into cDNA.

[0257] Host cell selection and transformation Host cells are transfected or transformed with the expression or cloning vectors described herein for anti-CTHRC1 antibody production and cultured in conventional nutrient media modified as appropriate to induce promoters, select transformants, or amplify genes encoding desired sequences. Culture conditions such as medium, temperature, and pH can be selected by those skilled in the art without excessive experimentation. Generally, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991) and Sambrook et al. (above).

[0258] Linker In any of the embodiments described above, the anti-CTHRC1 T cell engager may further include a peptide linker positioned between the first and second domains. In certain embodiments, the peptide linker directly links the first domain to the second domain. It should be understood that the first and second domains can be positioned at either end of the peptide linker. For example, the anti-CTHRC1 T cell engager may include (first domain-linker-second domain) or (second domain-linker-first domain).

[0259] In any of the embodiments described above, the peptide linker may be any suitable linker. For example, the peptide linker may 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).

[0260] 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 may 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).

[0261] Alternative methods and designs for constructing the anti-CTHRC1 T cell engager described herein are provided in U.S. Patent No. 10,392,445, which is incorporated herein by reference.

[0262] Pharmaceutical composition In some embodiments, the anti-CTHRC1 T cell engagers of the present disclosure may be formulated into pharmaceutically acceptable compositions comprising the anti-CTHRC1 T cell engager and a pharmaceutically acceptable carrier.

[0263] The anti-CTHRC1 T cell engagers and compositions of the present invention may be administered by any route appropriate to the pathological condition to be treated. The T cell engagers will typically be administered parenterally, i.e., by infusion, subcutaneously, intramuscularly, intravenously, intradermally, intrathecally, and epidurally.

[0264] In one embodiment, for the treatment of these cancers, the T-cell engager is administered via intravenous infusion. The dosage administered via infusion ranges from approximately 0.001 mg / kg to approximately 100 mg / kg per dose, based on the patient's body weight, and generally consists of one, two, three, or four doses per week. Alternatively, the dosage ranges are approximately 0.01 mg / kg to approximately 100 mg / kg, approximately 0.1 mg / kg to approximately 100 mg / kg, approximately 1 mg / kg to approximately 100 mg / kg, approximately 0.001 mg / kg to approximately 50 mg / kg, approximately 0.01 mg / kg to approximately 50 mg / kg, approximately 0.1 mg / kg to approximately 50 mg / kg, approximately 1 mg / kg to approximately 50 mg / kg, approximately 0.001 mg / kg to approximately 10 mg / kg, and approximately 0.01 mg / kg. The dosages are approximately ~10 mg / kg, approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 1 mg / kg to approximately 10 mg / kg, approximately 0.001 mg / kg to approximately 5 mg / kg, approximately 0.01 mg / kg to approximately 5 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 1 mg / kg to approximately 5 mg / kg, approximately 0.001 mg / kg to approximately 1 mg / kg, approximately 0.01 mg / kg to approximately 1 mg / kg, and approximately 0.1 mg / kg to approximately 1 mg / kg. The dosage may be once daily, once weekly, multiple times per week but less than once daily, multiple times per month but less than once daily, multiple times per month but less than once per week, once per month, or intermittently to reduce or alleviate the symptoms of the disease. Administration may be continued at any of the disclosed intervals until the symptoms of the treated tumor or cancer are in remission. Administration may be continued after remission or reduction of symptoms is achieved, if such remission or reduction is prolonged by such continued administration.

[0265] Therapeutic formulations containing the anti-CTHRC1 T cell engager used in the present invention are prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing the anti-CTHRC1 T cell engager of a desired degree of purity with a pharmaceutically acceptable optional carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used and include buffers such as acetic acid, tris, phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than approximately 10 residues) polypeptides; and tannins such as serum albumin, gelatin, or immunoglobulins. This includes 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; isotonic agents (tonicizers) such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose, or sorbitol; surfactants such as polysorbate; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Pharmaceutical preparations used for in vivo administration are generally sterile. This is easily achieved by filtration through a sterile filtration membrane.

[0266] The active ingredient may also be encapsulated in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules, 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).

[0267] Sustained-release preparations may be prepared. Preferred examples of sustained-release preparations include semipermeable matrices of hydrophobic solid polymers containing antibodies, which are in the form of molded articles, e.g., films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers (e.g., LUPRON DEPOT® (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate)), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid allow for molecular release for more than 100 days, while certain hydrogels release proteins over shorter periods. If encapsulated immunoglobulins remain in the body for extended periods, they may denature or aggregate as a result of exposure to moisture at 37°C, potentially leading to loss of biological activity and altered immunogenicity. Depending on the mechanism involved, reasonable strategies for stabilization can be employed. For example, if the aggregation mechanism is found to be the formation of intermolecular disulfide bonds through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, freeze-drying from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

[0268] The anti-CTHRC1 T cell engagers of this disclosure may be formulated in any form suitable for delivery to target cells / tissues. For example, the anti-CTHRC1 T cell engagers may be formulated as immunoliposomes. A “liposome” is a small vesicle composed of various types of lipids, phospholipids, and / or surfactants that are useful for drug delivery to mammals. The components of a liposome are generally arranged in a bilayer formation similar to the arrangement of lipids in 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 WO97 / 38731, published on October 23, 1997. Liposomes with improved circulation time are disclosed in U.S. Patent No. 5,013,556.

[0269] Particularly useful liposomes can be produced by reverse-phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). Liposomes of a desired diameter can be obtained by extruding the liposomes through a filter of a specified pore size. The Fab' fragment of the antibody of the present invention can be conjugated into the liposomes via a disulfide exchange reaction, as described in Martin et al., J. Biol. Chem. 257:286-8 (1982). Optionally, chemotherapeutic agents can be incorporated into the liposomes (see Gabizon et al., J. National Cancer Inst. 81(19):1484 (1989)).

[0270] Preparations used for in vivo administration must be sterile. This can be easily achieved by filtration through a sterile filtration membrane.

[0271] Manufacturing method Conventional methods for producing T cell engagers are known in the field.

[0272] Methods of eukaryotic cell transfection and prokaryotic cell transformation, meaning the introduction of DNA into a host either extrachromosomally or through chromosomal integration, are known to those skilled in the art, such as CaCl2, CaPO4, liposome-mediated, polyethylene glycol (gycol) / DMSO, and electroporation. Depending on the host cell used, transformation is carried out using standard techniques appropriate for such cells. Calcium treatment using calcium chloride, as described in Sambrook et al. (above), or electroporation are commonly used for prokaryotes. Infection with Agrobacterium tumefaciens is used for the transformation of certain plant cells, as described in Shaw et al., Gene, 23:315 (1983) and WO 89 / 05859 published on June 29, 1989. For mammalian cells that do not have such cell walls, the calcium phosphate precipitation method described in Graham and van der Eb, Virology, 52:456-457 (1978) may 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 carried out 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, such as nuclear microinjection, electroporation, bacterial plasmofusion with intact cells, or by polycations, e.g., polybren, polyornithine, etc., may also be used. For various techniques for transforming mammalian cells, see Keown et al., Methods in Enzymology, 185:527-537 (1990) and Mansour et al., Nature, 336:348-352 (1988).

[0273] In some embodiments, polynucleotides encoding the anti-CTHRC1 T cell engager of the present disclosure are provided. In some embodiments, the polynucleotides encoding the anti-CTHRC1 T cell engager may be incorporated into a vector suitable for expressing the anti-CTHRC1 T cell engager in host cells.

[0274] The vector may be in the form of, for example, a plasmid, cosmid, viral particle, or phage. A suitable nucleic acid sequence can be inserted into the vector by various procedures. Generally, DNA is inserted into a suitable restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, origin of replication, one or more marker genes, enhancer elements, promoter, and transcription termination sequence. Standard ligation techniques known to those skilled in the art are used to construct a suitable vector containing one or more of these components.

[0275] The anti-CTHRC1 T cell engager may be recombinantly produced and may further contain heterologous polypeptides, which may be signal sequences or other polypeptides having a specific cleavage site at the N-terminus of a mature protein or polypeptide, or affinity tags such as a 6×His tag. Generally, the signal sequence may be a component of the vector, or it may be part of the anti-CTHRC1 T cell engager coding DNA inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, lpp, or a thermostable enterotoxin II reader. In the case of yeast secretion, the signal sequence may be, for example, a yeast invertase reader, an alpha factor reader (including Saccharomyces and Kluyveromyces α-factor readers (the latter described in U.S. Patent No. 5,010,182)), or an acid phosphatase reader, a C. albicans glucoamylase reader (EP362,179 published April 4, 1990), or a signal described in WO90 / 13646 published November 15, 1990. In expression in mammalian cells, protein secretion may be induced simultaneously with a viral secretion reader using a mammalian signal sequence, such as a signal sequence from a secretory polypeptide of the same or closely related species.

[0276] In the recombinant production of T cell engagers according to the present invention, the nucleic acid encoding it (e.g., cDNA or genomic DNA) is isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. The DNA encoding the T cell engager can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of an antibody). Many vectors are available. Vector selection depends in part on the host cell used. Generally, preferred host cells are either of prokaryotic or eukaryotic (generally mammalian) origin. Suitable host cells for cloning or expressing DNA in the vectors herein include prokaryotic, yeast, or higher eukaryotic cells.

[0277] The vector may be in the form of, for example, a plasmid, cosmid, viral particle, or phage. A suitable nucleic acid sequence can be inserted into the vector by various procedures. Generally, DNA is inserted into a suitable restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, origin of replication, one or more marker genes, enhancer elements, promoter, and transcription termination sequence. Standard ligation techniques known to those skilled in the art are used to construct a suitable vector containing one or more of these components.

[0278] Anti-CTHRC1 T cell engagers may be recombinantly produced not only directly but also as fusion polypeptides with heterologous polypeptides, the heterologous polypeptides may be signal sequences or other polypeptides having a specific cleavage site at the N-terminus of a mature protein or polypeptide. Generally, the signal sequence may be a component of the vector, or it may be part of the anti-CTHRC1 T cell engager coding DNA inserted into the vector. The signal sequence may be a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, lpp, or a thermostable enterotoxin II reader. In the case of yeast secretion, the signal sequence may be, for example, a yeast invertase reader, an alpha factor reader (including Saccharomyces and Kluyveromyces α-factor readers (the latter described in U.S. Patent No. 5,010,182)), or an acid phosphatase reader, a C. albicans glucoamylase reader (EP362,179 published April 4, 1990), or a signal described in WO90 / 13646 published November 15, 1990. In expression in mammalian cells, protein secretion may be induced simultaneously with the viral secretion reader using mammalian signal sequences, such as signal sequences from secretory polypeptides of the same or closely related species. In several embodiments, anti-CTHRC1 T cell engagers may be produced tagged with a 6×His tag or the like.

[0279] vector Nucleic acid sequences encoding a desired molecule can be obtained by using recombinant methods known in the art, for example, by screening a library from cells expressing the gene using standard techniques, by inducing the gene from a vector known to contain the gene, or by directly isolating it from cells and tissues containing the gene. Alternatively, the gene of interest can be generated synthetically rather than by cloning.

[0280] The present invention also provides vectors into which the DNA of the present invention is inserted. Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they enable the long-term stable integration of the transgene and its proliferation in daughter cells. Lentiviral vectors have further advantages over vectors derived from onchoretroviruses such as mouse leukemia virus in that they can transduce non-proliferating cells such as hepatocytes. They also have the further advantage of having low immunogenicity.

[0281] In short, the expression of native or synthetic nucleic acids encoding CARs is typically carried out by operably ligating a nucleic acid encoding an anti-CTHRC1 T cell engager or a portion thereof to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and incorporation in eukaryotes. Typical cloning vectors contain transcriptional and translational terminators, start sequences, and promoters useful for regulating the expression of the desired nucleic acid sequence.

[0282] In addition to the methods described above, the following methods may also be used.

[0283] The expression constructs of the present invention may also be used in nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art (e.g., U.S. Patents 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety). In another embodiment, the present invention provides a gene therapy vector.

[0284] 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-generating vectors, and sequencing vectors.

[0285] Furthermore, the expression vector may be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology and molecular biology manuals. Useful viruses as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Generally, a suitable vector contains a replication origin that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selectable markers (e.g., WO01 / 96584, WO01 / 29058, and U.S. Patent No. 6,326,193).

[0286] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a favorable platform for gene delivery systems. Selected genes can be inserted into vectors using techniques known in the art and packaged into retroviral particles. Recombinant viruses can then be isolated and delivered to target cells in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenovirus vectors are used. Several adenovirus vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0287] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located 30–110 bp upstream of the initiation site, but in recent years, some promoters have been shown to contain functional elements downstream of the initiation site as well. The spacing between promoter elements is often flexible, so that promoter function is maintained even if the elements are reversed or moved relative to each other. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased up to 50 bp, beyond which activity begins to decrease. Depending on the promoter, individual elements may function cooperatively or independently to activate transcription.

[0288] A suitable promoter is the pre-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a potent constitutive promoter sequence that can promote high levels of expression of any polynucleotide sequence operatively linked to it. Another suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may be used, including, but are not limited to, the simian virus 40 (SV40) early promoter, mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus pre-early promoter, Roussarcoma virus promoter, and human gene promoters such as actin promoters, myosin promoters, hemoglobin promoters, and creatine kinase promoters. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also intended as part of the present invention. The use of inductive promoters provides molecular switches that can turn on the expression of the polynucleotide sequence to which they are operatively linked, when such expression is desired, or turn it off when such expression is not desired. Examples of inductive promoters include, but are not limited to, metallothione promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0289] To evaluate the expression of an anti-CTHRC1 T cell engager or a portion thereof, the expression vector introduced into cells may also contain either or both a selectable marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a cell population intended to be transfected or infected via the viral vector. In other embodiments, the selectable marker may be immobilized on a separate DNA fragment and used in the cotransfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences that enable expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as neo.

[0290] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and encodes a polypeptide whose expression is revealed by some readily detectable characteristic, such as enzymatic activity. Reporter gene expression is assayed at a suitable time after the DNA has been introduced into 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 commercially available. Generally, a construct exhibiting the highest expression level of the reporter gene in the smallest 5' facie region is identified as the promoter. Such promoter regions may be ligated to the reporter gene and used to evaluate the ability of a drug to regulate promoter-driven transcription.

[0291] Methods for introducing and expressing genes in cells are known in the art. In relation to expression vectors, vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0292] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, and electroporation. 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.

[0293] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, are the most widely used method for gene insertion into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses, among others. See, for example, U.S. Patents 5,350,674 and 5,585,362.

[0294] Chemical means for introducing polynucleotides into host cells include colloidal dispersions, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is liposomes (e.g., artificial membrane vesicles). When non-viral delivery systems are utilized, the exemplary delivery vehicle is liposomes. Lipid formulations are intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, nucleic acids may be associated with lipids. Nucleic acids associated with lipids can be encapsulated within the aqueous interior of liposomes, dispersed within the lipid bilayer of liposomes, bound to liposomes via linking molecules associated with both liposomes and oligonucleotides, contained within liposomes, complexed with liposomes, dispersed in lipid-containing solutions, mixed with lipids, combined with lipids, contained as a suspension in lipids, contained in or complexed with micelles, or otherwise associated with lipids. Lipid, lipid / DNA, or lipid / expression vector associated compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, as micelles, or in "broken-down" structures. They can also simply be dispersed in solution and possibly form aggregates of non-uniform size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include naturally occurring lipid droplets in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons such as fatty acids, alcohols, amines, amino alcohols, and aldehydes, and their derivatives.

[0295] Suitable lipids for use can be obtained from commercial sources. For example, dimyristylphosphatidylcholine ("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 dimyristylphosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid stock solutions in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it evaporates more readily than methanol. "Liposomes" is a general term encompassing various monolayer and multilayer lipid vehicles formed by the formation of encapsulated lipid bilayers or aggregates. Liposomes may be characterized by having a vesicle structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They spontaneously form when phospholipids are suspended in an excess aqueous solution. The lipid components undergo self-reorganization 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 different from the usual vesicle structure in solution are also included. For example, lipids may take the form of micellar structures or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also considered.

[0296] Various assays may be performed to confirm the presence of recombinant DNA sequences in host cells, regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors of the present invention. Such assays include, for example, “molecular biological” assays well known to those skilled in the art, such as Southern blotting and Northern blotting, RT-PCR and PCR; and “biochemical” assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blotting) or by assays described herein for identifying agents within the scope of the present invention.

[0297] prokaryotic host cells The polynucleotide sequences encoding the polypeptide components of the antibody of the present invention can be obtained using standard recombinant techniques. The desired polynucleotide sequence can be isolated and sequenced from antibody-producing cells, such as hybridoma cells. Alternatively, the polynucleotides can be synthesized using a nucleotide synthesizer or PCR technique. 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 a suitable vector depends primarily on the size of the nucleic acid to be inserted into the vector and the specific host cell to be transformed with the vector. Each vector contains various components depending on its function (amplification or expression of heterologous polynucleotides, or both) and its compatibility with the specific host cell in which it resides.

[0298] Generally, plasmid vectors containing replicons and regulatory sequences derived from species compatible with host cells are used in relation to these hosts. Both expression vectors 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 various bacteria, yeasts, and viruses. A replication origin from plasmid pBR322, which contains genes encoding ampicillin (Amp) and tetracycline (Tet) resistance and thus provides a convenient means of identifying transformed cells, is suitable for most Gram-negative bacteria; a 2μ plasmid origin is suitable for yeast; and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells. pBR322, its derivatives, or other microbial plasmids or bacteriophages may also contain, or be modified to contain, promoters that can be used by the microorganism for the expression of endogenous proteins. Examples of pBR322 derivatives used for the expression of specific antibodies are detailed in Carter et al.'s U.S. Patent No. 5,648,237.

[0299] In addition, phage vectors containing replicons and regulatory sequences compatible with host microorganisms can be used as transformation vectors in relation to these hosts. For example, bacteriophages such as λGEM(trademark)-11 may be used to create recombinant vectors, which can then be used to transform susceptible host cells such as E. coli LE392.

[0300] The expression vector of the present invention may contain two or more promoter-cistron pairs, each encoding a polypeptide component. The promoter is a non-translating regulatory sequence located upstream (5') of the cistron that regulates its expression. Prokaryotic promoters are typically divided into two classes: inductive promoters and constitutive promoters. Inductive promoters are promoters that initiate transcription of increased levels of cistron under their control in response to changes in culture conditions, such as the presence or absence of nutrients or changes in temperature.

[0301] Numerous promoters recognized by various potential host cells are well known. A selected promoter can be operably ligated to cistron DNA encoding a light or heavy chain by extracting the promoter from source DNA via restriction enzyme digestion and inserting the isolated promoter sequence into the vector of the present invention. Either a natural promoter sequence or one of many heterologous promoters may be used to lead to amplification and / or expression of the target gene. In some embodiments, heterologous promoters are utilized because they generally allow for greater transcription and higher yield of the expressed target gene compared to natural target polypeptide promoters.

[0302] Promoters recognized by various potential host cells are well known. Promoters suitable 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 and tryptophan (trp) promoter systems (Goeddel, Nucleic Acids Res., 8:4057 (1980), EP36, 776), and 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 include Shine-Dalgano (SD) sequences operably linked to DNA encoding anti-CTHRC1 antibodies. However, other functional promoters in bacteria (e.g., other known bacterial or phage promoters) are also suitable. Their nucleotide sequences are publicly available, so that those skilled in the art can operably link them to cistrons encoding target light and heavy chains using linkers or adapters to supply any required restriction sites (Siebenlist et al. (1980) Cell 20:269).

[0303] In one aspect of the present invention, each cistron in a recombinant vector contains a secretory signal sequence component that guides the transmembrane migration of the expressed polypeptide. Generally, the signal sequence can be a component of the vector, or it can be part of the target polypeptide DNA inserted into the vector. The signal sequence selected for the purposes of the present invention should be 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 native signal sequence in heterologous polypeptides, the signal sequence is replaced, for example, with a prokaryotic signal sequence selected from the group consisting of alkaline phosphatase, penicillinase, Ipp, or a thermostable enterotoxin II (STII) reader, LamB, PhoE, PelB, OmpA, and MBP. In one embodiment of the present invention, the signal sequences used in both cistrons of the expression system are STII signal sequences or variants thereof.

[0304] In another embodiment, immunoglobulin production according to the present invention may occur in the cytoplasm of the host cell and therefore does not require the presence of a secretory signaling sequence within each cistron. In this respect, the immunoglobulin light and heavy chains are expressed, folded, and assembled in the cytoplasm to form a functional immunoglobulin. Certain host strains (e.g., E. coli trxB- strain) provide cytoplasmic conditions favorable for disulfide bond formation, thereby enabling proper folding and assembly of the expressed protein subunits. Proba and Pluckthun Gene, 159:203 (1995).

[0305] The present invention provides an expression system in which the quantitative ratio of expressed polypeptide components can be adjusted to maximize the yield of secreted and appropriately assembled T cell engagers of the present invention. Such adjustment is carried out, at least in part, by simultaneously adjusting the translational intensity for the polypeptide components.

[0306] One technique for regulating translation intensity is disclosed in U.S. Patent No. 5,840,523 by Simmons et al. It utilizes variants of the translation initiation region (TIR) ​​within the cistron. For a given TIR, a series of amino acid or nucleic acid sequence variants can be generated at various translation intensities, thereby providing a convenient means for regulating this factor to a desired expression level for a particular strand. TIR variants can be generated by conventional mutagenesis techniques that result in codon changes that can alter the amino acid sequence, but silent changes in the nucleotide sequence are preferred. TIR changes may include, for example, changes in the signal sequence as well as changes in the number or spacing of Shine-Dalgano sequences. One method for generating variant signal sequences is to generate a "codon bank" at the beginning of the coding sequence in which the amino acid sequence of the signal sequence is not altered (i.e., the changes are silent). This can be accomplished by altering 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 bank's construction. This mutagenesis method is described in detail in Yansura et al. (1992) METHODS: A Companion to Methods in Enzymol. 4:151-158.

[0307] Preferably, a set of vectors is generated with varying TIR intensities for each cistron within it. 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 intensities. The TIR intensity can be determined by quantifying the expression level of the reporter gene, as detailed in U.S. Patent No. 5,840,523 by Simmons et al. Based on the comparison of translation intensities, the desired individual TIRs are selected and combined in the expression vector construct of the present invention.

[0308] Suitable prokaryotes include, but are not limited to, archaea and bacteria, such as Gram-negative or Gram-positive organisms, including Enterobacteriaceae such as E. coli. Various E. coli strains are generally available, such as K12 strain MM294 (ATCC31,446), X1776 (ATCC31,537), W3110 (ATCC27,325), and K5 772 (ATCC53,635). 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 DD266,710 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 parental host because it is a common host strain for the fermentation of recombinant DNA products. Preferably, the host cell secretes a minimal amount of proteolytic enzymes.For example, strain W3110 (Bachmann, Cellular and Molecular Biology, vol.2 (Washington, DC: American Society for Microbiology, 1987), pp.1190-1219; ATCC deposit numbers 27,325) can be modified to induce gene mutations in genes encoding endogenous proteins in the host, and examples of such hosts include E. coli strain 1A2 with complete genotype tonA; E. coli strain 9E4 with complete genotype tonA ptr3; E. coli strain 27C7 (ATCC 55,244) with complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kanr; E. coli strain 27C7 with complete genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT rbs7 ilvG kanr Examples include E. coli strain W3110 strain 37D6; E. coli strain W3110 strain 40B4, which is strain 37D6 having a degP deletion mutation that does not make it kanamycin resistant; E. coli strain W3110 strain 33D3 (U.S. Patent No. 5,639,635) having the genotype W3110ΔfhuA(ΔtonA)ptr3 lac Iq lacL8ΔompTΔ(nmpc-fepE)degP41 kanR; and E. coli strains having the mutant periplasmic protease disclosed in U.S. Patent No. 4,946,783 issued on August 7, 1990. Other strains and their derivatives, such as E. coli 294 (ATCC31,446), E. coli B, E. coli λ 1776 (ATCC31,537), and E. coli RV308 (ATCC31,608), are also preferred. These examples are illustrative and not limiting. Methods for constructing derivatives of any of the aforementioned bacteria having 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 appropriate bacteria considering the replication potential of the replicons in bacterial cells.For example, when supplying replicons using well-known plasmids such as pBR322, pBR325, pACYC177, or pKN410, E. coli, Serratia, or Salmonella species can be suitably used as hosts. Typically, the amount of proteolytic enzymes secreted by the host cells should be minimal, and preferably, additional protease inhibitors may be incorporated into the cell culture. Alternatively, in vitro cloning methods, such as PCR or other nucleic acid polymerase reactions, are preferred.

[0309] Antibody T-cell engagers can be produced in bacteria, particularly when glycosylation and Fc effector function are not required. Full-length antibodies have a longer half-life in the bloodstream. Production in E. coli is more rapid and cost-effective. For the expression of antibody fragments and polypeptides in bacteria, see, for example, US5,648,237, US5,789,199, and US5,840,523, which describe translation initiation regions (TIRs) and signal sequences for optimizing expression and secretion, and these patents are incorporated herein by reference. After expression, T-cell engagers can be isolated from E. coli cell paste and purified, for example, by passing them through a protein A or G column depending on the isotype. Final purification can be carried out in the same way as the process for purifying antibodies expressed in, for example, CHO cells.

[0310] eukaryotic host cell In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for anti-CTHRC1 T cell engager-coding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Other microorganisms include Schizosaccharomyces pombe (Beach and Nurse, Nature, 290:140 (1981), EP139,383 published May 2, 1985); for example, K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 (1983)), K. fragilis (ATCC12,424), K. bulgaricus (ATCC16,045), K. wickeramii (ATCC24,178), K. waltii (ATCC56,500), and K. drosophilarum (ATCC36,906; Van den Berg et al. 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 occidentalis (EP394,538, published October 31, 1990); as well as, for example, Neurospora, Penicillium, Tolypocladium (WO91 / 00357, published January 10, 1991), and Aspergillus hosts, such as A. nidulans (Ballance et al.).This includes filamentous fungi such as 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)). Methylotrope yeasts are preferred herein and include, but are not limited to, methanol-grown yeasts selected from the genera Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. A list of specific species representative of this class of yeast can be found in C. Anthony, *The Biochemistry of Methylotrophs*, 269 (1982).

[0311] Suitable host cells for the expression of glycosylated T cell engagers are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells such as cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco. Numerous baculovirus strains and variants, as well as corresponding permissible insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori, have been identified. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are generally available, and such viruses may be used as the viruses of this specification according to the present invention, particularly for the transfection of Spodoptera frugiperda cells.

[0312] However, vertebrate cells have received the most attention, and the proliferation of vertebrate cells in cultured media (tissue cultures) is the standard procedure. Examples of useful mammalian host cell lines include the monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCCCRL1651), human fetal kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)), baby hamster kidney cells (BHK, ATCC 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), and human cervical cancer cells (HELA, ATCC These include 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 cancer cells (MMT060562, ATCC CCL51), TRI cells (Mather et al., Annals NYAcad.Sci.383:44-68(1982)), MRC5 cells, FS4 cells, and human hepatocellular carcinoma cell line (Hep G2).

[0313] Vector components generally include, but are not limited to, one or more of the following: signal sequences, origins of replication, one or more marker genes, enhancer elements, promoters, and transcription termination sequences.

[0314] Signal array components Vectors for use in eukaryotic host cells may also contain signal sequences or other polypeptides having a specific cleavage site at the N-terminus of the mature protein or polypeptide of interest. The selected heterologous signal sequence is preferably 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 secretion leaders, such as the herpes simplex gD signal, are available.

[0315] The DNA in this precursor region is ligated within the reading frame to the DNA encoding the T cell engager.

[0316] origin of replication Generally, origin of replication components are not required for mammalian expression vectors. For example, the SV40 origin may be used simply because it contains the initial promoter.

[0317] Selected gene components Expression and cloning vectors typically contain selectable genes, also known as selectable markers. Typical selectable genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) compensate for nutritional deficiencies; or (c) supply essential nutrients unavailable from complex media, such as the gene encoding D-alanine racemase for Bacilli.

[0318] One example of a selection scheme involves using drugs that halt the growth of host cells. Cells successfully transformed with heterologous genes produce proteins that confer drug resistance and therefore survive the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolate, and hygromycin.

[0319] Examples of selectable markers suitable for mammalian cells include DHFR, or thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc., which allow for the identification of cells competent to take up anti-CTHRC1 T cell engager coding nucleic acids. When wild-type DHFR is used, suitable host cells are DHFR-deficient CHO cell lines (e.g., ATCC CRL-9096) prepared and grown as described by Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). For example, cells transformed with a DHFR selector gene are identified by first culturing all transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Alternatively, host cells transformed or co-transformed with an anti-CTHRC1 T cell engager, wild-type DHFR protein, and another selectable marker, such as a DNA sequence encoding aminoglycoside 3'-phosphotransferase (APH) (particularly wild-type hosts containing endogenous DHFR), may be selected by cell growth in a medium containing a selector for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.

[0320] The trp1 gene, located in the yeast plasmid YRp7, is a suitable select gene for use in yeast (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 select marker for yeast mutants lacking the ability to grow on tryptophan, such as ATCC number 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)).

[0321] Promoter components Expression and cloning vectors typically contain a promoter operably linked to an anti-CTHRC1 T cell engager coding nucleic acid sequence to guide mRNA synthesis. Promoters recognized by a variety of potential host cells are well known.

[0322] Virtually all eukaryotic genes have an AT-rich region located approximately 25–30 base pairs upstream from the transcription start site. Another sequence found 70–80 base pairs upstream from the transcription start site of many genes is the CNCAAT region (where N can be any nucleotide). The 3' end of most eukaryotic genes contains an AATAAA sequence, which can be a signal for the addition of a poly(A) tail to the 3' end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors.

[0323] Examples of promoters suitable for use with yeast hosts include promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem., 255:2073 (1980)) or other glycoseptic 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, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0324] Other yeast promoters that are inducible promoters, offering the additional advantage of transcription being controlled by growth conditions, include promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatases, nitrogen metabolism-related degrading enzymes, 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 EP73,657.

[0325] The transcription of anti-CTHRC1 T cell engagers from vectors in mammalian host cells is controlled by promoters derived from the genomes of viruses such as polyomavirus, fowlpox virus (UK2,211,504, published July 5, 1989), adenovirus (adenovirus 2, etc.), bovine papillomavirus, aerosarcoma virus, cytomegalovirus, retrovirus, hepatitis B virus, and Simian virus 40 (SV40), heterozoan promoters, such as actinp promoters or immunoglobulin promoters, and heat shock promoters, provided that such promoters are compatible with the host cell line.

[0326] Early and late promoters of the SV40 virus can be conveniently obtained as SV40 restriction fragments that also contain the SV40 virus replication origin. The very early promoter of human cytomegalovirus can be conveniently obtained as the HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papillomavirus as a vector is disclosed in U.S. Patent No. 4,419,446. Modified forms of this system are described in U.S. Patent 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 a herpes simplex virus-derived thymidine kinase promoter. Alternatively, the long-terminal repeat sequence of Roussarcoma virus can be used as a promoter.

[0327] Enhancer element components Transcription of DNA encoding the anti-CTHRC1 T cell engager by higher eukaryotes may be increased by inserting enhancer sequences into the vector. Enhancers are typically cis-acting elements of DNA, usually around 10–300 bp in length, that act on the promoter to increase transcription. Many enhancer sequences derived from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin) are now known. However, typically, enhancers derived from eukaryotic viruses will be used. Examples include the SV40 enhancer located posteriorly (base pairs 100–270) of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer located posteriorly of the origin of replication, and the adenovirus enhancer. See also Yaniv, Nature 297:17–18 (1982) for enhancing elements for eukaryotic promoter activation. The enhancer may be ligated into the vector at a 5' or 3' position on the anti-CTHRC1 T cell engager coding sequence, but is preferably located at a 5' site from the promoter.

[0328] Transcription termination components Expression vectors used in eukaryotic host cells (nucleated cells derived from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) will also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are generally available from the 5' untranslated region, and sometimes the 3' untranslated region, of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments in the untranslated portion of mRNA encoding the anti-CTHRC1 T cell engager. One useful transcription termination component is the bovine growth hormone polyadenylated region. See WO94 / 11026 and the expression vectors disclosed therein.

[0329] Other methods, vectors, and host cells suitable for the synthesis of anti-CTHRC1 T cell engagers in recombinant vertebrate cell cultures are described in Gething et al., Nature, 293:620-625 (1981), Mantei et al., Nature, 281:40-46 (1979), EP117,060, and EP117,058.

[0330] Host cells are transformed with the aforementioned expression or cloning vectors for anti-CTHRC1 T cell engager production and cultured in conventional nutrient media modified as appropriate for promoter induction, transformant selection, or amplification of genes encoding desired sequences.

[0331] Culture of host cells The host cells used to produce the anti-CTHRC1 T cell engager of the present invention can be cultured in a variety of media.

[0332] prokaryotic host cells The prokaryotic cells used to produce the polypeptides of the present invention are grown in a culture medium known in the art and suitable for culturing selected host cells. Examples of suitable media include Luria broth (LB) with necessary nutritional supplements. In some embodiments, the medium also contains a selectant 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.

[0333] In addition to carbon, nitrogen, and inorganic phosphate sources, any necessary auxiliary agents may also be included in appropriate concentrations, either alone or as mixtures with other auxiliary agents such as a complex nitrogen source or with the medium. Optionally, the medium may contain one or more reducing agents selected from the group consisting of glutathione, cysteine, cystamine, thioglycolates, dithioerythritol, and dithiothreitol.

[0334] Prokaryotic host cells are cultured at a suitable temperature. For example, in the case of E. coli growth, the preferred temperature is in the range of about 20°C to about 39°C, more preferably about 25°C to about 37°C, and even more preferably about 30°C. The pH of the culture medium may be any pH in the range of about 5 to about 9, mainly depending on the host organism. In the case of E. coli, the pH is preferably about 6.8 to about 7.4, more preferably about 7.0.

[0335] When an inducible promoter is used in the expression vector of the present invention, protein expression is induced under conditions suitable for promoter activation. In one embodiment of the present invention, a PhoA promoter is used to control polypeptide transcription. Thus, transformed host cells are cultured in phosphate-restricted medium for induction. In some embodiments, the phosphate-restricted medium is CRAP medium (see, for example, Simmons et al., J. Immunol. Methods (2002), 263:133-47). As is known in the art, various other inducers may be used depending on the vector construct employed.

[0336] In one embodiment, the expressed polypeptide of the present invention is secreted into the periplasm of host cells and recovered therefrom. Protein recovery typically involves the destruction of microorganisms by means such as osmotic shock, sonication, or lysis. Once the cells are destroyed, cell debris or whole cells can be removed by centrifugation or filtration. This protein can be further purified, for example, by affinity resin chromatography. Alternatively, the protein can be transported to a culture medium and isolated therein. The cells can be removed from the culture, and the culture supernatant is 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 blotting assays.

[0337] In one aspect of the present invention, the production of fusion proteins is carried out on a large scale by a fermentation process. Various large-scale fed-batch fermentation procedures are available for the production of recombinant proteins. Large-scale fermentation has a volume of at least 1,000 liters, preferably about 1,000 to 100,000 liters. These fermenters use agitator impellers to distribute oxygen and nutrients, especially glucose (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.

[0338] In the fermentation process, the induction of protein expression is typically initiated after the cells have been grown under suitable conditions to a desired density, e.g., an OD550 of approximately 180–220, at which point the cells are in an initial quiescent phase. Various inducers known in the art and described above can be used depending on the vector construct employed. The cells may be grown for a shorter period before induction. Cells are usually induced for about 12–50 hours, but longer or shorter induction times can also be used.

[0339] Various fermentation conditions can be modified to improve the production yield and quality of the polypeptides of the present invention. For example, to improve the proper assembly and folding of secreted T cell engager polypeptides, host prokaryotic cells can be co-transformed using 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 shown 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.

[0340] To minimize the proteolysis of expressed heterologous proteins (particularly those sensitive to proteolysis), certain host strains lacking proteolytic enzymes may be used in the present invention. For example, host cell lines may be modified to produce gene mutations(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, for example, described in Joly et al. (1998) (above), 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).

[0341] In one embodiment, an E. coli strain transformed with a plasmid lacking a proteolytic enzyme and overexpressing one or more chaperone proteins is used as the host cell in the expression system of the present invention.

[0342] 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 Medium ((DMEM), Sigma) are suitable for culturing host cells. In addition, Ham et al., Meth. Enz. 58:44 (1979), Barnes et al. Any of the media described in al., Analyst Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704, 4,657,866, 4,927,762, 4,560,655, or 5,122,469, WO90 / 03430, WO87 / 00195, or U.S. Patent Reissue 30,985 may be used as a culture medium for 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), etc. The culture may be supplemented with minerals (aluminum, calcium, magnesium, and phosphates), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), antibiotics (e.g., GENTAMYCIN® drugs), trace elements (defined as inorganic compounds normally present at final concentrations in the micromolar concentration range), and glucose or equivalent energy sources. Any other necessary adjuvants may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those that have been used in conjunction with the host cells selected for expression and will be apparent to those skilled in the art.

[0343] Detection of gene amplification / expression Gene amplification and / or expression may be measured directly in the sample using probes appropriately labeled based on the sequences provided herein, for example, by conventional Southern blotting for quantifying mRNA transcription, Northern blotting (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5 (1980)), dot blotting (DNA analysis), or in situ hybridization. Alternatively, antibodies capable of recognizing specific double strands, including DNA double strands, RNA double strands, and DNA-RNA hybrid double strands, or DNA-protein double strands, may be used. The antibodies may then be labeled, and assays may be performed such that the double strands are bound to the surface, thereby allowing the presence of the double-strand-bound antibody to be detected upon the formation of the double strands on the surface.

[0344] Alternatively, gene expression may be measured by immunological methods such as immunohistochemical staining of cell or tissue sections and assays of cell cultures or body fluids to directly quantify the expression of gene products. Antibodies useful for immunohistochemical staining and / or assays of sample fluids may be monoclonal or polyclonal and may be prepared in any mammal. Conveniently, antibodies may be prepared against the natural CTHRC1 polypeptide sequence, or against synthetic peptides based on the DNA sequences provided herein, or against exogenous sequences fused to CTHRC1 DNA and encoding specific antibody epitopes.

[0345] Purification of anti-CTHRC1 T cell engagers The anti-CTHRC1 T cell engager morphology may be recovered from the culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a suitable surfactant solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells used for the expression of anti-CTHRC1 T cell engagers may be destroyed by various physical or chemical means such as freeze-thaw cycles, sonication, mechanical disruption, or cell lysants.

[0346] It may be desirable to purify anti-CTHRC1 T cell engagers from recombinant cell proteins or polypeptides. The following procedure is representative of a suitable purification procedure: fractionation on an ion-exchange column; ethanol precipitation; reverse-phase HPLC; chromatography on a cation-exchange resin such as silica or DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; Protein A Sepharose column for removing contaminants such as IgG; and metal chelate column for binding epitope tag morphology of anti-CTHRC1 T cell engagers. Various protein purification methods may be used, and such methods are known in the art, for example, described in Deutscher, Methods in Enzymology, 182 (1990) and Scopes, Protein Purification: Principles and Practice, Springer-Verlag, New York (1982). The selected purification step(s) will depend, for example, on the nature of the production process used and the specific anti-CTHRC1 T cell engager being produced.

[0347] When recombinant techniques are used, T cell engagers can be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If T cell engagers are produced intracellularly, the first step is to remove particulate debris (either host cells or lysed fragments) by, for example, centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-7 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. If T cell engagers are secreted into the culture medium, the supernatant of such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the aforementioned steps to inhibit protein degradation, and antibiotics may be included to prevent the growth of foreign contaminants.

[0348] T cell engager compositions prepared from cells can be purified using, for example, hydroxyl apatite 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 T cell engager. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J.Immunol.Meth.62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J.5:15671575 (1986)). The matrix on which affinity ligands are attached is most often agarose, but other matrices are also available. Mechanically stable matrices such as pore-controlled glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than those achievable with agarose. If the CTHRC1 binding region contains a CH3 domain, Bakerbond ABX® resin (JTBaker, Phillipsburg, NJ) is useful for purification. Depending on the T cell engager to be recovered, other techniques for protein purification are also available, such as fractionation on ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin-SEPHAROSE®, chromatography on anion or cation exchange resins (e.g., polyaspartate columns), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation.

[0349] After any optional pre-purification step(s), the mixture containing the target T cell engager and contaminants may be subjected to low-pH hydrophobic interaction chromatography using a generally low-salt elution buffer (e.g., about 0–0.25 M salt) at a pH of about 2.5–4.5.

[0350] Assay Activity assay In one embodiment, an assay is provided for identifying an anti-CTHRC1 antibody having biological activity. Biological activity may include, for example, the ability to inhibit cell growth or proliferation (e.g., "cytotoxic" 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.

[0351] In certain embodiments, an anti-CTHRC1 antibody is tested for its ability to inhibit cell growth or proliferation in vitro. Assays relating to the inhibition of cell growth or proliferation are well known in the art. Certain assays relating to cell proliferation, exemplified by the “cell killing” assays described herein, measure cell viability. One such assay is the CellTiter-Glo® Luminescent Cell Viability Assay, commercially available from Promega (Madison, WI). This assay determines the number of viable cells in a culture based on the quantification of ATP present, an indicator of metabolically active cells. See Crouch et al. (1993) J.Immunol.Meth. 160:81-8, U.S. Patent No. 6602677. 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 directly adding a single reagent (CellTiter-Glo® reagent) to cultured cells. This results in cell lysis and the generation of a luminescence signal produced by the luciferase reaction. The luminescence signal is proportional to the amount of ATP present, which is directly proportional to the number of viable cells in the culture. Data can be recorded using a luminometer or CCD camera imaging device. The luminescence output is expressed as relative luminescence (RLU).

[0352] Another assay related to cell proliferation is the "MTT" assay, a colorimetric assay that measures the oxidation of 3-(4,5-dimethylthiazole-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 the cell culture (see, e.g., Mosmann (1983) J. Immunol. Meth. 65:55-63 and Zhang et al. (2005) Cancer Res. 65:3877-82).

[0353] In one embodiment, an anti-CTHRC1 antibody is tested for its ability to induce cell death in vitro. Assays for inducing cell death are well known in the art. In some embodiments, such assays measure loss of membrane integrity, indicated by the uptake of, for example, 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 immunoeffector cells. Cells are seeded in 100 × 20 mm dishes at a density of 3 × 10⁶ cells per dish and allowed to adhere overnight. The culture medium is removed and replaced with fresh medium alone or medium containing varying concentrations of antibodies. The cells are incubated for a period of 3 days. Following the treatment, the monolayer is washed with PBS and detached by trypsin treatment. The cells are then centrifuged at 1200 rpm at 4°C for 5 minutes, and the pellet is resuspended in 3 mL of cold Ca2+ binding buffer (10 mM Hepes (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2) and aliquoted into 12 × 75 mm tubes with 35 mm strainer caps to remove cell clumps (1 mL per tube, 3 tubes per treatment group). PI (10 μg / mL) is then added to the tubes. The samples are analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (Becton Dickinson). Antibodies that induce statistically significant levels of cell death when determined by PI uptake are thus identified.

[0354] In one embodiment, an anti-CTHRC1 antibody is tested for its ability to induce apoptosis (programmed cell death) in vitro. An exemplary assay for an apoptosis-inducing antibody is the annexin binding assay. In an exemplary annexin binding assay, cells are cultured and seeded in a dish 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 trypsin treatment. The cells are then centrifuged as discussed in the previous paragraph, resuspended in Ca2+ binding buffer, and ali-coated in tubes. Labeled annexin (e.g., annexin V-FITC) (1 μg / mL) is then added to the tubes. The samples are analyzed using a FACSCAN® flow cytometer and FACSCONVERT® CellQuest software (BD Biosciences). Antibodies that induce statistically significant annexin binding levels compared to controls are thus identified. Another exemplary assay for antibodies that induce apoptosis is a histone DNA ELISA colorimetric assay for detecting internucleosome degradation of genomic DNA. Such assays can be performed, for example, using a cell death detection ELISA kit (Roche, Palo Alto, CA).

[0355] Cells intended for use in any of the in vitro assays described above include cells or cell lines that naturally express CTHRC1 or 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 nucleic acids that encode CTHRC1.

[0356] In one embodiment, the anti-CTHRC1 antibody is tested for its ability to inhibit cell growth or proliferation in vivo. In a particular embodiment, the anti-CTHRC1 antibody is tested for its ability to inhibit tumor growth in vivo. In vivo model systems, such as xenograft models, can be used for such tests. 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 antibody's ability to inhibit or reduce tumor growth is measured. In a particular embodiment of the xenograft system described above, the human tumor cells are tumor cells derived from a human patient. In a particular embodiment, the human tumor cells are introduced into a suitable immunodeficient non-human animal by subcutaneous injection or by transplantation into a suitable site, such as a mammary fat body.

[0357] Binding assays and other assays In one embodiment, an anti-CTHRC1 antibody is tested for its antigen-binding activity. For example, in a particular embodiment, an anti-CTHRC1 antibody is tested for its ability to bind to CTHRC1 expressed on the surface of a cell. A FACS assay may be used for such testing.

[0358] In one embodiment, a competitive assay may be used to identify monoclonal antibodies that compete for binding to CTHRC1 with monoclonal antibodies containing any one of the variable domains of SEQ ID NOs: 81-82, or with chimeric antibodies containing the variable domain of a monoclonal antibody containing the sequences of Tables 2 and 3, plus a constant domain from IgG1 or IgG4. In certain embodiments, such competitive antibodies bind to the same epitope (e.g., a linear or structural epitope) to which the monoclonal antibody containing any one of the variable domains of SEQ ID NOs: 81-82, or the chimeric antibody containing the variable domain of a monoclonal antibody containing the sequences of Tables 2 and 3, plus a constant domain from IgG1 or IgG4, binds. Exemplary competitive assays include, but are not limited to, conventional assays such as those provided in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed exemplary methods for mapping the epitopes to which antibodies bind are provided in Morris (1996) “Epitope Mapping Protocols,” in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). Two antibodies are said to bind to the same epitope if each blocks the binding of the other by more than 50%.

[0359] In one embodiment, the purified anti-CTHRC1 antibody may be further characterized by a series 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.

[0360] Treatment method The anti-CTHRC1 T cell engagers of the present invention may be used, for example, in vitro, ex vivo, and in vivo therapeutic methods. In one embodiment, the present invention provides a method for inhibiting cell growth or proliferation, either in vivo or in vitro, the method comprising exposing cells to the anti-CTHRC1 T cell engager or a composition thereof under conditions that allow the binding of the anti-CTHRC1 T cell engager 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%, and includes inducing cell death. In certain embodiments, the cells are tumor cells. Anti-CTHRC1 T cell engagers or compositions thereof may, as an addition or alternative, (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 cell division inhibitory 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) inhibit the suppression of immune cells in the tumor microenvironment in vivo.

[0361] Cancers that can be treated include tumors in which angiogenesis has not occurred or has not yet substantially occurred, as well as tumors in which angiogenesis has occurred. Cancers may include non-solid tumors (hematological malignancies, e.g., leukemia and lymphoma) or solid tumors. The types of cancer treated with the T cell engager 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 may be used therapeutically in patients with non-hematological malignancies, such as solid tumors arising from malignancies of the breast, CNS, and skin.

[0362] Blood cancers are cancers of the blood or bone marrow. Examples of blood (or hematogenous) cancers include acute leukemia (acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia, etc.), leukemia including chronic leukemia (chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia, etc.), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (low-grade and high-grade types), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0363] Solid tumors are abnormal masses of tissue that do not usually contain cysts or fluid areas. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells that form them (sarcomas, carcinomas, and lymphomas, etc.). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synoviomas, 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, and Wilms' tumor. These include cervical cancer, testicular tumors, seminomas, bladder cancer, melanoma, and CNS tumors (gliomas (brainstem gliomas and mixed gliomas, etc.), glioblastomas (also known as glioblastoma multiforme), astrocytomas, CNS lymphomas, germ cell tumors, medulloblastomas, Schwann cell tumors, craniopharyogiomas, ependymomas, pineal gland tumors, hemangioblastomas, acoustic neuromas, oligodendrogliomas, meningiomas, neuroblastomas, retinoblastomas, and metastatic brain tumors, etc.).

[0364] In several embodiments, the subject anti-CTHRC1 T-cell engager is advantageously utilized in the treatment of breast cancer, lung cancer, ovarian cancer, pancreatic cancer, and colorectal cancer, including MSI and MSS colorectal cancer. In several embodiments, the subject anti-CTHRC1 T-cell engager is advantageously utilized in the treatment of breast cancer, colon cancer, pancreatic cancer, lung cancer, gastric cancer, and liver cancer. See, for example, Liu et al. CTHRC1, a novel gene with multiple functions in physiology, disease and solid tumors (Review), Oncol Lett. 2023 Jun;25(6):266.

[0365] In some embodiments, a method for treating a cell proliferation disorder may include administering to a subject a therapeutically effective amount of an anti-CTHRC1 T cell engager or a pharmaceutical composition of any of the embodiments described above. 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 cells, either directly or as a complex. In certain embodiments, the cell proliferation disorder is a tumor or cancer. In certain embodiments, the T cell engager may be administered in doses ranging from 0.001 mg / kg to about 100 mg / kg based on the patient's body weight.

[0366] In some embodiments, a method for treating cancer may include administering to a subject a therapeutically effective amount of any anti-CTHRC1 T-cell engager or pharmaceutical composition from the embodiments described above. For example, cancer may be selected from the group consisting of, but is not limited to, breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, 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 proliferation disorder is a tumor or cancer. In certain embodiments, the T-cell engager may be administered in doses ranging from 0.001 mg / kg to about 100 mg / kg based on the patient's body weight.

[0367] In some embodiments, the subject anti-CTHRC1 T-cell engager may be used to treat immune disorders such as autoimmune diseases. Inflammatory diseases, including autoimmune diseases, are also a class of diseases associated with B-cell dysfunction. Examples of immune diseases or conditions, including autoimmune conditions, include rheumatoid arthritis, rheumatic fever, multiple sclerosis, experimental autoimmune encephalomyelitis, psoriasis, uveitis, diabetes mellitus, lupus, systemic lupus erythematosus (SLE), lupus nephritis, eczema, scleroderma, polymyositis / scleroderma, polymyositis / dermatomyositis, ulcerative proctitis, ulcerative colitis, severe combined immunodeficiency (SCID), DiGeorge syndrome, ataxia telangiectasia, seasonal allergies, perennial allergies, food allergies, anaphylaxis, mastocytosis, allergic rhinitis, atopic dermatitis, Parkinson's disease, Alzheimer's disease, hypersplenicity, leukocyte adhesion deficiency, X-linked lymphoproliferative disorder, X-linked agammaglobulinemia, selective immunoglobulin A deficiency, hyper-IgM syndrome, HIV, and autoimmune lymphoglobulinemia. Pachypnea syndrome, Wiscott-Aldrich syndrome, chronic granulomatous disease, unclassified immunodeficiency (CVID), hyperimmune globulin E syndrome, Hashimoto's thyroiditis, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, polyglandular syndrome, bullous pemphigoid, Henoch-Schönlein purpura, post-streptococcal nephritis, erythema nodosum, erythema multiforme, gA nephropathy, Takayasu's arteritis, Addison's disease, sarcoidosis, ulcerative colitis, polyarteritis nodosa, ankylosing spondylitis, Goodpasture syndrome, thromboangiitis obliterans, Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, chronic active hepatitis, polychondritis, pemphigus vulgaris Examples include vulgaris, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, tabes dorsalis, giant cell arteritis / polymyalgia, pernicious anemia, rapidly progressive glomerulonephritis, psoriasis, fibrotic alveolitis, and cancer.

[0368] The pharmaceutical compositions 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 may be determined by factors such as the patient's condition and the type and severity of the patient's disease, but the appropriate dosage may be determined by clinical trials.

[0369] Where an "immunologically effective dose," "antitumor effective dose," "tumor inhibitory effective dose," or "therapeutic dose" is indicated, the exact amount of the composition of the present invention to be administered may be determined by a physician, taking into account age, individual differences in weight, tumor size, degree of infection or metastasis, and the patient's (subject's) condition.

[0370] In any of the embodiments described above, the step of administering the anti-CTHRC1 T cell engager or its pharmaceutical composition (and any additional therapeutic agents or adjuvants) may be carried out by any preferred means, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal, and, if desired, intrafocal administration for local treatment. Parenteral infusions include intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous administration. Further examples, though not limited to, administration may be carried out by known methods, such as intramuscular, intraperitoneal, intratracherobrospinal, subcutaneous, intra-articular, synovial, intrathecal, oral, topical, or inhalation routes, for example, by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time. In addition, anti-CTHRC1 T cell engagers are preferably administered by pulse infusion, particularly using tapered dose anti-CTHRC1 T cell engagers. The medication may be administered via any preferred route, such as intravenous or subcutaneous injection, depending in part whether the administration is short-term or chronic. In some embodiments, intravenous or subcutaneous administration of the anti-CTHRC1 T cell engager is preferred.

[0371] The subject composition may be administered in any convenient manner, including aerosol inhalation, injection, ingestion, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intramedullary, intramuscular, intravenous (IV) injection, or intraperitoneal injection.

[0372] As discussed, anti-CTHRC1 T-cell engagers are administered to human patients according to known methods, such as intramuscular, intraperitoneal, intraracerobrospinal, subcutaneous, intra-articular, synovial, intrathecal, oral, topical, or inhalation routes, for example, by intravenous administration, e.g., as a bolus or by continuous infusion over a period of time. In some embodiments, intravenous or subcutaneous administration of anti-CTHRC1 T-cell engagers is preferred.

[0373] The anti-CTHRC1 T cell engager and / or its composition of the present invention shall be formulated, administered, and given in a manner consistent with the principles of quality medicine. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of drug delivery, the method of administration, the schedule of administration, and other factors known to healthcare professionals.

[0374] The CTHRC1 binding portion of the present invention can be in different forms, encompassed by the definition of “antibody” as herein. Therefore, 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. The Fc region of an antibody may be modified to provide a desired effector function. As discussed in more detail in the sections herein, a naked antibody bound to a cell surface, having a suitable Fc region, can induce cytotoxicity, for example, via antibody-dependent cytotoxicity (ADCC), or by complement recruitment in complement-dependent cytotoxicity, or by some other mechanism. Alternatively, certain other Fc regions may be used when it is desirable to eliminate or reduce effector function to minimize side effects or complications from the procedure.

[0375] In one embodiment, the antibody (i) competes with the antibody of the present invention for binding to the same epitope, and / or (ii) substantially binds to the same epitope.

[0376] Methods for producing the above-mentioned T cell engagers are described in detail herein.

[0377] The anti-CTHRC1 T-cell engager is useful in treating CTHRC1-presenting cancers or alleviating one or more symptoms of cancer in mammals. Cancer encompasses any metastatic cancer among those described herein. The T-cell engager can bind to at least a portion of cancer cells that present CTHRC1 directly or as a complex in mammals. In preferred embodiments, when the T-cell engager binds to a CTHRC1 epitope on a cell, it is effective in destroying or killing CTHRC1-presenting tumor cells or inhibiting the growth of such tumor cells in vitro or in vivo. In other preferred embodiments, T cell engagers are effective in (i) inhibiting tumor metastasis in vivo, (ii) inhibiting tumor growth in vivo, (iii) reducing tumor size in vivo, (iv) inhibiting tumor angiogenesis in vivo, (v) exhibiting cytotoxic activity against tumor cells and cancer-associated fibroblasts expressing and / or presenting CTHRC1 in vivo, (vi) exhibiting cell division inhibitory activity against tumor cells or cancer-associated fibroblasts expressing and / or presenting CTHRC1 in vivo, (vii) enhancing the infiltration of anti-tumor immune cells in vivo, or (viii) preventing the suppression of immune cells in the tumor microenvironment in vivo.

[0378] The present invention provides a composition comprising the anti-CTHRC1 T cell engager of the present invention and a carrier. The present invention also provides a formulation comprising the anti-CTHRC1 T cell engager of the present invention and a carrier. In one embodiment, the formulation is a therapeutic formulation comprising a pharmaceutically acceptable carrier.

[0379] Another aspect of the present invention is an isolated nucleic acid encoding an anti-CTHRC1 T cell engager. This includes, for example, nucleic acids encoding both the H difference and L chain, as well as particularly hypervariable region residues, the chain encoding the native CTHRC1 binding moiety, and variants, variants, and humanized versions of the antibody.

[0380] The present invention also provides a method useful for treating CTHRC1 polypeptide-presenting cancer in mammals or for alleviating one or more symptoms of cancer, the method comprising administering a therapeutically effective amount of anti-CTHRC1 T cell engager to a mammal. The therapeutic antibody composition may be administered short-term (acute), chronically, or intermittently as directed by a physician. Methods for inhibiting the growth of CTHRC1 polypeptide-presenting cells and methods for killing them are also provided.

[0381] 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 anti-CTHRC1 T-cell engager will depend on the type of disease to be treated, the severity and course of the disease, whether the anti-CTHRC1 T-cell engager 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. Anti-CTHRC1 T-cell engager is preferably administered to the patient as a single dose or over a series of treatments. Preferably, the anti-CTHRC1 T-cell engager or its pharmaceutical composition is administered by intravenous infusion or subcutaneous injection. Depending on the type and severity of the disease, for example, whether by one or more separate doses or by sequential infusions, an initial candidate dosage of anti-CTHRC1 T-cell engager of about 1 μg / kg to about 100 mg / kg body weight (e.g., about 0.1 to 30 mg / kg / dose) may be administered to the patient. The drug regimen may include administering an initial loading dose of approximately 4 mg / kg of anti-CTHRC1 T-cell engager, followed by a maintenance dose of approximately 2 mg / kg weekly. However, other drug regimens may be useful. Typical daily doses can range from approximately 1 μg / kg to over 1000 mg / kg, depending on the factors mentioned above. In cases of repeated administration over several days or more, the treatment is continued, depending on the patient's condition, until the desired suppression of disease symptoms occurs. The progress of this therapy can be readily monitored by conventional methods and assays, as well as based on criteria known to physicians or others skilled in the art.

[0382] In several embodiments, the anti-CTHRC1 T cell engager of the present invention may be advantageously administered in combination with adoptive cell therapy (ACT), which is the first-line treatment for many malignancies, including allogeneic and autologous hematopoietic stem cell transplantation (HSCT) and recombinant cell (i.e., CAR T) therapy (see Rager & Porter, Ther. Adv. Hematol. (2011), 2(6) 409-428; Roddie & Peggs, Expert Opin. Biol. Ther. (2011) 11(4): 473-487, Wang et al. Int. J. Cancer: (2015) 136, 1751-1768, and Chang, Y. Jand X. Huang, Blood Rev, 2013. 27(1): 55-62). Such adoptive cell therapies include, but are not limited to, allogeneic and autologous hematopoietic stem cell transplantation, donor leukocyte (or lymphocyte) infusion (DLI), adoptive transfer of tumor-infiltrating lymphocytes, or adoptive transfer of T cells or NK cells (including recombinant cells, i.e., CAR T and CAR NK). In addition to the need for donor-derived cells to reconstruct hematopoiesis after radiotherapy or chemotherapy, immunological reconstruction from transferred cells is important for eliminating residual tumor cells. The effectiveness of ACT as a curative option for malignant tumors is influenced by several factors, including the origin, composition, and phenotype (lymphocyte subset, activation status) of donor cells, the underlying disease, pre-transplant conditioning regimens and post-transplant immune support (i.e., IL-2 therapy), and the graft-versus-tumor (GVT) effect mediated by donor cells within the graft. Additionally, these factors must be considered in comparison to transplant-related mortality, which typically arises from pre-conditioning regimens and / or excessive immune activity of donor cells within the host (i.e., graft-versus-host disease, cytokine release syndrome, etc.).

[0383] Methods for preparing and administering adoptive cell therapy, i.e., cell-based therapy, are known in the art and can be carried out by standard practices in the art.

[0384] In any of the embodiments described above, the treatment method may further include cell-based therapy. In any of the embodiments described above, the cell-based therapy may include, but is not limited to, administering allogeneic or autologous T-cell therapy, CAR-T-cell therapy, macrophage therapy such as CAR macrophages, and / or NK cell therapy. In certain embodiments, the cell-based therapy may include administering allogeneic or autologous T-cell therapy.

[0385] In any of the embodiments described above, the amount of cell-based therapy may be an effective amount. A person skilled in the art can determine the effective amount using ordinary skill.

[0386] In any of the embodiments described above, the subject may be a human or a non-human mammal.

[0387] In certain embodiments, the pharmaceutical compositions of this disclosure can be used in the preparation of pharmaceuticals for the treatment of cell proliferation disorders, preferably cancer.

[0388] Products and kits Another embodiment of the present invention is a product comprising materials useful for the treatment, prevention, and / or diagnosis of CTHRC1-presenting cancer. The product comprises a container and a label or accompanying documentation on or attached to the container. Suitable containers include, for example, bottles, vials, syringes, etc. Containers may be formed from a variety of materials such as glass or plastic. The container may hold a composition effective for treating, preventing, and / or diagnosing cancerous conditions and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper puncturable by a subcutaneous needle). At least one active agent in the composition is the anti-CTHRC1 T-cell engager of the present invention, or the CAR-modified immune cells of the present invention, or the nucleic acid of the present invention. Optionally, the composition further comprises a carrier, for example, a pharmaceutically acceptable carrier. The label or accompanying documentation indicates that the composition is used for the treatment of cancer. The label or accompanying documentation further comprises instructions for administering the antibody composition to a cancer patient. In addition, the product may further include a second container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It may further include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0389] Kits useful for various purposes, such as CTHRC1-presenting cell killing assays, purification of CTHRC1 polypeptides from cells, or immunoprecipitation methods, are also provided. For the isolation and purification of CTHRC1 polypeptides, the kit may include an anti-CTHRC1 antibody conjugated to beads (e.g., Sepharose beads). Kits may also be provided that include antibodies for in vitro detection and quantification of CTHRC1 polypeptides, for example, in ELISA or Western blotting. Similar to manufactured products, the kit includes a container and a label or accompanying documentation on or attached to the container. The container holds a composition comprising at least one anti-CTHRC1 T cell engager of the present invention. Additional containers may include, for example, diluents and buffers, and control antibodies. The label or accompanying documentation may provide a description of the composition, as well as instructions for the intended in vitro or detection application.

[0390] For example, the kit may comprise a first container containing a composition comprising one or more CTHRC1 T cell engagers of the present 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 pharmaceutically acceptable.

[0391] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0392] All patents, patent applications, and references cited herein are incorporated herein by reference in their entirety. [Examples]

[0393] Example 1: CTHRC1 expression in tumor and stroma of 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 an 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 antibodies at the recommended dilutions for immunohistochemical examination. After incubation with the primary antibody, the slides were washed and bound mAbs were detected by common colorimetric methods. As shown in Figure 1A, the tumor stroma is stained with FAP, while the tumor lesions (blue) lack FAP staining. However, as shown in Figure 1B, CTHRC1 is localized in both the tumor stroma and tumor lesions, which is consistent with scRNA data (not shown).

[0394] 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 the mouse EMT6 breast cancer line, were selected. Cell lines were incubated with 0.25 ug / mL recombinant CTHRC1 to form a complex on the cell surface. Following incubation, the cells were washed, and anti-CTHRC1 mAb was added at 10 ug / mL and incubated for 30 minutes. The cells were then washed again and incubated with an anti-mouse secondary antibody diluted 1:250 in FACS buffer for 20 minutes. The cells were washed again and resuspended in FACS buffer for analysis on a Sony cell analyzer. The results are shown in Figures 2A-2B, which support the idea that anti-CTHRC1 antibody can bind to the surface of tumor cells.

[0395] Figure 2C is from the Encyclopedia of Cancer Cell Lines. sites.broadinstitute.org / ccle / datasetsUsing [a specific method / tool], we determined the expression of CTHRC1 across multiple human cancer cell lines. High expression of CTHRC1 was observed in several cell lines, including SKOV3 ovarian cancer. In these experiments, SKOV3 cells were incubated with 1 ug / mL recombinant CTHRC1 to form a complex on the cell surface. Following incubation, the cells were washed, and 1 ug / mL of anti-CTHRC1 mAb was added and incubated for 30 minutes. The cells were then washed again and incubated for 20 minutes with an anti-mouse secondary antibody diluted 1:250 in FACS buffer. The cells were washed again and resuspended in FACS buffer containing DAPI for analysis on a Sony cell analyzer. This analysis identified CTHRC1S-M3 (PAI-0303) and CTHRC1S-M23 (PAI-0323) as binding to SKOV3 ovarian cancer cells (Figure 2C). Data for other cell lines are also shown in Figure 2C.

[0396] To evaluate whether CTHRC1S-M3 (PAI-0303) is internalized by cancer cells, the antibody was first labeled with pHrodo, a pH-sensitive dye that covalently binds to free lysine present in the antibody. pHrodo is nonfluorescent outside the cell (neutral pH) and fluoresces once the antibody is internalized in the acidic environment of phagosomes and endosomes. Following antibody labeling with pHrodo, SKOV3 ovarian cancer cell lines were treated for 24 hours with two concentrations (0.1 and 1 ug / mL) of pHrodo-labeled anti-CTHRC1 antibody, as well as pHrodo-labeled isotype control (negative control) and pHrodo-labeled nesitumumab (positive control). The following day, the cells were detached and washed using cell dissociation buffer, and the internalization level was measured by flow cytometry using a Sony cell analyzer. CTHRC1S-M3 (PAI-0303) was not internalized by human cancer cell lines, while CTHRC1S-M23 (PAI-0323) showed the highest level of internalization within 24 hours (Figure 2D).

[0397] Bridging ELISA was performed to determine the relative affinity of each CTHRC1-targeted immunoengager. Recombinant human CTHRC1 protein was coated onto maxisorp plates overnight at 4°C. The following day, the plates were washed once with PBST, then blocked with 1% BSA blocking buffer at room temperature for 90 minutes with shaking, followed by further washing with PBST. Eight titrations of each CTHRC1-targeted immunoengager were prepared, added to the plates, and conjugated at room temperature for 45 minutes. The plates were then washed three times with PBST, followed by incubation with biotinylated antigen (either human CD3ε or human 4-1BB) at room temperature for 45 minutes. The plates were then washed three times with PBST, followed by incubation with HRP-labeled streptavidin secondary antibody at room temperature for 45 minutes. Finally, the plates were washed six times with PBST and stained with TMB substrate and 0.5 M phosphate. In PAI-03023-hu41BBL_bsIgG(1:1 KiH), the orientation of the bridging ELISA was reversed (His-tagged human CTHRC1 was captured and detected as an antigen in biotinylated human 4-1BB). The anti-CTHRC1 T cell (CD3) engager antibody based on M3 (including SEQ ID NOs. 81 and 82) contained a T cell engager moiety (PAI-SP34) including SEQ ID NOs. 7 and 8, and the results are shown in Figure 2E. In particular, antibody M3 was shown to bind to the cell surface and not be internalized.

[0398] Example 3: In vivo efficacy: Combination with cell therapy NK cells and CD8 T cells

[0399] Performed using xenografts derived from cell lines.

[0400] In vivo efficacy will be evaluated in xenograft-carrying tumor-bearing mice (SCID / nude) derived from common cell lines. Tumor cells will be subcutaneously inoculated into naive mice, and tumor growth will be monitored. The tumor will reach approximately 150 mm. 3When this is reached, mice will be randomly assigned to a treatment group. Mice will be injected with CAR-T cells or CAR-NK cells and treated with an anti-CTHRC1 T cell engager or a relevant control protein. Tumor growth inhibition and survival will be evaluated over the duration of the study. The anti-CTHRC1 T cell engager should enhance the antitumor activity of CAR-T cells and CAR-NK cells.

[0401] Example 4: CTHRC1 is highly selectively localized to cancer, associated with poor outcomes, and most upregulated on cancer-associated fibroblasts (CAFs) in the immunocold tumor microenvironment. While not bound by theory, it is expected that, in the context of cancer, inhibition of CTHRC1 may confer therapeutic benefits by disrupting CTHRC1-mediated immunosuppression and blocking CAF and autocrine pro-survivability signaling to cancer cells. Targeting CTHRC1 with toxin-bound or immune cell-bound antibodies may further drive antitumor activity. We have gathered data supporting the idea that CTHRC1 is selectively upregulated in cancer and contributes to cancer progression.

[0402] Binding data (from biolayer interferometry) for exemplary anti-CTHRC1 antibodies are provided in the table below. Anti-CTHRC1 antibodies were screened in cell adhesion assays to evaluate their functional activity. Of the 12 clones identified as selective CTHRC1 conjugates by ELISA, three clones (CTHRC1S-M5, CTHRC1S-M11, and CTHRC1S-M23) showed functional activity. [Table 16]

[0403] CTHRC1 mRNA is a leading marker of CAF in cancer-rich, immunocold tumor samples. In this example, CTHRC1 is demonstrated to be specifically upregulated in cancer-rich, immunocold samples compared to CAFs in T-cell-rich, immunohot samples (Figures 3A-3B). Cancer-rich, T-cell-poor tumors, i.e., immune excluded or immunodesert tumors, are also associated with poor outcomes, treatment resistance, and immunosuppression (Gooden et al. British J. of Cancer, 2011). Therefore, therapeutic targeting of CTHRC1 presents an opportunity to target these challenging tumor types. Cancer cells transform fibroblasts into CAFs, which in turn promote cancer progression, treatment resistance, and immunosuppression (Sahai et al. Nat. Rev. Cancer, 2020). Thus, targets closely related to CAFs in cancer-rich samples compared to CAFs in T-cell-rich samples present an opportunity for therapeutic intervention in these cancers. To demonstrate the association of CTHRC1 with CAF in these tumor types, samples were divided into two groups based on the scRNA atlas described by Swechha et al. (bioRxiv 2021): those containing at least 50% cancer cells and less than 25% T cells (immunocold), and those containing more than 50% T cells and less than 25% cancer cells (immunohot) (Figure 3A). Four types of cancer were included in this analysis: pancreatic cancer, lung cancer, breast cancer, and colorectal cancer. These represent solid tumors in the scRNA atlas (Swechha et al., bioRxiv, 2021). Next, CAF-specific gene expression levels in cancer cell-rich and T cell-poor samples were ranked by the number of samples showing significantly higher expression (P<0.05) than in T cell-rich and cancer cell-poor samples, as well as by the overall P-value of this finding (Wilcoxon rank; Navon, Roy, et al., PLoS One, 2009). Finally, targets were narrowed down to those that are upregulated by CAFs in all or most of the samples but not upregulated in other cell types (top 500 CAF genes as measured by Wilcoxon rank).These results rank CTHRC1 as a highly localized target in CAFs in tumor samples that are rich in cancer cells and poor in T cells (Figure 3B), thus demonstrating an opportunity for therapeutic intervention.

[0404] CTHRC1 mRNA is upregulated in cancer compared to adjacent tissues and correlates with disease progression. This example demonstrates that CTHRC1 expression is highly upregulated in many solid tumors. Specifically, analysis of bulk-scRNA data obtained from the Cancer Genome Atlas (TCGA) shows that CTHRC1 is highly upregulated in cancerous tissue samples compared to normal adjacent tissue samples across numerous solid cancers, including breast cancer, lung cancer, ovarian cancer, pancreatic cancer, sarcoma, melanoma, and uterine carcinosarcoma (Figure 4). This indicates that CTHRC1 selectively localizes to cancerous regions within these organs. Furthermore, analysis of TCGA data using the GEPIA online tool (Tang, Z. et al. Nucleic Acids Res, 2017) demonstrates that CTHRC1 is a prognostic indicator of survival in many solid tumors (Figure 5). Patients with high CTHRC1 levels in liver cancer, gastric cancer, and sarcoma show significantly lower survival rates. All of these cancers are stromal-rich fibrous cancers, consistent with the above examples demonstrating that CTHRC1 localizes to CAFs in cancer-rich, immunopoor tumor samples. Furthermore, it was demonstrated that CTHRC1 expression increases with cancer stage in colorectal and hepatic cancers (Figure 6), suggesting that targeting CTHRC1 may be beneficial in patients with advanced, aggressive cancers and a poor prognosis.

[0405] CTHRC mRNA has a favorable expression profile in normal tissues. In addition to being upregulated in cancer compared to adjacent tissues, this example also demonstrates that CTHRC1 expression is highly selective for cancer tissue and is expressed at relatively very low levels in normal, healthy tissues in the body. This indicates that CTHRC1 targeting is accomplished with a significant therapeutic range and can be used to target payloads to the tumor microenvironment. For example, a comparison of CTHRC1 bulk-RNA expression (TCGA) in pancreatic cancer samples with CTHRC1 expression in normal tissue samples (GTEX data; both re-analyzed by UCSC Xena project, Goldman et al., Nat. Biotech, 2020) highlights a significant therapeutic range in almost all pancreatic cancer samples analyzed (Figures 7A-7C). At the single-cell level, CTHRC1 expression was observed to be localized to CAF and epithelial cancer cells in cancer samples and not in any cell type in the tissue (Figure 8). This level of localization is comparable to, if not better than, previous mAb targets such as LRRC15, which have been used to target antibody-drug conjugates (ADCs) to the tumor microenvironment and have been shown to be safe and non-toxic in clinical trials (Figure 9). Note that cancer epithelial cells have been observed to present CTHRC1 in breast, ovarian, pancreatic, and lung cancers, as well as melanoma. Expression is not found in normal epithelial cells, indicating that the mesenchymal program is switched on in cancer, and further linking CTHRC1 to a tumor-promoting role in contrast to antitumor, given the significant evolutionary pressures that cause cancer cells to downregulate antitumor targets and mechanisms. Overall, this indicates that CTHRC1 expression is sufficiently cancer-selective compared to normal tissues to allow for targeting of payloads to cancer using mAbs, and that CTHRC1 expression by cancer cells demonstrates the tumor-promoting role of this protein in humans. In summary, this embodiment therefore demonstrates the value of CTHRC1 mAb as a method for treating cancer in humans.

[0406] The CTHRC1 protein is expressed in cancer-fibroblast co-cultures and in mouse tumors. This example also demonstrated that CTHRC1 is upregulated under experimental conditions in which fibroblasts are co-cultured with cancer cells compared to monocultures of the same cells (Figure 10), indicating that CTHRC1 induction depends on fibroblast-cancer cell interactions that exhibit specificity to cancer tissue. Furthermore, it was found in vivo that the CTHRC1 protein is selectively expressed in tumor sections using an mAb that selectively binds to CTHRC1 (Figure 11). Finally, CTHRC1 was observed to be expressed in human cancer samples either on cancer cells at the stromal boundary (melanoma and head and neck cancer) or in CAF-rich regions (pancreatic cancer) (Figure 12). Overall, this analysis shows that CTHRC1 expression is observed at the protein level in cancer, as well as CTHRC1 mRNA.

[0407] Example 5: T cell proliferation Naive CD8 T cells are isolated from PBMCs obtained from normal healthy donors by negative selection using a commercially available magnetic isolation kit (StemCell Technologies). After isolation, CD8 T cells are labeled with CFSE and incubated for 72 hours on plates coated with dose-response anti-CTHRC1 T cell (CD3) engagers. After 72 hours, T cell proliferation is quantified by flow cytometry by measuring the CFSE dilution ratio.

[0408] Example 6: Tumor cell killing Naive CD8 T cells are isolated from PBMCs obtained from normal healthy donors by negative selection using a commercially available magnetic isolation kit (StemCell Technologies). After isolation, CD8 T cells are incubated with CTHRC1-expressing tumor cells (such as SKOV3) and either dose-response anti-CTHRC1 T cells (CD3) or isotype-targeted CD3 engagers for a period of 6–24 hours. Post-incubation tumor cell killing is evaluated by flow cytometry and by measuring LDH release in the culture supernatant.

[0409] Example 7: In vivo experiment The antitumor efficacy will be evaluated in a CT26 syngenic model. CT26 tumor cells will be subcutaneously inoculated into Balb / c transgenic mice expressing human CD3edg chains. One week after inoculation, mice will be randomly assigned to a treatment group with an average tumor volume of 120 mm3. Treatment with an anti-CTHRC1 T cell (CD3) engager will be initiated on the day of randomization and continued for two weeks. Tumor volume will be measured every three days, and tumor growth and survival rates will be reported as endpoints.

[0410] Example 8: In vivo model The efficacy of anti-CTHRC1 was tested in the syngenic mouse mammary tumor model EMT6. Briefly, 100,000 EMT6 cells were injected into the mammary fat pads (MFPs) of female Balb / c mice. Ten days after inoculation, the size was 120–250 mm. 3 When the range was reached, the mice were divided into groups according to tumor volume. Following group division, the mice were administered 2.5 mg / kg of anti-CTHRC1 or isotype control, 5 mg / kg of aPD-1, and / or 10 mg / kg of α-TGFb(SR) according to the group treatment. Tumor volume was assessed twice weekly by caliper measurement, (length × width 2 The dosage was calculated as ) / 2. The initial dose was administered intravenously (iv), and the remaining dose was administered intraperitoneally (ip) three times a week for three weeks. Mice were euthanized when the tumor size exceeded 1500 mm3 or due to tumor ulceration. Figure 14 illustrates the tumor growth curve obtained from measurements taken 31 days after inoculation of tumor cells. As shown in Figure 14A, anti-CTHRC1 clone M5 combined with anti-PD-1 resulted in tumor growth inhibition equivalent to that obtained with anti-PD-1 combined with anti-TGFb. Similar results were obtained for anti-CTHRC1 clone M23 (Figure 14B), while anti-CTHRC1 clone M14 combined with anti-PD-1 showed only moderate concomitant activity (Figure 14C). The control data are identical for Figures 14A-C. Data are plotted as the mean + / - standard deviation of each data point across nine mice per group.

[0411] Example 9: In vivo model Female C57BL / 6J mice were subcutaneously inoculated with Pan02 cells in Matrigel. Tumors were measured, and if a tumor was 100 mm, 3 Mice were randomly assigned to treatment groups when they reached an average tumor volume. Twenty-four hours after randomization, treatment with either an isotype control or an anti-CTHRC1 mAb (clone M5) at a dose of 10 mg / kg was initiated and continued at three doses / week for the indicated treatment duration. Mice were monitored for tumor growth (Figure 15A) and overall survival (Figure 15B). Tumor growth inhibition was considered significantly more pronounced by ANOVA. As shown in Figure 15A, anti-CTHRC1 clone M5 resulted in tumor growth inhibition compared to the isotype control. Data are plotted as the mean tumor volume + / - standard deviation of each data point across nine mice per group. As shown in Figure 15B, anti-CTHRC1 clone M5 resulted in improved survival compared to the isotype control.

[0412] Example 10: Anti-CTHRC1 pretreatment leads to CD8 T cell recruitment in the tumor microenvironment EMT6 tumor cells in Matrigel were orthotopically inoculated into Balb / c mice. The tumor was 200 mm. 3 When the average volume was reached, the animals were assigned to treatment groups. Mice were treated with 10 mg / kg isotype or anti-CTHRC1 mAb (M5 clone) for 1 week (3 doses). Following administration, tumors were isolated and processed onto slides. Slides were stained with anti-CD8 antibody, and the level of CD8 invasion into the tumor lesion was quantified using HALO image analysis software. Data were plotted as the number of infiltrating CD8 T cells versus the distance from the tumor boundary (Figure 16). As shown in Figure 16, cell pretreatment with anti-CTHRC1 antibody resulted in improved CD8 T cell recruitment in the tumor microenvironment. These results suggest that treatment as a pretreatment with anti-CTHRC1 antibody according to the present invention may result in CD8 T cell recruitment that could be advantageous for cellular immunotherapies such as CAR-T cell therapy.

[0413] Example 11: Cell Killing Assay Co-cultures of pancreatic cancer-associated fibroblasts (CAF) and indication-matched KP4 tumor cells were established by plated with both cell types in predetermined ratios 24 hours prior to drug application. The following day, adherent co-culture wells or single-cell type control culture wells were treated with either a 1:1 or 2:1 cis-oriented CTHRC1-T cell engager molecule or a matched bispecific control molecule of the same form at a single concentration, together with PBMCs as effector cells. The control was identical to the CTHRC1×CD3 engager, except that the anti-CTHRC1 variable domain sequence was replaced with an anti-Dengue variable domain sequence obtained from US9073981B2. The treated culture plates were placed in an Incucyte S3 live-cell imaging instrument (Sartorius) for 72 hours, where cell killing was evaluated by detection and quantification of the caspase-3 / 7 dye (Sartorius catalog number 4440) fluorescence signal related to apoptosis at the assay's terminal endpoint.

[0414] The tested anti-CTHRC1 T cell engagers are schematically illustrated in Figure 17A, and the corresponding reduced and annotated sequences are provided below.

[0415] 1:1 anti-CTHRC1 x anti-CD3 engager [simplified] [Table 17]

[0416] 1:1 Anti-CTHRC1 × Anti-...

Claims

1. An anti-CTHRC1 T cell engager comprising at least one first domain containing a CTHRC1 binding moiety that selectively binds to human CTHRC1 and cells presenting the CTHRC1 epitope without being internalized, and at least one second domain containing a T cell engager moiety.

2. The anti-CTHRC1 T cell engager according to claim 1, comprising an anti-CTHRC1 antibody, wherein the CTHRC1 binding portion comprises heavy chain complementarity-determining regions (HCDRs) 1, HCDR2, and HCDR3 of SEQ ID NOs. 48, 53, and 58, and light chain complementarity-determining regions (LCDRs) 1, LCDR2, and LCDR3 of SEQ ID NOs. 63, 68, and 73, respectively.

3. The anti-CTHRC1 T cell engager according to claim 1 or 2, wherein the T cell engager portion comprises an anti-CD3 antibody or a fragment thereof.

4. The anti-CTHRC1 T cell engager according to claim 2, wherein the CTHRC1 binding portion includes a heavy chain variable region (HCVR) containing SEQ ID NO: 81 and a light chain variable region (LCVR) containing SEQ ID NO:

82.

5. The anti-CTHRC1 T cell engager according to any of the prior claims, wherein the anti-CD3 antibody or the fragment thereof comprises heavy chain complementarity-determining regions (HCDRs) 1, 2, and 3 of SEQ ID NOs: 1, 2, and 3, and light chain complementarity-determining regions (LCDRs) 1, 5, and 6 of SEQ ID NOs: 4, 5, and 6, respectively.

6. The anti-CTHRC1 T cell engager according to any of the prior claims, wherein the anti-CD3 antibody or the fragment thereof comprises a heavy chain variable region (HCVR) sequence having at least 80% sequence identity with respect to the sequence of SEQ ID NO: 7 and / or a light chain variable region (LCVR) sequence having at least 80% sequence identity with respect to the sequence of SEQ ID NO:

8.

7. The anti-CTHRC1 T cell engager according to any of the prior claims, wherein the anti-CD3 antibody or the fragment thereof comprises a heavy chain variable region (HCVR) sequence having the sequence of SEQ ID NO: 7 and / or a light chain variable region (LCVR) sequence having the sequence of SEQ ID NO:

8.

8. The anti-CTHRC1 T cell engager according to any of the prior claims, wherein the CTHRC1 binding moiety comprises an anti-CTHRC1 antibody that binds to CTHRC1, and preferably the CTHRC1 antibody binds to CTHRC1 with a binding affinity of less than 10 nM.

9. The anti-CTHRC1 T cell engager according to claim 8, wherein the anti-CTHRC1 antibody is a chimeric, humanized, or human antibody.

10. The anti-CTHRC1 T cell engager according to claim 8, wherein the anti-CTHRC1 antibody is a monoclonal antibody.

11. The anti-CTHRC1 T cell engager according to claim 8, wherein the anti-CTHRC1 antibody comprises an anti-CTHRC1 antibody fragment, preferably the antibody fragment is selected from the group consisting of Fab, F(ab')2, Fv, scFv, dsFv, and a single-domain antibody.

12. The anti-CTHRC1 T cell engager according to claim 11, wherein the anti-CTHRC1 antibody comprises a single-chain antibody.

13. The anti-CTHRC1 T cell engager according to claim 11, wherein the anti-CTHRC1 antibody comprises an antibody consisting only of the heavy chain (single-domain antibody).

14. The anti-CTHRC1 T cell engager according to any of the prior claims, comprising two first domains and / or two second domains, wherein the anti-CTHRC1 T cell engager has the first domains and the second domains in a ratio of 2:1, 1:2, or 2:

2.

15. The anti-CTHRC1 T cell engager according to claim 14, wherein the anti-CTHRC1 T cell engager has a 2:1 ratio in cis configuration.

16. A method for activating T cells in a tumor microenvironment, comprising contacting a tumor with an anti-CTHRC1 T cell engager according to any one of claims 1 to 15.

17. A method for inhibiting the growth of cells presenting a CTHRC1 tumor epitope in a patient requiring inhibition of the growth of such cells, comprising administering to the patient a therapeutically effective amount of the anti-CTHRC1 T-cell engager according to any one of claims 1 to 15.

18. The method according to claim 16 or 17, wherein the cancer is selected from the group consisting of breast cancer, lung cancer, colon cancer, ovarian cancer, melanoma, 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.

19. The method according to any one of claims 16 to 18, further comprising administering allogeneic or autologous T-cell therapy in combination with a cell-based therapy.

20. The method according to claim 19, wherein the cell-based therapy is selected from the group comprising administering allogeneic or autologous T-cell therapy, NK cell therapy, or macrophage therapy.

21. The method according to any one of claims 16 to 18, further comprising administering allogeneic or autologous T-cell therapy in combination with the anti-CTHRC1 T-cell engager.

22. The method according to any one of claims 16 to 21, wherein the subject is a human.

23. A pharmaceutical composition comprising an anti-CTHRC1 T cell engager according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier.

24. Use of the pharmaceutical composition according to claim 23 in the preparation of a pharmaceutical for the treatment of cell proliferation disorders, preferably cancer, or fibrous diseases.

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