Transmembrane and coiled-coil domain family 3 specific antibodies and uses thereof

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof having specificity for transmembrane and coiled-coil domain family 3 (TMCC3). The present invention also relates to a pharmaceutical composition for treating and / or preventing a disease associated with TMCC3-mediated signaling in an individual in need thereof, and a method for detecting TMCC, cancer stem cells, or cancer in a sample.

Description

[Technical Field]

[0001] The present invention relates to antibodies or antigen-binding fragments thereof having specificity for transmembrane and coiled-coil domain family 3 (TMCC3) and uses thereof. [Background technology]

[0002] Cancer is a leading cause of death worldwide and a major obstacle to extending life expectancy. Most cancer deaths are due to recurrent or metastatic disease, not the effects of the primary tumor. Increasing evidence indicates that cancer stem cell (CSC) populations are a major cause of chemotherapy resistance and cancer recurrence because these cells possess the ability to self-renew and differentiate into cancer cells.

[0003] Since the first identification of CSCs in leukemia in 1994, cancer stem cells have been shown to possess metastatic properties and resistance to chemotherapy and radiation therapy, which contribute to tumor recurrence several years after clinical remission (Phi LTH, et al. Stem Cells Int. 2018, 5416923 (2018)).

[0004] The plasticity of CSCs is supported by diverse signaling pathways involved in the induction and maintenance of CSCs (Zhang, S., et al., Identification and characterization of ovarian cancer-initiating cells from primary human tumors. Cancer research 68, 4311-4320 (2008)).

[0005] Given the functional relationship between CSCs and normal stem cells, signal transduction pathways involved in normal stem cell physiology have received particular attention, such as the actions of WNT, Notch, and Hedgehog (Hh) (Matsui, W.H. Medicine (Baltimore) 95, S8-S19 (2016); Cochrane, C.R. et al. Cancers (Basel) 7, 1554-1585 (2015)). Indeed, many CSC markers are also expressed in normal stem cell populations and play important roles in tissue homeostasis and self-renewal (Yang, W., et al. Int J Mol Sci 22 (2021); Terraneo, N. et al. Front Oncol 10, 319 (2020)).

[0006] Therefore, identifying unique molecular targets that are expressed only in CSCs but not in normal cells is of great importance and will be useful for the research and development of novel CSC-targeting therapeutic agents. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention provides antibodies with specificity for unique molecular targets expressed in CSCs and not expressed in normal cells, and uses thereof for treating, preventing, or detecting cancer. [Means for solving the problem]

[0008] Therefore, the present invention provides an antibody or antigen-binding fragment thereof having specificity for an epitope in TMCC3, the antibody or antigen-binding fragment thereof comprising a complementarity-determining region (CDR) of a heavy chain variable region and a CDR of a light chain variable region, The CDRs of the heavy chain variable region are a CDRH1 having the amino acid sequence of SEQ ID NO:3, a CDRH2 having the amino acid sequence of SEQ ID NO:4, and a CDRH3 having the amino acid sequence of SEQ ID NO:5; and The CDRs of the light chain variable region are It comprises CDRL1 having the amino acid sequence of SEQ ID NO:6, CDRL2 having the amino acid sequence of SEQ ID NO:7, and CDRL3 having the amino acid sequence of SEQ ID NO:8.

[0009] In some examples, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO:1 or a substantially similar sequence with at least 95% sequence identity, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO:2 or a substantially similar sequence with at least 95% sequence identity.

[0010] In some embodiments of the invention, the antibody or antigen-binding fragment thereof is a Fab fragment, a F(ab')2 fragment, a ScFv fragment, a monoclonal antibody, a chimeric antibody, a nanobody, a humanized antibody, or a human antibody.

[0011] In some embodiments of the invention, the antibody or antigen-binding fragment thereof is multispecific, ie, linked to a second antibody or antigen-binding fragment thereof having specificity for a second epitope.

[0012] In some embodiments of the invention, the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent, examples of which include, but are not limited to, antimetabolites, alkylating agents, alkylating-like agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicins, antimitotic agents, topoisomerase inhibitors, proteasome inhibitors, and radioisotopes.

[0013] In some embodiments of the invention, the therapeutic agent is selected from DM1, DM3, DM4, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

[0014] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof is expressed on the surface of a cell. The cell may be an immune cell, a cancer stem cell, or a stem cell. In one embodiment of the present invention, the immune cell is a T cell.

[0015] The present invention provides vectors encoding the antibodies or antigen-binding fragments thereof disclosed herein.

[0016] The present invention provides genetically engineered cells that express the antibodies or antigen-binding fragments thereof disclosed herein or that contain the vectors disclosed herein.

[0017] The present invention also provides methods for producing an antibody or antigen-binding fragment thereof disclosed herein, the methods comprising: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions favorable for expression of the one or more polynucleotides; and (c) optionally, isolating the antibody or antigen-binding fragment from the host cell and / or the medium in which the host cell is cultured.

[0018] The present invention provides pharmaceutical compositions comprising the antibodies or antigen-binding fragments thereof disclosed herein, or genetically engineered cells, and a pharmaceutically acceptable carrier.

[0019] The invention provides a container or injection device comprising the antibody or antigen-binding fragment thereof, or genetically engineered cell disclosed herein.

[0020] The present invention provides methods for treating or preventing a disease associated with TMCC3-mediated signaling in an individual in need thereof, the method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or genetically engineered cell disclosed herein.

[0021] Alternatively, the present invention provides a pharmaceutical composition for treating or preventing a disease associated with TMCC3-mediated signaling in an individual in need thereof, the pharmaceutical composition comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or a genetically engineered cell used herein, and a pharmaceutically acceptable carrier.

[0022] The present invention further provides methods for treating, prophylactically treating, and / or preventing cancer in an individual suffering from cancer, the methods comprising administering a pharmaceutical composition to the individual. In some embodiments of the present invention, the tumor is a solid tumor. Examples of tumors include, but are not limited to, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, prostate cancer, or colorectal cancer.

[0023] Alternatively, the present invention provides a pharmaceutical composition for treating, prophylactically treating and / or preventing cancer in an individual suffering from cancer, said pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof or genetically engineered cell disclosed herein.

[0024] In some embodiments of the invention, the pharmaceutical composition is in a form suitable for injection. Alternatively, the invention provides a method for administering an antibody or antigen-binding fragment thereof disclosed herein to the body of an individual, the method comprising injecting the antibody or antigen-binding fragment into the body of the individual.

[0025] In some embodiments of the invention, the injection is subcutaneous, intravenous, or intramuscular. Alternatively, the antibody or antigen-binding fragment is injected into the individual's body via subcutaneous, intravenous, or intramuscular injection.

[0026] The present invention provides a method for detecting TMCC3, cancer stem cells, or cancer in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof disclosed herein.

[0027] In some embodiments of the present invention, the method further comprises assessing the expression level of TMCC3 in the sample, wherein an increase in the expression level of TMCC3 compared to the standard indicates the presence of cancer stem cells in the sample.

[0028] Examples of said cancer stem cells include, but are not limited to, hematopoietic, epidermal, breast, ovarian, lung, pancreatic, prostate, brain, colon, bone marrow or lymphatic cancer stem cells.

[0029] The present invention provides a kit for detecting TMCC3 or cancer in a sample, the kit comprising an antibody or antigen-binding fragment thereof disclosed herein.

[0030] The present invention provides a method for detecting a propensity for cancer or predicting the likelihood, treatment response, prognosis or recurrence of said cancer in an individual, said method comprising: The expression level of TMCC3 in a sample is assessed using an antibody or antigen-binding fragment thereof disclosed herein, and an increase in the expression level of TMCC3 compared to the standard indicates the presence of cancer stem cells in the sample.

[0031] The present invention is described in detail below. Other features, objects, and advantages of the present invention are found in the following detailed description and claims. [Brief explanation of the drawings]

[0032] [Figure 1] Figure 1 shows the expression level of TMCC3 mRNA in tumor tissues of breast cancer patients. High expression of TMCC3 is associated with poor clinical outcomes in breast cancer patients (including early-stage patients). [Figure 2] We show that TMCC3 expression in tumors is higher than that in normal tissues and contributes to cancer progression. [Figure 3] Binding affinity of anti-TMCC3 mAb by ELISA analysis is shown. [Figure 4] Binding activity of anti-TMCC3 mAb in MCF7 cells overexpressing TMCC3 is shown. [Figure 5]Shows TMCC3 expression in breast, pancreatic and ovarian cancer PDX tumors. [Figure 6A] 1 shows FACS analysis using mAb8D6, which demonstrates TMCC3 expression on the surface of serous (OC042) and clear cell (OC085) ovarian cancer PDXs. [Figure 6B] IHC analysis of TMCC3 expression, which was also detected in these PDXs, is shown. [Figure 7A-7C] Figure 7 shows the expression of TMCC3 in primary ovarian cancer specimens. Figure 7A shows the results of IHC analysis. Figure 7B shows the expression levels in three ovarian cancer subtypes: mucinous, clear cell, and endometrioid. Figure 7C shows the expression levels in early (stage I-II) and late (stage III-IV) ovarian cancers. [Figures 8A-8J] 1 shows a survival curve related to TMCC3 expression level in ovarian cancer patients. [Figures 9A-9D] TMCC3 antibody enrichment of breast and ovarian cancer stem cells in PDXs was demonstrated, as assessed by in vitro sphere formation (A, C) or in vivo tumor growth (B, D). [Figures 10A-10E] 1 shows the in vitro and in vivo tumor suppressive potential of MMAF-conjugated ADC-8D6. [Figure 11] 1 shows the results of reduced mass spectrometry of trimannosyl-anti-TMCC3 8D6 mAb. [Figures 12A-12B] 1 shows the results of reduced mass spectrometry of anti-TMCC3 8D6 mAb-4Az. [Figure 13] 1 shows the results of reduced mass spectrometry of anti-TMCC3 8D6 mAb-4 (DBCO-vc-MMAE). [Figure 14] 1 shows the clinical information of ovarian cancer patient OC085 and the TMCC3 expression in his tumor. [Figures 15A-15G] Figure 1 shows the in vivo anticancer activity of ADC-trimannosyl-MMAE (8D6) against OC085 PDX. [Figures 16A-16B] Figure 1 shows the in vivo anticancer activity of trimannosyl ADC (8 mpk) against OC085 PDX. [Figures 17A-17B]1 shows the in vivo chemotherapy and anticancer activity of ADC-8D6 (8 mg / kg / dose) against OC085 PDX. [Figures 18A-18B] Figure 1 shows that 50mpk niraparib failed to suppress tumor growth of OC085 PDX in vivo. DETAILED DESCRIPTION OF THE INVENTION

[0033] It is to be understood that this invention is not limited to the particular materials and methods described herein. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0034] It should be noted that as used herein and throughout the appended patent application, "a / an" and "the" include plural referents unless the context clearly dictates otherwise.

[0035] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex that contains at least one complementarity-determining region (CDR) that has specificity for or interacts with a particular antigen (TMCC3). The term "antibody" includes immunoglobulin molecules and multimers thereof (e.g., IgM), which contain four polypeptide chains: two heavy (H) chains and two light (L) chains linked together via disulfide bonds.

[0036] Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region comprises three regions, CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region.

[0037] The light chain constant region contains one region (CL1). The VH and VL regions can be subdivided into highly variable regions called complementarity-determining regions (CDRs) and more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0038] In different embodiments of the present invention, the FRs of an anti-SARS-CoV-2 spike protein antibody (or antigen-binding portion thereof) may correspond to human germline sequences or may be naturally or artificially modified. An amino acid consensus sequence can be defined based on a parallel analysis of two or more CDRs.

[0039] As used herein, the terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, and similar terms include any naturally occurring, enzymatically derived, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.

[0040] As used herein, the terms "specific for" or "specifically binds" indicate that the antibody does not cross-react to a significant extent with other epitopes.

[0041] As used herein, the term "epitope" refers to the site at which an antibody binds to an antigen.

[0042] As used herein, the term "complementarity determining region" (CDR) refers to the non-contiguous antigen-binding sites found within the variable regions of heavy and light chain polypeptides.

[0043] CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), and the definitions include overlapping or subsets of amino acid residues when compared against each other.

[0044] When applied to polypeptides, the terms "substantial similarity" or "substantially similar" mean that two peptide sequences share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity when optimally aligned, for example, by the programs GAP or BESTFIT using default gap weights. Differences at mismatched residue positions are preferably conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced with another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) in its side chain (R group).

[0045] Generally, conservative amino acid substitutions do not substantially alter the functional properties of a protein. When the differences between two or more amino acid sequences are conservative substitutions, the percentage of sequence identity or similarity can be increased to correct for the nature of the conservative substitution.

[0046] This method of preparation is well known to those skilled in the art. Examples of amino acids with side chains of similar chemical properties include: (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartic acid and glutamic acid; and (7) sulfur-containing side chains: cysteine ​​and methionine.

[0047] Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic-aspartic, and asparagine-glutamine.

[0048] Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443-1445, which is incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0049] As used herein, the term "monoclonal antibody" is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies may be derived from a single line by any method available or known in the art, including eukaryotic, prokaryotic, and bacteriophage lines.

[0050] As used herein, the term "chimeric" antibody refers to an antibody having variable sequences derived from a non-human immunoglobulin and a human immunoglobulin constant region, usually selected from a human immunoglobulin template.

[0051] As used herein, the term "nanobody" refers to antibodies that contain a small single variable region (VHH for antibodies obtained from camels and dromedaries).

[0052] Antibody proteins from members of the camel and dromedary (Camelus bactrianus and Calelus dromaderius) family, including New World members such as llama species (alpaca, Lama pacos, Lama glama, and Lama vicugna), have already been characterized for size, structural complexity, and antigenicity to human individuals.

[0053] As found in nature, some IgG antibodies from this mammalian family lack light chains and are therefore structurally distinct from the typical four-chain quaternary structure of other animal antibodies, with two heavy chains and two light chains.

[0054] "Humanized" forms of non-human antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. Generally, a humanized antibody will contain substantially all of at least one, and usually two, variable regions, in which all or nearly all of the CDR regions correspond to those of a non-human immunoglobulin, and all or nearly all of the FR regions are those of a human immunoglobulin sequence.

[0055] As used herein, the term "nanobody" refers to antibodies that contain a small single variable region (VHH of antibodies obtained from camels and dromedaries). Antibody proteins obtained from members of the camel and dromedary (Bactrian and dromedary) family (including New World members such as llama species, alpacas, llamas, and vicuñas) have already been characterized for size, structural complexity, and antigenicity to human individuals.

[0056] As found in nature, some IgG antibodies from this mammalian family lack light chains and are therefore structurally distinct from the typical four-chain quaternary structure of other animal antibodies, with two heavy chains and two light chains.

[0057] As used herein, the term "therapeutic agent" refers to any compound, substance, drug, medicament or active ingredient having a therapeutic or pharmacological effect suitable for administration to a mammal (e.g., a human).

[0058] As used herein, the term "immune cell" refers to a cell that functions in an immune response. Immune cells are of hematopoietic origin and include lymphocytes such as B cells and T cells, natural killer cells, myeloid cells such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0059] As used herein, the term "T cells" refers to CD4 + T cells and CD8 + Includes T cells. The term T cells includes T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells and suppressor T cells.

[0060] As used herein, the term "stem cell" refers to a cell in an undifferentiated or partially differentiated state, possessing the property of self-renewal and the developmental potential to differentiate spontaneously into more differentiated cell types, without any specific connotation of developmental potential (i.e., totipotency, pluripotency, multipotency, etc.). Self-renewal refers to the ability of a stem cell to proliferate and produce more stem cells while maintaining its developmental potential. Thus, the term "stem cell" refers to any cell subpopulation that, under certain circumstances, has the developmental potential to differentiate into a more specialized or differentiated phenotype, and that, under certain circumstances, retains the ability to proliferate substantially without differentiation.

[0061] As used herein, the term "immunoconjugate" refers to an antigen-binding protein, such as an antibody or antigen-binding fragment, that is chemically or biologically linked to a radiopharmaceutical, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antigen-binding protein can be linked to the radiopharmaceutical, cytokine, interferon, target or reporter moiety, enzyme, peptide, or therapeutic agent at any position along the molecule, so long as it is capable of binding to its target (TMCC3).

[0062] Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment of the invention, the drug may be a second, different antibody that specifically binds to TMCC3. The type of therapeutic moiety that can be conjugated to the anti-TMCC3 protein (e.g., antibody or fragment) will depend on the condition to be treated and the therapeutic effect to be achieved.

[0063] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0064] The terms "genetically engineered" or "genetic engineering" of a cell refer to the manipulation of genes using genetic material to alter the gene copy number and / or expression level in the cell. The genetic material may be in the form of DNA or RNA. Genetic material can be transferred to cells in a variety of ways, including viral transduction and non-viral transduction. After being genetically engineered, the expression level of some genes in the cell can be altered permanently or temporarily.

[0065] As used herein, the term "pharmaceutical composition" refers to a mixture containing a therapeutic agent that is administered to a mammal (e.g., a human) to prevent, treat, or eliminate a particular disease or pathological condition from which the mammal is afflicted.

[0066] As used herein, the terms "therapeutically effective amount" or "effective amount" refer to the amount of an antibody that, when administered to a mammal or other individual for treating a disease, is sufficient to effect such treatment for the disease.

[0067] As used herein, the term "treatment" and like terms encompass any treatment of a disease in a mammal (particularly a human) and includes: (a) preventing an individual who is susceptible to, but has not yet been diagnosed with, the disease from acquiring the disease; (b) suppressing the disease, i.e., arresting its progression; and (c) palliating the disease, i.e., causing the disease to regress.

[0068] The term "preventing" or "prevention," when used in conjunction with a medical condition, is known in the art and includes reducing the incidence or severity of a medical condition in an individual, or delaying the onset of symptoms, by administering a drug to an individual prior to the onset of the condition, where the individual has not previously been administered the drug.

[0069] As used interchangeably herein, the terms "individual," "subject," and "patient" refer to mammals, including, but not limited to, murines (rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), etc. Specifically, an individual is receiving a vaccination.

[0070] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver (e.g., in the case of a human, a doctor, nurse, caregiver, or individual; in the case of an animal (including a non-human mammal), a veterinarian) that an individual requires or would benefit from treatment. This judgment is made within the caregiver's area of ​​expertise and based on multiple factors, including knowledge that the individual is suffering from or will be suffering from a condition treatable with the compounds of the invention.

[0071] "Cancer," "tumor," and similar terms include precancerous, neoplastic, transformed, and cancerous cells, and may refer to solid tumors or non-solid cancers (see, e.g., Edge et al. AJCC Cancer Staging Manual (7th ed. 2009); Cibas and Ducatman Cytology: Diagnostic principles and clinical correlates (3rd ed. 2009)). Cancer includes both benign and malignant neoplasms (abnormal growths).

[0072] "Transformation" refers to spontaneous or induced phenotypic changes, such as cellular immortalization, morphological changes, abnormal cell growth, loss of contact inhibition and colonization, and / or malignant disease (see Freshney, Culture of Animal Cells: A Manual of Basic Technique (3rd ed. 1994)). Transformation is caused by infection with a transforming virus and integration of de novo genomic DNA or uptake of foreign DNA, but can also occur spontaneously or after exposure to carcinogens.

[0073] As used herein, the term "cancer stem cell" refers to a cell capable of self-renewal and differentiation into cancer cell lineages, including solid tumors and / or hematologic malignancies. Cancer stem cells can specifically induce and maintain disease. Mutated neoplastic stem cells are pluripotent cells that exhibit the properties of cancer stem cells.

[0074] As used herein, the term "sample" encompasses multiple sample types obtained from an individual, substance, or patient and can be used for diagnostic or monitoring analysis. This definition includes blood and other liquid samples of biological origin, solid tissue samples such as biopsy specimens and tissue cultures, or cells derived therefrom, and the progeny thereof.

[0075] The present invention studies and develops antibodies or antigen-binding fragments thereof that have specificity for epitopes in TMCC3.

[0076] Specifically, the antibody or antigen-binding fragment thereof has specificity for an epitope in TMCC3, and the antibody or antigen-binding fragment thereof comprises a CDR of a heavy chain variable region and a CDR of a light chain variable region, the CDR of the heavy chain variable region comprising CDRH1, CDRH2, and CDRH3 regions, and the CDR of the light chain variable region comprising CDRL1, CDRL2, and CDRL3 regions, and the CDRH1 region comprises the amino acid sequence of SEQ ID NO:3 or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the CDRH2 region comprises the amino acid sequence of SEQ ID NO:4 or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the CDRH3 region comprises the amino acid sequence of SEQ ID NO:5 or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; and the CDRL1 region comprises the amino acid sequence of SEQ ID NO:6 or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; The CDRL3 region comprises the amino acid sequence of SEQ ID NO:7 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity, and the CDRL3 region comprises the amino acid sequence of SEQ ID NO:8 or a substantially similar sequence having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0077] In some examples, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1, or a substantially similar sequence, and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or a substantially similar sequence. In some other examples, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:1, or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity. In some other examples, the antibody or antigen-binding fragment thereof comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:2, or a substantially similar sequence with at least 90%, at least 95%, at least 98%, or at least 99% sequence identity.

[0078] The sequences of the present invention are shown in Table 1.

[0079] [Table 1]

[0080] In some embodiments of the present invention, TMCC3 was found to be more highly phosphorylated at serine 216 in bCSCs than in non-bCSCs. TMCC3 belongs to the TMCC family, which includes TMCC1-3, and contains two coiled-coil domains in the N-terminal region and two transmembrane domains in the C-terminal region. In some embodiments of the present invention, TMCC3 was most strongly expressed in lung metastatic lesions, followed by metastatic lymph nodes and primary breast cancer tumors.

[0081] Silencing of TMCC3 reduces CSCs, as reflected by reduced mammary sphere formation and ALDH activity in vitro and reduced tumor growth in vivo. In some examples of the present invention, higher TMCC3 mRNA content was observed in metastatic lymph nodes than in primary ovarian cancer tumors. Attenuation of TMCC3 reduces sphere formation in vitro (Figure 3B) and suppresses ovarian cancer tumor growth in vivo. Overexpression of TMCC3 increases ALDH activity in ovarian cancer cells.

[0082] Antibodies according to the invention may be full length (e.g., IgG1 or IgG4 antibodies) or may comprise only the antigen-binding portion (e.g., Fab, F(ab')2 or scFv fragments), and can be modified as needed to affect functionality.

[0083] Antibodies also include antigen-binding fragments of intact antibody molecules. Antigen-binding fragments of antibodies can be derived from intact antibody molecules using any suitable standard technique, such as protease digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable regions and, optionally, constant regions. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, bacteriophage-antibody libraries), or can be synthesized.

[0084] The DNA can be sequenced and manipulated using chemical means or molecular biology techniques, for example, to place one or more variable and / or constant regions in the appropriate configuration, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0085] Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) antibody mimetics (e.g., isolated complementarity-determining regions (CDRs), such as CDR3 peptides) or minimal recognition units consisting of amino acid residues from hypervariable regions of restricted FR3-CDR3-FR4 peptides. Other engineered molecules, such as domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, bifunctional antibodies, trifunctional antibodies, tetrafunctional antibodies, microantibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains, are also encompassed by the term "antigen-binding fragment" as used herein.

[0086] Antigen-binding fragments of antibodies typically contain at least one variable region, which may be of any size or amino acid composition and generally contains at least one CDR adjacent to, or in frame with, one or more framework sequences. L V related to the region HIn an antigen-binding fragment having a region, V H and V L The domains may be positioned relative to each other in any suitable arrangement. For example, the variable domain may be a dimer, and the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a dimer of monomer V H or V L It may include a region.

[0087] In some examples, an antigen-binding fragment of an antibody may comprise at least one variable region covalently linked to at least one constant region. Non-limiting exemplary configurations of variable and constant regions found in antigen-binding fragments of an antibody of the invention include: (i) a V H -C H1 , (ii) V H -C H2 , (iii) V H -C H3 , (iv) V H -C H1 -C H2 , (v) V H -C H1 -C H2 -C H3 , (vi) V H -C H2 -C H3 , (vii) V H -C L , (viii) V L -C H1 , (ix) V L -C H2 , (x)V L -C H3 , (xi) V L -C H1 -C H2 , (xii) V L -C H1 -C H2 -C H3 , (xiii) V L -C H2 -C H3 , and (xiv) V L -CL Includes:

[0088] In any configuration of variable and constant regions, including any of the exemplary configurations described herein, the variable and constant regions can be directly linked to each other or can be linked by a complete or partial hinge or linker region. The hinge region can be composed of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible bond between adjacent variable and / or constant regions within a single polypeptide molecule.

[0089] It should be noted that the antigen-binding fragment of the antibody of the present invention may be any of the variable and constant region configurations listed above, which may be identical to each other and / or one or more monomeric V H or V L It may include homodimers or heterodimers (or other multimers) non-covalently associated with the domain (eg, by disulfide bonds).

[0090] The anti-TMCC3 antibodies disclosed herein may contain one or more amino acid substitutions, insertions, and / or deletions in the framework and / or CDR regions of the heavy and light chain variable regions compared to the corresponding germline sequences from which they are derived. These mutations can be readily determined by comparing the amino acid sequences disclosed herein to germline sequences purchased, for example, from public antibody sequence databases.

[0091] The present invention includes antibodies and antigen-binding fragments thereof, derived from any of the amino acid sequences disclosed herein, in which one or more amino acids in one or more framework and / or CDR regions have been mutated to the corresponding residue in the germline sequence from which the antibody is derived, or to the corresponding residue in another mammalian germline sequence, or to a conservative amino acid substitution of the corresponding germline residue (such sequence changes are collectively referred to herein as "germline mutations"). Starting with the heavy and light chain variable region sequences disclosed herein, one of skill in the art can readily produce numerous antibodies and antigen-binding fragments containing one or more single germline mutations or combinations thereof.

[0092] In some embodiments, V H and / or V L All framework and / or CDR residues within a region are mutated to residues found in the original germline sequence from which the antibody is derived. In other examples, only some residues are mutated relative to the original germline sequence, e.g., mutated residues found only within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or mutated residues found only within CDR1, CDR2, or CDR3.

[0093] In other examples, one or more of the framework and / or CDR residues are mutated to the corresponding residue in a different germline sequence (i.e., a germline sequence different from the germline sequence from which the antibody was originally derived). It should be noted that the antibodies of the invention may contain any combination of two or more germline mutations in the framework and / or CDR regions, e.g., some individual residues of which are mutated to the corresponding residue in a particular germline sequence, while some other residues that differ from the original germline sequence are either unchanged or mutated to the corresponding residue in a different germline sequence.

[0094] Once obtained, antibodies and antigen-binding fragments containing one or more germline mutations can be readily tested for one or more desired properties, such as improved binding specificity, increased binding affinity, improved or enhanced antagonistic or agonistic biological properties (as the case may be), reduced immunogenicity, etc. Antibodies and antigen-binding fragments obtained in this general manner are encompassed by the present invention.

[0095] The present invention also includes anti-TMCC3 antibodies, which are H , V L and / or CDR amino acid sequences. For example, the present invention includes anti-TMCC3 antibodies that have one or more conservative substitutions in any of the V H , V L and / or CDR amino acid sequences, which amino acid sequences are H , V L and / or have, for example, 10 or less, 8 or less, 6 or less, 4 or less, etc. conservative amino acid substitutions relative to any of the CDR amino acid sequences.

[0096] In some embodiments of the invention, the antibodies according to the invention are humanized antibodies. To improve the binding affinity of the humanized antibodies according to the invention, some amino acid residues in the human framework regions are replaced by the corresponding amino acid residues in the species (e.g., rodent) of the CDRs.

[0097] The antibodies of the present invention may be monospecific, bispecific, or multispecific. Multispecific antibodies may have specificity for different epitopes of a single target polypeptide or may contain antigen-binding regions specific for two or more target polypeptides. In some embodiments of the present invention, the antibody or antigen-binding fragment thereof is multispecific. In some embodiments of the present invention, the antibody or antigen-binding fragment thereof is linked to a second antibody or antigen-binding fragment thereof having specificity for a second epitope. The anti-TMCC3 antibodies of the present invention can be linked to or co-expressed with another functional molecule, such as another peptide or protein.

[0098] For example, an antibody or fragment thereof can be operatively linked (e.g., by chemical coupling, genetic fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody or antibody fragment, to generate a bispecific or multispecific antibody having a second binding specificity.

[0099] For example, the present invention includes bispecific antibodies, where one arm of the immunoglobulin has specificity for TMCC3 or a fragment thereof, and the other arm of the immunoglobulin has specificity for a second therapeutic target or binds to a therapeutic moiety.

[0100] In some embodiments of the invention, the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent. Examples of therapeutic agents include, but are not limited to, antimetabolites, alkylating agents, alkylating agent-like agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicins, antimitotic agents, topoisomerase inhibitors, proteasome inhibitors, and radioisotopes. In some embodiments of the invention, the therapeutic agent is selected from DM1, DM3, DM4, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF).

[0101] In one embodiment of the invention, the antibody or antigen-binding fragment thereof is expressed on the surface of a cell, particularly an immune cell (e.g., a T cell), a cancer stem cell, or a stem cell.

[0102] In some embodiments of the invention, the antibody or antigen-binding fragment thereof is in the form of a chimeric antigen receptor.

[0103] The term "chimeric antigen receptor" or "CAR" refers to a recombinant polypeptide construct consisting of at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (herein referred to as an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule, as defined below. In some examples, the domains in a CAR polypeptide construct are in the same polypeptide chain, e.g., constituting a chimeric fusion protein.

[0104] In some embodiments, the domains within a CAR polypeptide construct are not adjacent to one another, e.g., are in different polypeptide chains. CAR production and construction are summarized in Jayarama et al., EBioMedicine 58(2020)102931; Zhang et al., Biomarker Research (2017)5:22; Feins et al., Am J Hematol. (2019)94:S3-S9; and Roselli et al., J Clin Invest. 2021;131(2):e142030.

[0105] In another aspect, the invention provides a genetically engineered cell that expresses the antibody or antigen-binding fragment thereof or that contains the vector. The genetically engineered cell may be an immune cell.

[0106] In one preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof may be produced using any number of expression systems, including prokaryotic and eukaryotic expression systems. In some embodiments, the expression system is a mammalian cell expression system (e.g., hybridoma) or a CHO cell expression system. Many such expression systems are widely available from commercial vendors. H and V L In embodiments including regions, V H and V L The regions may be expressed in a single vector, for example, in a bicistronic expression unit, or under the control of different promoters.

[0107] In other embodiments, V H and V L The regions may be expressed in a single vector. H or V L The region may optionally include a methionine located at the N-terminus.

[0108] Heavy and light chain genes encoding the antibody of interest can be cloned from cells; for example, genes encoding a monoclonal antibody can be cloned from a hybridoma and used to produce recombinant monoclonal antibodies. Gene banks of heavy and light chains encoding monoclonal antibodies can also be made from hybridomas or plasma cells.

[0109] Random combinations of heavy and light chain gene products generate a large pool of antibodies with different antigen specificities (see, eg, Kuby, Immunology (3rd [expanded] ed. 1997)).

[0110] An example method for producing an antibody or antigen-binding fragment includes (a) introducing into a host cell one or more polynucleotides encoding the antibody or antigen-binding fragment; (b) culturing the host cell under conditions favoring expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment from the host cell and / or the medium in which the host cell is cultured.

[0111] Vectors may be used to introduce polynucleotides encoding the antibodies or antigen-binding fragments of the invention into host cells. In one example, one type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome.

[0112] Some vectors can replicate autonomously in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors), while other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby replicate along with the host genome.

[0113] Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). Generally, expression vectors of utility in recombinant DNA techniques are always in the form of plasmids. As the plasmid is the most common form of vector, "plasmid" and "vector" can be used interchangeably herein.

[0114] However, the invention is intended to include such other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0115] The present invention provides pharmaceutical compositions comprising antibodies or antigen-binding fragments thereof or genetically engineered cells, which are formulated with suitable diluents, carriers, excipients, and other agents that provide improved transport, delivery, tolerability, and similar properties.

[0116] The compositions can be formulated for a particular use, for example, for veterinary or human pharmaceutical use. The form of the composition and the excipients, diluents, and / or carriers used will be determined by the intended use of the antibody and the mode of administration for therapeutic use. Many suitable formulations can be found in formularies known to all medicinal chemists, such as Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa.

[0117] These formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid (cationic or anionic) containing vesicles (e.g., LIPOFECTIN™, Life Technologies, Carlsbad, Calif.), DNA binders, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (polyethylene glycols of various molecular weights), semi-solid gels, and semi-solid mixtures containing polyethylene glycol. See also Powell et al., "Compendium of Excipients for Parenteral Formulations," PDA (1998) J Pharm Sci Technol 52:238-311.

[0118] The dosage of an antibody administered to a patient varies depending on the patient's age and size, the target disease, the condition, the route of administration, and the like. Preferred dosages are typically calculated based on body weight or body surface area. When using the antibodies of the present invention to treat conditions or diseases associated with EPHA10 in adult patients, it is advantageous to administer the antibodies of the present invention intravenously. The frequency and duration of treatment can be adjusted depending on the severity of the condition. Effective dosages and schedules for administering the antibody can be determined empirically and monitored, for example, by periodically assessing the patient's progress and adjusting the dosage accordingly. Scaling of dosages between species can be performed using methods well known in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8:1351).

[0119] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, including, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated pinocytosis (see Wu et al., 1987, J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compositions can be administered by any convenient route, for example, injected by infusion or infusion, absorbed through epithelial or mucocutaneous linings (e.g., oral, rectal, and intestinal mucosa), and administered together with other bioactive agents. Administration can be systemic or local.

[0120] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using a container and injection device such as a standard needle and syringe. Regarding subcutaneous delivery, a pen delivery device can be easily applied to deliver the pharmaceutical compositions of the present invention. Such pen delivery devices can be reusable or disposable. Reusable pen delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. After the entire pharmaceutical composition in the cartridge has been administered and emptied, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition.

[0121] The pen delivery device can then be reused. Disposable pen delivery devices do not have a replaceable cartridge. Instead, disposable pen delivery devices are pre-filled with a pharmaceutical composition contained in a reservoir within the device. Once the reservoir is emptied of pharmaceutical composition, the entire device is discarded.

[0122] In some cases, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump can be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201).

[0123] In another embodiment, polymeric materials can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla. In yet another embodiment, a controlled release system can be placed in proximity to the target of the composition, thereby requiring only a fraction of the systemic dose (see, e.g., Goodson, 1984, in Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer, 1990, Science 249:1527-1533.

[0124] Injectable preparations may include dosage forms used for intravenous, subcutaneous, intradermal, and intramuscular injections, infusions, etc. These injectable preparations can be prepared by known methods. For example, injectable preparations can be prepared by dissolving, suspending, or emulsifying the antibody or salt thereof described herein in a sterile aqueous or oily medium commonly used for injections.

[0125] Aqueous vehicles for injection include, for example, isotonic solutions containing physiological saline, glucose, and other auxiliary agents, which can be used in combination with appropriate solubilizers such as alcohols (e.g., ethanol), polyols (e.g., propylene glycol, polyethylene glycol), and nonionic surfactants [e.g., polysorbate 80, HCO-50 (hydrogenated castor oil polyoxyethylene (50 mol) adduct)]. Oily vehicles include, for example, sesame oil and soybean oil, which can be used in combination with solubilizers (e.g., benzyl benzoate, benzyl alcohol, etc.). The injection solutions prepared in this manner are preferably filled into appropriate ampoules.

[0126] The above-mentioned pharmaceutical compositions for oral or parenteral use are preferably prepared in a dosage form suitable for the formulation of the active ingredient, such as tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0127] The present invention provides a method for treating or preventing a disease associated with TMCC3-mediated signaling in an individual in need of such treatment, the method comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof, or genetically engineered cells disclosed herein. Alternatively, the present invention provides a pharmaceutical composition for treating or preventing a disease associated with TMCC3-mediated signaling in an individual in need of such treatment, the pharmaceutical composition comprising a therapeutically effective amount of the antibody or antigen-binding fragment thereof, or genetically engineered cells used herein, and a pharmaceutically acceptable carrier.

[0128] The present invention further provides a method for treating, prophylactically treating, and / or preventing cancer in an individual suffering from cancer, the method comprising administering a pharmaceutical composition to the individual. In some embodiments of the present invention, the tumor is a solid tumor. Examples of such tumors include, but are not limited to, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, prostate cancer, and colorectal cancer. Alternatively, the present invention provides a pharmaceutical composition for treating, prophylactically treating, and / or preventing cancer in an individual suffering from cancer, the pharmaceutical composition comprising an effective amount of an antibody or antigen-binding fragment thereof, or a genetically engineered cell disclosed herein.

[0129] The present invention provides a method for detecting TMCC3, cancer stem cells, or cancer in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof disclosed herein.

[0130] In some embodiments of the present invention, the method further comprises assessing the expression level of TMCC3 in the sample, wherein an increase in the expression level of TMCC3 compared to the standard indicates the presence of cancer stem cells in the sample.

[0131] Examples of said cancer stem cells include, but are not limited to, hematopoietic, epidermal, breast, ovarian, lung, pancreatic, prostate, brain, colon, bone marrow or lymphatic cancer stem cells.

[0132] The present invention provides a kit for detecting TMCC3 or cancer in a sample, the kit comprising an antibody or antigen-binding fragment thereof disclosed herein.

[0133] The anti-TMCC3 antibodies of the present invention may also be used, for example, for diagnostic purposes, to detect and / or measure cancer or TMCC3-expressing cells in a sample. For example, an anti-TMCC3 antibody or a fragment thereof may be used to diagnose a condition or disease characterized by coronavirus infection. An exemplary diagnostic assay for coronavirus may include, for example, contacting a sample obtained from a patient with an anti-TMCC3 antibody of the present invention, where the anti-TMCC3 antibody is labeled with a detectable label or reporter molecule.

[0134] Alternatively, unlabeled anti-TMCC3 antibodies can be used in diagnostic applications in combination with a secondary antibody that is detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S or 125 The antibody may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure coronavirus in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0135] The present invention provides a method for detecting a propensity for cancer or predicting the likelihood, treatment response, prognosis or recurrence of said cancer in an individual, said method comprising: The expression level of TMCC3 in a sample is assessed using an antibody or antigen-binding fragment thereof disclosed herein, and an increase in the expression level of TMCC3 compared to the standard indicates the presence of cancer stem cells in the sample.

[0136] The following examples are provided to aid those skilled in the art in practicing the present invention.

[0137] Example 1 Clinical relevance of TMCC3 mRNA expression in cancer tissues To assess the clinical relevance of TMCC3 expression, we examined the TMCC3 mRNA content in tumor samples from 202 breast cancer patients by qRT-PCR. Kaplan-Meier analysis and log-rank tests showed that patients with low TMCC3 expression in their tumors had significantly longer recurrence-free survival (RFS) and overall survival (OS) than patients with high TMCC3 expression.

[0138] This was also true for 161 early-stage breast cancer patients. Multivariate analysis showed that TMCC3 expression level in tumor tissue was a significant and independent predictor of RFS and OS in breast cancer patients (Figure 1). Using the online RNA microarray database on the ONCOMINE website, we found that TMCC3 mRNA content was higher in tumor tissues than in normal tissues in cervical cancer, prostate cancer, pancreatic cancer, lung cancer, glioblastoma, skin cancer, liver cancer, and papillary thyroid cancer.

[0139] We evaluated the clinical significance of TMCC3 in cancer using the Kaplan-Meier Plotter website and found that high TMCC3 expression was associated with poorer overall survival in patients with ovarian, lung, and gastric cancer (Figure 2). These findings further support the important role of TMCC3 in the progression of ovarian cancer and support the development of strategies targeting TMCC3 for cancer therapy.

[0140] Example 2 Production of anti-TMCC3 monoclonal antibodies The results of our efforts support the view that TMCC3 is a potential therapeutic diagnostic target for clinical prognosis and CSC eradication. To generate appropriate mAbs to further study this novel protein, we immunized mice with the extracellular domain of TMCC3. ELISA and FACS analysis were used to verify the binding activity and specificity of the anti-TMCC3 mAb.

[0141] The helix 1 and helix 2 domains of the TMCC3 protein were produced for ELISA analysis. The inventors found that the BA5 strain recognized the helix 1 domain, and the 8D6 and 5G1 strains recognized the helix 2 domain of the TMCC3 protein. D Each value is 1x10 -11 M and <1x10 -11 M (Fig. 3).

[0142] Fresh and formalin-fixed preparations of TMCC3-overexpressing MCF7 cells were used for FACS analysis to verify the binding activity of the mAbs to cell surface or intracellular TMCC3. Among the many mAb isolates produced, isolate 8D6 had the highest binding affinity (4.36% and 63.1%, respectively) to surface and intracellular TMCC3 in TMCC3-overexpressing MCF7 cells compared with other isolates (Figure 4).

[0143] Example 3 Sequence of the Mouse Anti-TMCC3 8D6 Monoclonal Antibody The method for obtaining mouse anti-TMCC3 antibodies is as follows. First, anti-TMCC3 hybridomas are generated. Such hybridomas can be produced by standard protocols for producing monoclonal antibodies. Then, for example, TRIZOL (R) Total RNA from the hybridoma was isolated using reagents, such as a First-Strand cDNA Synthesis Kit (Superscript III) and oligo(dT 20 ) primer or Ig-3′ constant region primer was used to synthesize cDNA from total RNA.

[0144] Subsequently, the heavy and light chain variable regions of immunoglobulin genes were cloned from the cDNA. For example, the V of the anti-TMCC3 mAb was cloned from the mouse TMCC3 hybridoma cDNA by PCR using the mouse Ig-5′ primer set. H and V L The variable region was amplified.

[0145] PCR products were analyzed using CLONEJET TM They can be directly cloned into an appropriate vector (e.g., pJET1.2 vector) using a PCR cloning kit. The pJET1.2 vector contains a lethal insert and can survive under selection conditions only if the required gene is cloned into this lethal region. This aids in the selection of recombinant colonies. Finally, the recombinant colonies are screened for the required pure lines, and the DNA of those pure lines is isolated and sequenced. Immunoglobulin (IG) nucleotide sequences can be analyzed on the International ImmunoGeneTics information system (IGMT) website.

[0146] Example 4 FACS and IHC analysis of TMCC3 protein expression in PDX tumors TMCC3 protein expression in breast cancer (BC0145, BC0350R1, and BC0634), pancreatic cancer (PC001, PC025, and PC038), and ovarian cancer (OC042 and OC057) PDXs can be detected using mAb 8D6 (Figure 5). FACS analysis using mAb 8D6 demonstrated TMCC3 expression on the surface of serous (OC042) and clear cell (OC085) ovarian cancer PDXs (Figure 6A). TMCC3 expression was also detected in these PDXs by IHC analysis (Figure 6B).

[0147] Example 5 TMCC3 Expression in Primary Ovarian Cancer Specimens To elucidate the clinical relevance of TMCC3 in ovarian cancer, we examined TMCC3 protein expression in 125 primary ovarian cancer specimens by IHC analysis and its correlation with clinical characteristics and patient outcomes. Results showed that TMCC3 was highly expressed in tumor epithelial cells of mucinous, clear cell, and endometrioid ovarian cancer tissues (Figure 7A). Compared with other subtypes of ovarian cancer, serous ovarian cancer tissues showed low TMCC3 protein expression (Figure 7B). In TMCC3-positive specimens, TMCC3 was detected primarily in the cytoplasm and membrane of tumor cells (Figure 7A).

[0148] Specifically, clear cell carcinoma showed higher TMCC3 expression than other subtypes (Figure 7A). As shown in Figure 7C, TMCC3 expression in early-stage (stage I-II) ovarian cancer tissues was found to be higher than that in late-stage (stage III-IV) ovarian cancer tissues (P < 0.05).

[0149] Example 6 High expression of TMCC3 is associated with poor clinical outcome in ovarian cancer We investigated whether high TMCC3 expression is an important predictor of ovarian cancer prognosis. Results showed that patients with low levels of TMCC3 expression in their tumors had significantly greater OS (P<0.0001) and RFS (P<0.0001) than patients with high levels (Figures 8A and 8B). We then analyzed the potential prognostic value of TMCC3 expression in patients with different subtypes. As shown in Figures 8H and 8J, patients with endometrial or mucinous subtypes whose tumors expressed low levels of TMCC3 had greater RFS than patients with high levels (P=0.1336 and P=0.01, respectively).

[0150] For patients with clear cell carcinoma, the improved survival time for patients with low TMCC3 expression was even more significant (OS and RFS P<0.0001 and P<0.0001, respectively) (Figures 8E and 8F). These results support the adverse impact of high TMCC3 expression on the clinical outcome of ovarian cancer, especially clear cell carcinoma.

[0151] Example 7 CSC Enrichment from Breast and Ovarian Cancer PDXs Using Anti-TMCC3 mAb As shown in Figure 9A, mAb8D6 inhibited the BCSC population (CD44 + , H2k d- , 7AAD - ) and can enrich the BCSC population from BC0145 PDX tumors, - Compared to BCSC, 8D6 + This was reflected by the higher in vitro mammary sphere-forming ability of BCSCs.

[0152] Also, CD44 + / TMCC3 - / H2k d- / 7AAD - Compared to cells, CD44 + / TMCC3 + / H2k d- / 7AAD - The cells grew faster and produced larger tumors (Figure 9B). Similarly, mAb8D6 can enrich CSC populations from ovarian cancer PDX tumors OC057 and OC085.

[0153] As shown in Figures 9C and 9D, 8D6 + Cells were 8D6 from OC057 and OC085 PDXs. - These results suggest that mAb 8D6 can enrich breast and ovarian CSCs from PDX tumors.

[0154] Example 8 Anti-cancer effects of prototype anti-TMCC3 antibody-drug conjugates (ADCs) In vitro, mAb 8D6 binds to surface TMCC3 and then TMCC3 + It was found that mAb 8D6 was internalized into cells (Fig. 10A). This finding suggests that mAb 8D6 inhibits TMCC3 + This study demonstrates the feasibility of designing antibody-drug conjugates (ADCs) that target cancer cells. To this end, we generated chimeric mAb 8D6 and conjugated it to the anti-tubulin agent MMAF at a drug-antibody ratio (DAR) of 3.8 (Figure 10B). MMAF conjugated to ADC-8D6 exhibited an in vitro activity of 10 μM (IC 50 = 102.6 nM) caused 60% cell death in BC0145 PDX tumor cells (Figure 10C).

[0155] The antitumor effect of this first-generation ADC-8D6 was evaluated in two types of breast cancer PDXs: those with tumors of ∼50 mm 3At the time of the initial immunization, mice bearing tumors from BC0145 or BC0634 PDXs were treated with MMAF-conjugated ADC-8D6 (4 mg / kg) twice weekly. As shown in Figures 10D and 10E, ADC-8D6 significantly suppressed tumor growth compared to mice treated with human IgG (hIgG) (P<0.0001). Furthermore, treatment with ADC-8D6 reduced cancer metastasis to lymph nodes and vital organs compared to the hIgG control group (Table 2). These findings indicate that ADC-8D6 conjugated with MMAF can suppress tumor growth in BC0145 and BC0634 breast cancer PDXs and inhibit cancer metastasis in vivo. However, the DAR is heterogeneous in this prototype ADC, which poses an inherent challenge for chemical conjugation in the development of first-generation ADCs.

[0156] [Table 2]

[0157] Example 9 Preparation of trimannosyl anti-TMCC3 8D6 mAb To generate ADCs with homogeneous DAR and specific drug-binding sites, an efficient glycoengineering platform was implemented to conjugate a trimannosyl core antibody to novel ADCs. This platform converted the trimannosyl antibody into a trimannosyl-4GlcNAz antibody, and then conjugated a useful payload to the terminal GlcNAz group at a specific site on the antibody via a scalable accelerated azide-alkyne click chemistry (SPAAC) reaction.

[0158] To remove galactose and sialic acid moieties from N-glycans, anti-TMCC3 8D6 mAb was treated with β1,4-galactosidase and α2-3,6,8-neuraminidase in 1x glycosylation buffer at 37°C for 24 hours. β1,4-galactosidase was then added to the reaction mixture, and the mixture was further reacted at 37°C for another 24 hours to obtain the G0F / G0 antibody sample. The antibody sample was purified and subjected to combined mass chromatography-reducing mass spectrometry.

[0159] As shown in Figure 11, the majority of the antibody in the sample was G0F (having a heavy chain with a molecular weight of 50,019 Da), and only a small amount was G0 (having a heavy chain with no fucose and a molecular weight of 49,875 Da).

[0160] Example 10 Preparation of anti-TMCC3 8D6 mAb-4Az Trimannosyl anti-TMCC3 8D6 mAb and UDP-GlcNAz in 1x buffer SP were incubated in the presence of rabbit MGAT-1 and rat MGAT-2 at 37°C for 16 hours. After incubation, reduced-mass spectrometry and intact mass spectrometry were performed on anti-TMCC3 8D6 mAb-4Az. As shown in Figure 12A, the trimannosyl-anti-TMCC3 8D6 mAb-4GlcNAz antibody product was obtained. The heavy chain contained two GlcNAz molecules (molecular weight 219 Da × 2 = 438), and each heavy chain had a molecular weight of 50,457 Da.

[0161] These results indicated that GlcNAz binds to the α-3 mannose and α-6 mannose of each heavy chain of the trimannosyl-core anti-TMCC3 8D6 mAb via MGAT-1 and MGAT-2. This result was further confirmed by intact mass spectrometry. As shown in Figure 12B, compared to the intact trimannosyl-core anti-TMCC3 8D6 mAb with a molecular weight of 147,355 Da, the G0F tritrimannosyl anti-TMCC3 8D6 mAb-4GlcNAz antibody product was obtained, which contained four GlcNAz molecules (molecular weight 244 Da × 4 = 976 Da) and had a molecular weight of 148,331 Da.

[0162] Example 11 Preparation of anti-TMCC3 8D6 mAb-4 (DBCO-vc-MMAE) DBCO-vc-MMAE (10 mM in DMSO) was slowly added over 18 hours to the trimannosyl anti-TMCC3 8D6 mAb-4GlcNAz antibody product solution dissolved in MES buffer (pH 6.5) at 37°C. The antibody preparation was desalted and concentrated using an Amicon Ultra-15 centrifugal filter device with a 30 kDa NMWL in sodium citrate pH 6.5 buffer to obtain anti-TMCC3 8D6 mAb-4 (DBCO-vc-MMAE).

[0163] As shown in Figure 13, a trimannosyl anti-TMCC3 8D6 mAb-4 (GlcNAc-triazole-DBCO-(PEG)-MMAE) ADC product was obtained containing four DBCO-(PEG)-MMAE molecules (molecular weight 1,614 Da × 4 = 6,456 Da) (Formula I). ​​The molecular weight of the ADC was 154,788 Da, and the drug-antibody ratio (DAR) was 3.78.

[0164] TIFF2026500717000004.tif73170

[0165] Example 12 Anticancer effect of trimannosyl-MMAE 8D6-ADC To evaluate the efficacy of trimannosyl ADC-8D6 against ovarian cancer, a four-arm animal study was conducted using OC085 ovarian cancer PDX tumors. Patient OC085's clinical information and medical history are outlined in Figure 14. Briefly, after being diagnosed with stage 4 ovarian cancer, she underwent debulking surgery followed by first-line chemotherapy with carboplatin and paclitaxel.

[0166] The patient had a very short disease stabilization period (2.5 months) and switched to second-line chemotherapy (carboplatin + lipodoxacin), followed by the addition of nivolumab monoclonal antibody and concurrent DC-CIK cell therapy, but neither treatment prevented disease progression. As shown in Figure 14, we found that TMCC3 was highly expressed in the patient's primary tumor and PDX tumors. Therefore, PDX OC085, derived from a patient with advanced ovarian cancer who had a poor response to first- and second-line therapies, is an ideal model for the target patient population.

[0167] In one study, trimannosyl ADC-8D6 conjugated MMAE was administered at 4 mg / kg twice weekly starting the same day after tumor inoculation. ADC treatment initiated the same day after tumor inoculation mimicked the minimal residual disease (MRD) state in high-risk ovarian cancer patients, e.g., complete response after chemotherapy and / or surgical resection.

[0168] As shown in Figure 15B, tumor growth was completely abrogated, in contrast to the progressive tumor growth in control mice treated with human IgG. In a second study, 4 mg / kg of trimannosyl ADC-8D6-conjugated MMAE prevented tumors from growing to ∼100 mm 3 Treatment was initiated when tumor growth reached 4 mg / kg / dose × 2 / week. Tumor growth in ADC-treated mice was slower than in control mice treated with human IgG (Figure 15D) (p<0.001). As shown in Figures 15C and 15E, the body weight of tumor-bearing mice was not affected by ADC treatment at 4 mg / kg / dose × 2 / week.

[0169] We also analyzed ki67 expression and CSC frequency in ADC-treated tumors after euthanasia. As shown in Figure 15F, ki67 expression was lower in ADC-treated tumors than in hIgG-treated tumors. For example, ADC-treated tumors exhibited reduced sphere formation compared with the hIgG control group (Figure 15G). In a third experiment, starting the day after tumor inoculation, mice bearing OC085 ovarian PDX tumors were treated with trimannosyl ADC-8D6-conjugated MMAE at a weekly dose of 8 mg / kg / dose (intravenous injection) for 4 weeks.

[0170] As shown in Figure 16A, tumor growth was completely eliminated in ADC-treated mice compared to control mice treated with hIgG. Treatment with trimannosyl ADC-8D6 induced sustained tumor suppression in OC085 PDX-bearing mice until euthanasia on day 128 (Figure 16A). Weight loss was observed in the human IgG-treated group, but not in ADC-treated mice (Figure 16B).

[0171] To explore the anticancer effects of trimannosyl ADC-8D6 as a single agent or in combination with niraparib (a PARP inhibitor), we investigated the efficacy of niraparib in patients with tumors of ∼100 mm 3 Once this was achieved, OC085 PDX tumor-bearing mice were treated with trimannosyl ADC-8D6 alone at 8 mg / kg / dose per week (intravenous injection), or niraparib at 25 mg / kg / day (oral gavage, 5 days / week) either alone or in combination.

[0172] As shown in Figure 17A, tumor growth in mice treated with trimannosyl ADC-8D6 was negligible (p<0.001) compared to stable tumor growth in mice treated with human IgG. The 25mpk niraparib dose was selected based on reported therapeutic doses in mice bearing ovarian and cholangiocarcinoma tumors (Bezrookove, V., et al. Cancers 13, 4405 (2021); Meng, J., et al. Journal of Translational Medicine 19 (2021)).

[0173] However, in mice bearing OC085 PDX, treatment with 25 mpk niraparib slightly reduced tumor growth, but did not reach statistical significance. The combination of ADC-8D6 and niraparib eliminated tumor growth, similar to that of mice treated with ADC-8D6 alone (Figure 17A). In this experiment, no weight loss was observed in mice treated with ADC-8D6 alone or in combination with niraparib (Figure 17B).

[0174] Further, higher doses of niraparib were tested. As shown in Figure 18, administration of 50 mg / kg / day of niraparib did not inhibit tumor growth in OC085 PDX tumor-bearing mice, but caused a state of weakness and significant weight loss. These results indicated that trimannosyl ADC-8D6 has greater anticancer activity and fewer side effects in vivo than niraparib.

[0175] TMCC3 has been shown to be crucial for CSCs in breast and ovarian cancer, and mAb8D6 has been shown to be a TMCC3-specific monoclonal antibody with therapeutic diagnostic potential. The six animal studies described above demonstrated that ADC-8D6 not only exhibited anticancer activity in breast cancer PDX (BC0145 and BC0634) and ovarian PDX (OC085) tumors, but also demonstrated the ability to target CSCs in vivo.

[0176] In a comparative study of ADC-8D6 and niraparib at therapeutic doses, ADC-8D6 clearly demonstrated superior antitumor efficacy to niraparib in ovarian PDX (OC085) tumors. These results provided impetus for further research and development of anti-TMCC3 / ADCs as anticancer agents targeting ovarian cancer stem cells.

[0177] While the present invention has been described in conjunction with specific disclosed embodiments, many alternatives, modifications, and variations thereof will be apparent to those skilled in the art, and all such alternatives, modifications, and variations are deemed to be within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof having specificity for an epitope in transmembrane and coiled-coil domain family 3 (TMCC3), wherein the antibody or antigen-binding fragment thereof comprises a complementarity-determining region (CDR) of a heavy chain variable region and a CDR of a light chain variable region; The CDRs of the heavy chain variable region a CDRH1 having the amino acid sequence of SEQ ID NO: 3, a CDRH2 having the amino acid sequence of SEQ ID NO: 4, and a CDRH3 having the amino acid sequence of SEQ ID NO: 5; and The CDRs of the light chain variable region An antibody or antigen-binding fragment thereof, comprising a CDRL1 having the amino acid sequence of SEQ ID NO:6, a CDRL2 having the amino acid sequence of SEQ ID NO:7, and a CDRL3 having the amino acid sequence of SEQ ID NO:

8.

2. 2. The antibody or antigen-binding fragment thereof of claim 1, wherein the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1 or a substantially similar sequence with at least 95% sequence identity, and / or the light chain variable region comprises the amino acid sequence of SEQ ID NO: 2 or a substantially similar sequence with at least 95% sequence identity.

3. The antibody may be a Fab fragment, F(ab') ２ The antibody or antigen-binding fragment thereof of claim 1, which is a fragment, an ScFv fragment, a monoclonal antibody, a chimeric antibody, a nanobody, a humanized antibody or a human antibody.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody is multispecific.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the antibody or antigen-binding fragment thereof is linked to a second antibody or antigen-binding fragment thereof having specificity for a second epitope.

6. The antibody or antigen-binding fragment thereof of claim 1 , conjugated to a therapeutic agent.

7. The antibody or antigen-binding fragment thereof of claim 6, wherein the therapeutic agent is selected from antimetabolites, alkylating agents, alkylating agent-like agents, DNA minor groove alkylating agents, anthracyclines, antibiotics, calicheamicins, antimitotic agents, topoisomerase inhibitors, proteasome inhibitors, and radioactive isotopes.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the therapeutic agent is selected from DM1, DM3, DM4, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF).

9. The antibody or antigen-binding fragment thereof of claim 1 , wherein the antibody or antigen-binding fragment thereof is expressed on the surface of a cell.

10. The antibody or antigen-binding fragment thereof of claim 9, wherein the cell is an immune cell, a cancer stem cell, or a stem cell.

11. The antibody or antigen-binding fragment thereof of claim 10, wherein the cell is a T cell.

12. A vector encoding the antibody or antigen-binding fragment thereof of claim 1.

13. A genetically engineered cell that expresses the antibody or antigen-binding fragment thereof of claim 1 or contains the vector of claim 12.

14. 12. A method for producing an antibody or antigen-binding fragment thereof described in any one of claims 1 to 11, comprising: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions favorable for expression of the one or more polynucleotides; and (c) optionally, isolating the antibody or antigen-binding fragment from the host cell and / or the medium in which the host cell is cultured.

15. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or a genetically engineered cell according to claim 13, and a pharmaceutically acceptable carrier.

16. A container or injection device comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, or a genetically engineered cell according to claim 13.

17. A pharmaceutical composition for treating, prophylactically treating, and / or preventing a disease associated with TMCC3 signaling in an individual in need thereof, comprising an effective amount of an antibody or antigen-binding fragment thereof described in any one of claims 1 to 11 or a genetically engineered cell described in claim 13.

18. The pharmaceutical composition of claim 17, wherein the disease is cancer.

19. The pharmaceutical composition of claim 18 , wherein the cancer is a solid cancer.

20. 18. The pharmaceutical composition of claim 17, wherein the cancer is lung cancer, breast cancer, ovarian cancer, pancreatic cancer, bile duct cancer, gallbladder cancer, prostate cancer or colorectal cancer.

21. 18. The pharmaceutical composition of claim 17, wherein the pharmaceutical composition is in a form suitable for injection.

22. 22. The pharmaceutical composition of claim 21, wherein the injection is subcutaneous, intravenous, or intramuscular.

23. A method for detecting TMCC3, cancer stem cells or cancer in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof described in any one of claims 1 to 11.

24. The method of claim 23, further comprising assessing the expression level of TMCC3 in a sample using the antibody or its antigen-binding fragment, and an increase in the expression level of TMCC3 compared to the standard indicates the presence of cancer stem cells in the sample.

25. 25. The method of claim 24, wherein the cancer stem cells are cancer stem cells of a hematopoietic, epidermal, breast, ovarian, lung, pancreatic, prostate, brain, colon, bone marrow, or lymphatic cancer.

26. A kit for detecting TMCC or cancer in a sample, comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

27. 1. A method for detecting a propensity for, or predicting the likelihood, treatment response, prognosis, or recurrence of, cancer in an individual, comprising: A method comprising assessing the expression level of TMCC3 in a sample using an antibody or its antigen-binding fragment described in any one of claims 1 to 11, and an increase in the expression level of TMCC3 compared to the standard indicates the presence of cancer stem cells in the sample.

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