Specific antibodies across membrane and coiled domain family 3 and uses thereof

Abstract

The present invention relates to an antibody or antigen-binding fragment thereof specific for transmembrane and coiled domain family 3 (TMCC3). The invention also relates to a pharmaceutical composition for treating and / or preventing a disease associated with a TMCC3-mediated signal in a subject 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 an antibody or antigen-binding fragment thereof specific for transmembrane and coiled coil domain family 3 (TMCC3) and uses thereof. Background Art

[0002] Cancer is the leading cause of death worldwide and a major obstacle to improving life expectancy. Most cancer deaths are due to recurrent or metastatic disease, rather than the effects of the primary tumor. Increasing evidence suggests that cancer stem cell (CSC) populations are the primary cause of chemotherapy resistance and cancer relapse, as these cells possess the ability to self-renew and differentiate into cancer cells.

[0003] Since CSCs were first identified in leukemia in 1994, they have been shown to possess metastatic properties and resistance to chemotherapy and radiation, which can lead to tumor relapse years after clinical remission (Phi LTH, et al. Stem Cells Int. 2018, 5416923 (2018)). The plasticity of CSCs is also due to a large number of 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)). Given the functional correlation between CSCs and normal stem cells, the signaling pathways involved in the physiological functions of normal stem cells have received particular attention (such as the roles of WNT, Notch, and Hedgehog (Hh)) (Matsui, WH Medicine (Baltimore) 95, S8-S19 (2016); Cochrane, CR et al. Cancers (Basel) 7, 1554-1585 (2015)). In fact, many CSC markers are also expressed in normal stem cell populations, playing an important role 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)).

[0004] Therefore, it is very important to identify unique molecular targets expressed only in CSCs but not in normal cells, which will help develop novel CSC-targeted therapeutics. Summary of the Invention

[0005] The present invention provides antibodies specific for unique molecular markers expressed on CSCs but not on normal cells, and their use in treating, preventing, or detecting cancer.

[0006] Therefore, the present invention provides an antibody or antigen-binding fragment thereof that is specific for an antigenic determinant in 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,

[0007] wherein the CDRs of the heavy chain variable region comprise:

[0008] CDRH1 of the amino acid sequence of SEQ ID NO:3, CDRH2 of the amino acid sequence of SEQ ID NO:4, and CDRH3 of the amino acid sequence of SEQ ID NO:5; and

[0009] wherein the CDRs of the light chain variable region comprise:

[0010] CDRL1 of the amino acid sequence of SEQ ID NO:6, CDRL2 of the amino acid sequence of SEQ ID NO:7, and CDRL3 of the amino acid sequence of SEQ ID NO:8.

[0011] In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO: 1, or a substantially similar sequence having 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 having at least 95% sequence identity.

[0012] In some embodiments of the present 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.

[0013] In some embodiments of the invention, the antibody or antigen-binding fragment thereof is multispecific. In some embodiments of the invention, the antibody or antigen-binding fragment thereof is linked to a second antibody or antigen-binding fragment thereof that is specific for a second antigenic determinant.

[0014] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent. Examples of such therapeutic agents 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. In some embodiments of the present invention, the therapeutic agent is selected from DM1, DM3, DM4, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF).

[0015] 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.

[0016] The present invention provides a vector encoding an antibody or antigen-binding fragment thereof as disclosed herein.

[0017] The present invention provides a genetically engineered cell that expresses the antibody or antigen-binding fragment thereof disclosed herein or contains the vector disclosed herein.

[0018] The present invention also provides a method for producing an antibody or antigen-binding fragment thereof as disclosed herein, 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 conducive to the expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment from the host cell and / or the culture medium in which the host cell is grown.

[0019] The present invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof or genetically engineered cells disclosed herein and a pharmaceutically acceptable carrier.

[0020] The present invention provides a container or injection device comprising the antibody or antigen-binding fragment thereof or genetically engineered cells disclosed herein.

[0021] The present invention provides a method for treating or preventing a disease associated with TMCC3-mediated signaling in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or genetically engineered cells as disclosed herein. Alternatively, the present invention provides a pharmaceutical composition for treating or preventing a disease associated with TMCC3-mediated signaling in a subject in need thereof, comprising a therapeutically effective amount of an antibody or antigen-binding fragment thereof, or genetically engineered cells as used herein, and a pharmaceutically acceptable carrier. The present invention further provides a method for treating, prophylactically treating, and / or preventing cancer in a subject suffering from cancer, comprising administering a pharmaceutical composition to the subject. 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, or colorectal cancer. Alternatively, the present invention provides a pharmaceutical composition for treating, prophylactically treating, and / or preventing cancer in a subject suffering from cancer, comprising an effective amount of an antibody or antigen-binding fragment thereof, or genetically engineered cells as disclosed herein.

[0022] In some embodiments of the present invention, the pharmaceutical composition is in a form suitable for injection. Alternatively, the present invention provides a method for administering an antibody or antigen-binding fragment thereof as disclosed herein to a subject, comprising injecting the antibody or antigen-binding fragment into the subject.

[0023] In some embodiments of the invention, the injection is subcutaneous, intravenous or intramuscular. Alternatively, the antibody or antigen-binding fragment is injected into the subject subcutaneously, intravenously or intramuscularly.

[0024] The present invention provides 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 as disclosed herein.

[0025] In some embodiments of the present invention, the method further comprises evaluating the expression level of TMCC3 in the sample, wherein an increase in the expression level of TMCC3 compared to a standard indicates the presence of cancer stem cells in the sample.

[0026] Examples of such cancer stem cells include, but are not limited to, hematopoietic, epidermal, breast, ovarian, lung, pancreatic, prostate, brain, colon, bone marrow, or lymphoid cancer stem cells.

[0027] The present invention provides a kit for detecting TMCC3 or cancer in a sample, wherein the kit comprises the antibody or antigen-binding fragment thereof disclosed herein.

[0028] The present invention provides a method for detecting cancer predisposition or predicting the likelihood, treatment response, prognosis or recurrence of cancer in an individual, comprising

[0029] The expression level of TMCC3 in a sample is assessed using the antibody or antigen-binding fragment thereof as disclosed herein, wherein an increase in the expression level of TMCC3 compared to a standard indicates the presence of cancer stem cells in the sample.

[0030] The present invention is described in detail in the following sections. Other features, objectives and advantages of the present invention can be found in the embodiments and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 The expression level of TMCC3 mRNA in tumor tissues of breast cancer patients is shown. High expression of TMCC3 is associated with poor clinical outcomes in breast cancer patients (including early-stage patients).

[0032] Figure 2 Higher TMCC3 expression in tumors compared to normal tissues was shown to contribute to cancer progression.

[0033] Figure 3 The binding affinity of anti-TMCC3 mAbs analyzed by ELISA is shown.

[0034] Figure 4 Showing the binding activity of anti-TMCC3 mAb in TMCC3-overexpressing MCF7 cells.

[0035] Figure 5 Demonstrating TMCC3 expression in breast, pancreatic, and ovarian cancer PDX tumors.

[0036] Figure 6A Shown are FACS analyses using mAb 8D6 showing TMCC3 expression on the surface of serous (OC042) and clear cell (OC085) ovarian cancer PDXs. Figure 6B IHC analysis showed that TMCC3 expression was also detected in these PDXs.

[0037] 7A to 7C The expression of TMCC3 in primary ovarian cancer specimens is shown. Figure 7A The results of IHC analysis are shown. Figure 7B Expression levels are shown in the following three ovarian cancer subtypes: mucinous, clear cell, and endometrioid types. Figure 7C The expression levels in the early stage (stage I-II) and late stage (stage III-IV) are shown.

[0038] Figures 8A to 8J Showing the survival curve of ovarian cancer patients associated with TMCC3 expression levels.

[0039] 9A to 9D Shown are the enrichment of breast and ovarian cancer stem cells in PDXs using TMCC3 antibodies, as assessed by in vitro spheroid formation (A, C) or in vivo tumor growth (B, D).

[0040] Figures 10A to 10E The tumor inhibitory ability of ADC-8D6 combined with MMAF in vitro and in vivo was shown.

[0041] Figure 11 Shown are the results of reduced mass chromatography analysis of trimannosyl-anti-TMCC3 8D6 mAb.

[0042] FIG. 12A to FIG. 12B Shown are the results of reduced mass chromatography analysis of anti-TMCC3 8D6 mAb-4Az.

[0043] Figure 13 Shown are the results of reduced mass chromatography analysis of anti-TMCC3 8D6 mAb-4 (DBCO-vc-MMAE).

[0044] Figure 14 Shown are the clinical information of ovarian cancer patient OC085 and TMCC3 expression in her tumor.

[0045] Figures 15A to 15G The in vivo anticancer activity of ADC-trimannosyl-MMAE (8D6) against OC085PDX was shown.

[0046] 16A to 16B In vivo anticancer activity of trimannosyl ADC (8mpk) against OC085 PDX was demonstrated.

[0047] 17A to 17B In vivo anticancer activity of chemotherapy and ADC-8D6 (8 mg / kg / dose) against OC085 PDX was demonstrated.

[0048] 18A to 18B Results showed that 50 mpk of niraparib failed to inhibit tumor growth of OC085PDX in vivo. DETAILED DESCRIPTION

[0049] It should be understood that the present invention is not limited to the specific materials and methods described herein. It should also be understood that the terminology used herein is only for the purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

[0050] It must be noted that, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0051] As used herein, the term "antibody" refers to any antigen-binding molecule or molecular complex comprising at least one complementary determining region (CDR) that is specific for or interacts with a particular antigen (TMCC3). The term "antibody" includes immunoglobulin molecules and multimers thereof (e.g., IgM), which comprise four polypeptide chains, namely, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as HCVR or V H ) and the heavy chain constant region. The heavy chain constant region contains three domains C H1 、C H2 and C H3 Each light chain comprises a light chain variable region (abbreviated herein as LCVR or V L ) and the light chain constant region. The light chain constant region contains a domain (C L1 ). V H and V L The V domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V L It is composed of three CDRs and four FRs arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In various embodiments of the present invention, the FRs of anti-SARS-CoV-2 spike protein antibodies (or antigen-binding portions thereof) may be identical to human germline sequences or may be naturally occurring or artificially modified. The amino acid consensus sequence can be defined based on a side-by-side analysis of two or more CDRs.

[0052] 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 obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex.

[0053] As used herein, the term "specific for" or "specifically binds" means that the antibody does not cross-react to a significant extent with other antigenic determinants.

[0054] As used herein, the term "antigenic determinant" refers to the site where an antibody binds to an antigen.

[0055] As used herein, the term "complementarity determining region" (CDR) refers to the non-contiguous antigen combining sites found within the variable regions of heavy and light chain polypeptides. Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabat et al., US Pat. 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) have described CDRs, where the definition includes overlap or subsets of amino acid residues when compared against each other.

[0056] When applied to polypeptides, the term "substantial similarity" or "substantially similar" means that two peptide sequences, when optimally aligned using preset gap weights, such as by the programs GAP or BESTFIT, share at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. Differences in the positions of inconsistent residues are preferably conservative amino acid substitutions. A "conservative amino acid substitution" is an amino acid substitution in which an amino acid residue is replaced by another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity) through a side chain (R group). In general, conservative amino acid substitutions do not substantially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the sequence identity or similarity percentages may be adjusted upward to correct for the nature of the conservative substitution. The manner in which this adjustment is made is well known to those skilled in the art. Examples of amino acid groups with chemically similar side chains 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. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. 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, incorporated herein by reference. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

[0057] As used herein, the term "monoclonal antibody" is not limited to antibodies produced via hybridoma technology. A monoclonal antibody is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, by any means available or known in the art.

[0058] As used herein, the term "chimeric" antibody refers to antibodies having variable sequences derived from a non-human immunoglobulin and a human immunoglobulin constant region, typically selected from a human immunoglobulin template.

[0059] As used herein, the term "nanobody" refers to antibodies comprising small single variable domains (VHHs of antibodies obtained from camels and dromedaries). Antibody proteins obtained from camel and dromedary (Camelus bactrianus and Calelus dromaderius) family members (including New World members, such as llama species (Lama paccos, Lama glama and Lama vicugna)) have been characterized in terms of size, structural complexity and antigenicity to human individuals. Certain IgG antibodies from this mammalian family as found in nature lack light chains and are therefore structurally different from the typical four-chain quaternary structure with two heavy chains and two light chains of other animal antibodies.

[0060] "Humanized" forms of non-human antibodies are chimeric immunoglobulins that contain minimal sequence derived from non-human immunoglobulin. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence.

[0061] As used herein, the term "nanobody" refers to antibodies comprising small single variable domains (VHHs of antibodies obtained from camels and dromedaries). Antibody proteins obtained from camel and dromedary (Bactrian and dromedary) family members (including New World members, such as llama species (alpaca, llama and llama)) have been characterized in terms of size, structural complexity and antigenicity to human individuals. Certain IgG antibodies from this mammalian family as found in nature lack light chains and are therefore structurally different from the typical four-chain quaternary structure with two heavy chains and two light chains of other animal antibodies.

[0062] As used herein, the term "therapeutic agent" refers to any compound, substance, drug, medicament, or active ingredient having a therapeutic or pharmacological effect that is suitable for administration to a mammal, such as a human.

[0063] As used herein, the term "immune cell" refers to a cell that plays a role in an immune response. Immune cells are of hematopoietic origin and include lymphocytes, such as B cells and T cells; natural killer cells; and myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.

[0064] As used herein, the term "T cells" includes CD4 + T cells and CD8 + T cells. The term T cell also includes T helper type 1 T cells, T helper type 2 T cells, T helper type 17 T cells, and suppressor T cells.

[0065] As used herein, the term "stem cell" refers to a cell in an undifferentiated or partially differentiated state, which has self-renewal properties and has the developmental potential of naturally differentiating into more differentiated cell types, without specific implicit meaning (that is, full potential, rich potential, multipotency, etc.) about developmental potential. Self-renewal means that stem cells can proliferate and produce more of these stem cells while maintaining their developmental potential. Therefore, the term "stem cell" refers to the developmental potential of being differentiated into more specific or differentiated phenotypes in certain circumstances, and retains any cell subpopulation of the ability to propagate and not actually differentiate in some cases.

[0066] 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 protein, or therapeutic agent at any position along the molecule, as long as it is capable of binding to its target (TMCC3). Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment of the present invention, the agent may be a second, different antibody that specifically binds to TMCC3. The type of therapeutic moiety that can be bound to the anti-TMCC3 protein (e.g., antibody or fragment) will take into account the condition to be treated and the desired therapeutic effect to be achieved.

[0067] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0068] The term "genetically engineered" or "genetic engineering" refers to the manipulation of genes using genetic material to alter gene replication and / or gene expression in a cell. Genetic material can be in the form of DNA or RNA. Genetic material can be transferred into cells by various means, including viral transduction and non-viral transfection. Following genetic engineering, the expression of certain genes in a cell can be permanently or temporarily altered.

[0069] As used herein, the term "pharmaceutical composition" refers to a mixture containing therapeutic agents that is administered to a mammal (eg, a human) to prevent, treat, or eliminate a specific disease or pathological condition suffered by the mammal.

[0070] As used herein, the term "therapeutically effective amount" or "effective amount" refers 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.

[0071] As used herein, the terms "treatment" and "treating" and the like encompass any treatment of a disease in a mammal, particularly a human, and include: (a) preventing the development of a disease in an individual who may be susceptible to the disease but has not yet been diagnosed with the disease; (b) inhibiting the disease, that is, arresting its development; and (c) ameliorating the disease, that is, causing regression of the disease.

[0072] The terms "preventing" and "prevention" are recognized in the art and, when used in conjunction with a condition, include administering an agent to reduce the incidence or severity of a medical condition in a subject prior to the onset of the condition or to delay the onset of its symptoms relative to a subject who has not received the agent.

[0073] As used interchangeably herein, the terms "individual," "subject," "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, goats), etc. Specifically, the individual is vaccinated.

[0074] As used herein, the term "in need of treatment" refers to a judgment made by a caregiver (e.g., a physician, nurse, care practitioner, or individual in the case of humans; a veterinarian in the case of animals (including non-human mammals)) that an individual requires or will benefit from treatment. This judgment is based on a variety of factors within the caregiver's area of ​​expertise, including knowledge that the individual is or will become ill due to a condition that can be treated by the compounds of the present invention.

[0075] "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 tumors (abnormal growth). "Transformation" refers to a spontaneous or induced phenotypic change, e.g., immortalization of cells, morphological changes, abnormal cell growth, reduced contact inhibition and fixation, and / or malignancy (see Freshney, Culture of Animal Cells a Manual of Basic Technique (3rd ed. 1994)). Although transformation can result from infection with a transforming virus and the incorporation of new genomic DNA or the uptake of exogenous DNA, it can also occur spontaneously or following exposure to a carcinogen.

[0076] As used herein, the term "cancer stem cell" refers to cells that are capable of self-renewal and differentiation into cancer cell lineages comprising solid tumors and / or hematological malignancies. Cancer stem cells are uniquely capable of initiating and maintaining disease. Variant neoplastic stem cells are highly potent cells that exhibit properties of cancer stem cells.

[0077] As used herein, the term "sample" encompasses a variety of sample types obtained from an individual, subject, or patient and that can be used for diagnostic or monitoring analysis. This definition encompasses blood and other liquid samples of biological origin; solid tissue samples, such as biopsies or tissue cultures or cells derived therefrom, and their progeny.

[0078] The present invention develops an antibody or an antigen-binding fragment thereof that is specific to an antigenic determinant in TMCC3.

[0079] In particular, the antibody or antigen-binding fragment thereof is specific for an antigenic determinant in TMCC3; wherein the antibody or antigen-binding fragment thereof comprises CDRs of a heavy chain variable region and CDRs of a light chain variable region, wherein the CDRs of the heavy chain variable region comprise CDRH1, CDRH2, and CDRH3 regions, and the CDRs of the light chain variable region comprise CDRL1, CDRL2, and CDRL3 regions, and wherein:

[0080] The CDRH1 region comprises the amino acid sequence of SEQ ID NO:3, or a substantially similar sequence having 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 having 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 having 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 having at least 90%, at least 95%, at least 98%, or at least 99% sequence identity; the CDRL2 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; 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. The amino acid sequence of NO: 8 or a substantially similar sequence having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0081] In some embodiments, 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 thereof; and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 2 or a substantially similar sequence thereof. In some other embodiments, 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity. In some other embodiments, 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 thereof having at least 90%, at least 95%, at least 98% or at least 99% sequence identity.

[0082] The sequences of the present invention are shown in Table 1.

[0083] Table 1

[0084]

[0085] In some embodiments of the present invention, it is noted that phosphorylated TMCC3 at serine 216 is higher in bCSCs than in non-bCSCs. TMCC3 belongs to the TMCC family that includes TMCC1-3 and contains two coiled domains in the N-terminal region and two transmembrane domains in the C-terminal region. In some embodiments of the present invention, TMCC3 expression is strongest in metastatic lesions of the lungs, followed by metastatic lymph nodes, and then primary tumors of breast cancer. 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 embodiments of the present invention, higher TMCC3 mRNA levels were observed in metastatic lymph nodes than in primary tumors of ovarian cancer. TMCC3 attenuation reduces sphere formation ability in vitro ( Figure 3 B) and inhibited ovarian cancer tumor growth in vivo. TMCC3 overexpression increased ALDH activity in ovarian cancer cells.

[0086] Antibodies according to the invention may be full length (eg, IgG1 or IgG4 antibodies) or may comprise only an antigen binding portion (eg, Fab, F(ab')2, or scFv fragments), and may be modified as desired to affect functionality.

[0087] Antibodies also include antigen-binding fragments of complete antibody molecules.Antigen-binding fragments of antibodies can use any suitable standard techniques, such as proteolytic digestion or the manipulation and expression of DNA encoding antibody variable domains and optionally constant domains, for example, derived from complete antibody molecules.Such DNA is known and / or can be easily obtained from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized.DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, one or more variable domains and / or constant domains are arranged into a suitable configuration, or codons are introduced to produce cysteine ​​residues, modify, add or delete amino acids, etc.

[0088] 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) the smallest recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR), such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. 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, minibodies, nanobodies (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed by the expression "antigen-binding fragment" as used herein.

[0089] The antigen-binding fragment of an antibody typically comprises at least one variable domain. The variable domain can be of any size or amino acid composition and will generally comprise at least one CDR adjacent to or in frame with one or more framework sequences. L Domain associated V H In the antigen-binding fragment of the domain, V H and V L The domains can be positioned relative to each other in any suitable arrangement. For example, the variable region can be a dimer and contain V H -V H 、V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may contain a monomer V H or V L domain.

[0090] In certain embodiments, the antigen-binding fragment of an antibody may contain at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within the antigen-binding fragment of an antibody of the invention include: (i) 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 -C L . In any configuration of variable and constant domains, including any of the exemplary configurations listed herein, the variable and constant domains may be directly connected to each other or may be connected by a complete or partial hinge region or connecting region. The hinge region may be composed of at least 2 (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that create a flexible or semi-flexible bond between adjacent variable and / or constant domains in a single polypeptide molecule. In addition, the antigen-binding fragments of the antibodies of the present invention may comprise any of the variable and constant domain configurations listed above, linked to each other and / or to one or more monomeric V H or V L A homodimer or heterodimer (or other multimer) in which the domains are non-covalently associated (e.g., through disulfide bonds).

[0091] The anti-TMCC3 antibodies disclosed herein may comprise one or more amino acid substitutions, insertions and / or deletions in the framework and / or CDR regions of the heavy and light chain variable domains compared to the corresponding germline sequences from which the antibodies were derived. Such mutations can be easily determined by comparing the amino acid sequences disclosed herein with germline sequences purchased from, for example, public antibody sequence databases. The present invention includes an antibody and an antigen-binding fragment thereof, which are derived from any of the amino acid sequences disclosed herein, wherein one or more amino acids within one or more framework and / or CDR regions are mutated to the corresponding residues of the germline sequence from which the antibody was derived, or the corresponding residues of another mammalian germline sequence, or conservative amino acid substitutions of the corresponding germline residues (such sequence changes are collectively referred to herein as "germline mutations"). A person skilled in the art can easily prepare a number of antibodies and antigen-binding fragments comprising one or more individual germline mutations or combinations thereof using the heavy and light chain variable region sequences disclosed herein as starting materials. In certain embodiments, V H and / or V LIn some embodiments, all framework and / or CDR residues in the domain are all mutated back to the residues found in the original germline sequence from which the antibody was derived. In other embodiments, only certain residues are mutated back to the original germline sequence, such as only the mutated residues found within the first 8 amino acids of FR1 or the last 8 amino acids of FR4, or only the mutated residues found within CDR1, CDR2 or CDR3. In other embodiments, one or more of the framework and / or CDR residues are mutated into corresponding residues of different germline sequences (i.e., germline sequences different from the germline sequence from which the antibody was initially derived). In addition, the antibodies of the present invention may contain any combination of two or more germline mutations in the framework and / or CDR regions, such as where certain individual residues are mutated into corresponding residues of a specific germline sequence, while certain other residues different from the original germline sequence remain unchanged or mutate into corresponding residues of different germline sequences. 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.

[0092] The present invention also includes an anti-TMCC3 antibody comprising the V disclosed herein. H 、V L and / or CDR amino acid sequence variants with one or more conservative substitutions. For example, the present invention includes an anti-TMCC3 antibody having V H 、V L and / or CDR amino acid sequences relative to the V and / or CDR amino acid sequences disclosed herein. H 、V L and / or any of the CDR amino acid sequences have, for example, 10 or fewer, 8 or fewer, 6 or fewer, 4 or fewer, etc., conservative amino acid substitutions.

[0093] In some embodiments of the present invention, the antibodies according to the present invention are humanized antibodies. To improve the binding affinity of the humanized antibodies according to the present invention, some amino acid residues in the human framework regions are replaced with corresponding amino acid residues in species (e.g., rodents) of the CDRs.

[0094] The antibodies of the present invention may be monospecific, bispecific or multispecific. A multispecific antibody may be specific for different antigenic determinants of a target polypeptide or may contain antigen binding domains that are specific for more than one target polypeptide. In some embodiments of the present invention, the antibody or its antigen binding fragment is multispecific. In some embodiments of the present invention, the antibody or its antigen binding fragment is linked to a second antibody or its antigen binding fragment that is specific for a second antigenic determinant. The anti-TMCC3 antibodies of the present invention may be linked or co-expressed with another functional molecule, such as another peptide or protein. For example, the antibody or its fragment may be functionally 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 produce a bispecific or multispecific antibody with a second binding specificity. For example, the present invention includes bispecific antibodies in which one arm of the immunoglobulin is specific for TMCC3 or a fragment thereof, and the other arm of the immunoglobulin is specific for a second therapeutic target or is bound to a therapeutic moiety.

[0095] In some embodiments of the present invention, the antibody or antigen-binding fragment thereof is conjugated to a therapeutic agent. Examples of such therapeutic agents 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. In some embodiments of the present invention, the therapeutic agent is selected from DM1, DM3, DM4, monomethyl auristatin E (MMAE), and monomethyl auristatin F (MMAF).

[0096] In one embodiment of the present invention, the antibody or antigen-binding fragment thereof is expressed on the surface of a cell, particularly an immune cell (such as a T cell), a cancer stem cell, or a stem cell.

[0097] In some embodiments of the invention, the antibody or antigen-binding fragment thereof is in the form of a chimeric antigen receptor.

[0098] The term "chimeric antigen receptor" or alternatively "CAR" refers to a recombinant polypeptide construct comprising at least an extracellular antigen binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling domain") comprising a functional signaling domain derived from a stimulatory molecule as defined below. In some embodiments, the domains in the CAR polypeptide construct are in the same polypeptide chain, for example, constituting a chimeric fusion protein. In some embodiments, the domains in the CAR polypeptide construct are not adjacent to each other, for example, in different polypeptide chains. The generation and construction of CAR are summarized by: Jayarama et al., EBioMedicine58(2020)102931; Zhang et al., Biomarker Research(2017)5:22; Feins et al., Am JHematol.(2019)94:S3-S9; and Roselli et al., J Clin Invest.2021;131(2):e142030.

[0099] In another aspect, the present invention provides a genetically engineered cell that expresses an antibody or antigen-binding fragment thereof or contains a vector. The genetically engineered cell may be an immune cell.

[0100] In a preferred embodiment of the present invention, the antibody or antigen-binding fragment thereof can 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 (such as a hybridoma) or a CHO cell expression system. Many such systems are widely available from commercial suppliers. H and V L In the embodiment of the region, V H and V L The regions can be expressed using a single vector, for example as a bicistronic expression unit, or under the control of different promoters. H and V L The V region can be expressed using a separate vector. H or V L The region may optionally include a methionine at the N-terminus.

[0101] The heavy chain of the antibody of coding attention and the gene of light chain can be cloned from cell, and the gene of such as coding monoclonal antibody can be cloned from fusion tumor and be used to produce recombinant monoclonal antibody.The heavy chain of coding monoclonal antibody and the gene bank of light chain also can be made by fusion tumor or plasma cell.The random combination of heavy chain and light chain gene product produces the large antibody pool with different antigen specificity (referring to such as Kuby, Immunology (3rd [supplement] edition 1997)).

[0102] Examples of methods for producing an antibody or antigen-binding fragment include: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions conducive to expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment from the host cell and / or the culture medium in which the host cell is grown.

[0103] Vectors can be used to introduce polynucleotides encoding the antibodies or antigen-binding fragments of the present invention into host cells. In one embodiment, one type of vector is a "plastid," which refers to a circular double-stranded DNA loop that can be joined to an additional DNA segment. Another type of vector is a viral vector, in which the additional DNA segment can be joined to a viral genome. Certain vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and replicate together with the host genome through this. In addition, certain vectors can cause the expression of genes to which they are operably connected. Such vectors are referred to herein as "recombinant expression vectors" (or, in short, "expression vectors"). Generally speaking, expression vectors used in recombinant DNA technology are often in the form of plastids. Since plastids are the most commonly used form of vectors, in this specification, "plastids" and "vectors" are used interchangeably. However, the present invention is intended to include expression vectors of these other forms that perform equivalent functions, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses).

[0104] The present invention provides pharmaceutical compositions comprising antibodies or antigen-binding fragments thereof or genetically engineered cells. The pharmaceutical compositions of the present invention are formulated with suitable diluents, carriers, excipients, and other agents that provide improved transfer, delivery, tolerability, and similar properties. These compositions can be formulated for specific uses, such as veterinary or human pharmaceutical applications. The form of the composition and excipients, diluents, and / or carriers used will depend on the intended use of the antibody and the mode of administration for therapeutic use. Numerous suitable formulations can be found in the formulary known to all medicinal chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa. Such formulations include, for example, powders, pastes, ointments, gels, waxes, oils, lipids, lipid-containing vesicles (cationic or anionic) (such as LIPOFECTIN.™, Life Technologies, Carlsbad, Calif.), DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, polyethylene glycol emulsions (with polyethylene glycol of various molecular weights), semisolid gels, and semisolid mixtures containing polyethylene glycol. See also Powell et al. "Compendium of excipients for parenteral formulations," PDA (1998) J Pharm Sci Technol 52: 238-311.

[0105] The dosage of the antibody administered to the patient may vary depending on the patient's age and size, target disease, condition, route of administration, and the like. The preferred dosage is typically calculated based on body weight or body surface area. When the antibodies of the present invention are used to treat conditions or diseases associated with EPHA10 in adult patients, intravenous administration of the antibodies of the present invention may be advantageous. Depending on the severity of the condition, the frequency and duration of treatment may be adjusted. The effective dosage and time course for administering the antibody can be determined empirically; for example, patient progress can be monitored by periodic assessments, and the dosage adjusted accordingly. In addition, interspecies ratio adjustments of dosages may be performed using well-known methods in the art (e.g., Mordenti et al., 1991, Pharmaceut. Res. 8: 1351).

[0106] Various delivery systems are known and can be used to administer the pharmaceutical compositions of the present invention, such as encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing mutant viruses, and receptor-mediated endocytosis (see, for example, 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, such as by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal mucosa, intestinal mucosa, etc.), and can be administered with other biologically active agents. Administration can be systemic or local.

[0107] The pharmaceutical compositions of the present invention can be delivered subcutaneously or intravenously using a container or injection device such as a standard needle and syringe. In addition, with respect to subcutaneous delivery, pen-type delivery devices are readily applicable for delivering the pharmaceutical compositions of the present invention. Such pen-type delivery devices can be reusable or disposable. Reusable pen-type delivery devices typically utilize a replaceable cartridge containing the pharmaceutical composition. After all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can be easily discarded and replaced with a new cartridge containing the pharmaceutical composition. The pen-type delivery device can then be reused. In disposable pen-type delivery devices, there is no replaceable cartridge. In fact, disposable pen-type delivery devices are pre-filled with the pharmaceutical composition contained in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0108] 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). In another embodiment, a polymeric material can be used; see Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press., Boca Raton, Fla. In yet another embodiment, a controlled release system can be placed close to the target of the composition, thus 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.

[0109] Injectable preparations may include dosage forms for intravenous, subcutaneous, intradermal, and intramuscular injections, drips, and the like. Such injectable preparations may be prepared by publicly known methods. For example, injectable preparations may be prepared by dissolving, suspending, or emulsifying the antibodies described herein or their salts in a conventional sterile aqueous medium or oily medium for injection. Aqueous media for injection include, for example, physiological saline, isotonic solutions containing glucose and other adjuvants, and the like, which may 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 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)]. For example, sesame oil, soybean oil, and the like are used as oily media, which may be used in combination with solubilizers (e.g., benzyl benzoate, benzyl alcohol, and the like). The injection thus prepared is preferably filled into an appropriate ampoule.

[0110] The pharmaceutical compositions for oral or parenteral use described above are preferably prepared into unit dosage forms suitable for the dosage of the active ingredient. Such unit dosage forms include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc.

[0111] The present invention provides a method for treating or preventing a disease associated with TMCC3-mediated signaling in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, or genetically engineered cell as disclosed herein. Alternatively, the present invention provides a pharmaceutical composition for treating or preventing a disease associated with TMCC3-mediated signaling in a subject in need thereof, comprising a therapeutically effective amount of an antibody, or antigen-binding fragment thereof, or genetically engineered cell as used herein and a pharmaceutically acceptable carrier.

[0112] The present invention further provides a method for treating, prophylactically treating, and / or preventing cancer in an individual suffering from cancer, 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, or colorectal cancer. Alternatively, the present invention provides a pharmaceutical composition for treating, prophylactically treating, and / or preventing cancer in an individual suffering from cancer, comprising an effective amount of an antibody or antigen-binding fragment thereof, or a genetically engineered cell as disclosed herein.

[0113] The present invention provides 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 as disclosed herein.

[0114] In some embodiments of the present invention, the method further comprises evaluating the expression level of TMCC3 in the sample, wherein an increase in the expression level of TMCC3 compared to a standard indicates the presence of cancer stem cells in the sample.

[0115] Examples of such cancer stem cells include, but are not limited to, hematopoietic, epidermal, breast, ovarian, lung, pancreatic, prostate, brain, colon, bone marrow, or lymphoid cancer stem cells.

[0116] The present invention provides a kit for detecting TMCC3 or cancer in a sample, wherein the kit comprises the antibody or antigen-binding fragment thereof disclosed herein.

[0117] The anti-TMCC3 antibodies of the present invention can also be used, for example, to detect and / or measure cancer or cells expressing TMCC3 in a sample for diagnostic purposes. For example, anti-TMCC3 antibodies or fragments thereof can be used to diagnose a condition or disease characterized by a coronavirus infection. An exemplary diagnostic assay for a coronavirus can comprise, for example, contacting a sample obtained from a patient with an anti-TMCC3 antibody of the present invention, wherein the anti-TMCC3 antibody is labeled with a detectable label or reporter molecule. Alternatively, an unlabeled anti-TMCC3 antibody can be used in diagnostic applications in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be a radioactive isotope, such as 3 H. 14 C. 32 P. 35 S or 125 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 coronaviruses in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0118] The present invention provides a method for detecting cancer predisposition or predicting the likelihood, treatment response, prognosis or recurrence of cancer in an individual, comprising

[0119] The expression level of TMCC3 in a sample is assessed using the antibody or antigen-binding fragment thereof as disclosed herein, wherein an increase in the expression level of TMCC3 compared to a standard indicates the presence of cancer stem cells in the sample.

[0120] The following examples are provided to help those skilled in the art to practice the present invention.

[0121] Example 1 Clinical relevance of TMCC3 mRNA expression in cancer tissues

[0122] To evaluate the clinical relevance of TMCC3 expression, we examined the mRNA content of TMCC3 in tumor specimens from 202 breast cancer patients by qRT-PCR. Kaplan-Meier analysis and log-rank test showed that patients with low TMCC3 expression in tumors had significantly longer recurrence-free survival (RFS) and overall survival (OS) than patients with high expression. This was also true for 161 early-stage breast cancer patients. Multivariate analysis indicated that TMCC3 expression in tumor tissue was an important and independent predictor of RFS and OS in breast cancer patients ( Figure 1 Using the online RNA microarray database of the ONCOMINE website, we found that the mRNA content of TMCC3 in cancerous tissues was higher than that in normal tissues in cervical cancer, prostate cancer, pancreatic cancer, lung cancer, glioblastoma, skin cancer, liver cancer, and papillary thyroid cancer. Using the Kaplan-Meier Plotter website to evaluate the clinical significance of TMCC3 in cancer, we found that higher expression of TMCC3 was associated with worse overall survival in patients with ovarian cancer, lung cancer, and gastric cancer ( Figure 2 These findings further support the critical role of TMCC3 in ovarian cancer progression and support the development of strategies targeting TMCC3 for cancer therapy.

[0123] Example 2 Production of anti-TMCC3 monoclonal antibodies

[0124] Our efforts have supported the view that TMCC3 is a potential therapeutic diagnostic target for clinical prognosis and CSC eradication. To generate suitable mAbs to further investigate this novel protein, mice were immunized with the extracellular domain of TMCC3. The binding activity and specificity of the anti-TMCC3 mAbs were verified using ELISA and FACS analysis. The helix 1 and helix 2 domains of the TMCC3 protein were generated for ELISA analysis. We found that clone BA5 recognized the helix 1 domain, while clones 8D6 and 5G1 recognized the helix 2 domain of the TMCC3 protein. D The values ​​are 1x10 -11 M and <1x10 -11 M( Figure 3 In addition, fresh and formalin-fixed TMCC3-overexpressing MCF7 cells were prepared for FACS analysis to verify the binding activity of mAbs to cell surface or intracellular TMCC3. Among the many mAb clones generated, clone 8D6 had the highest binding affinity to surface and intracellular TMCC3 in TMCC3-overexpressing MCF7 cells (4.36% and 63.1%, respectively) compared to other clones ( Figure 4 ).

[0125] Example 3 Sequence of mouse anti-TMCC3 8D6 monoclonal antibody

[0126] A method for obtaining mouse anti-TMCC3 antibodies is as follows. First, generate anti-TMCC3 hybridomas. Such hybridomas can be generated by standard protocols for generating monoclonal antibodies. Then, for example, TRIZOL is used to generate the antibody. Then, for example, a first strand cDNA synthesis kit (Superscript III) and oligo (dT 20 cDNA was synthesized from total RNA using primers containing either IgG1 or Ig-3' constant region.

[0127] The heavy and light chain variable regions of the immunoglobulin genes were then cloned from the cDNA. For example, the VH and VL variable regions of the anti-TMCC3 mAb were amplified from the mouse TMCC3 fusion tumor cDNA by PCR using the mouse Ig-5' primer set. The PCR products were cloned using CLONEJET TM The PCR cloning kit is directly cloned into a suitable vector (e.g., pJET1.2 vector). The pJET1.2 vector contains a lethal insertion, and only when the desired gene is cloned into this lethal region can it survive under the selection conditions. This helps in the selection of recombinant colonies. Finally, the recombinant colonies are screened for the desired pure lines, and their DNA is isolated and sequenced. Immunoglobulin (IG) nucleotide sequences can be analyzed on the International ImMunoGeneTics Information System (IGMT) website.

[0128] Example 4 FACS and IHC analysis of TMCC3 protein expression in PDX tumors

[0129] TMCC3 protein expression in breast cancer (BC0145, BC0350R1, and BC0634), pancreatic cancer (PC001, PC025, and PC038), and ovarian cancer (OC042 and OC057) PDXs was detected using mAb 8D6 ( Figure 5 FACS analysis using mAb8D6 showed 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 ).

[0130] Example 5 TMCC3 expression in primary ovarian cancer specimens

[0131] To address 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. Our 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 express lower levels of TMCC3 protein ( Figure 7B In TMCC3-positive specimens, TMCC3 was detected mainly in the cytoplasm and membrane of tumor cells ( Figure 7A In particular, clear cell carcinoma showed higher TMCC3 expression compared with other subtypes ( Figure 7A ).like Figure 7C As shown in , the expression of TMCC3 was found to be higher in early-stage (stage I-II) ovarian cancer tissues than in late-stage (stage III-IV) ovarian cancer tissues (P<0.05).

[0132] Example 6 High expression of TMCC3 is associated with poor clinical outcomes in ovarian cancer

[0133] We investigated whether high expression of TMCC3 is an important predictor of prognosis in ovarian cancer. Our results showed that patients with low expression of TMCC3 in their tumors had significantly greater OS (P < 0.0001) and RFS (P < 0.0001) compared with patients with high expression ( Figure 8A and Figure 8B We then analyzed the potential prognostic value of TMCC3 expression in patients with different subtypes. Figure 8H and Figure 8J As shown in the , patients with endometrial or mucinous subtypes whose tumors expressed low amounts of TMCC3 had greater RFS than those with high amounts (P = 0.1336 and P = 0.01, respectively). For patients with clear cell carcinoma, the survival benefit of patients with lower TMCC3 expression was even more striking (OS and RFS, P < 0.0001 and P < 0.0001, respectively) ( Figure 8E and Figure 8F These results demonstrate that high expression of TMCC3 has an adverse effect on the clinical outcome of ovarian cancer, particularly in the case of clear cell carcinoma.

[0134] Example 7 CSC Enrichment of Breast and Ovarian Cancer PDX Using Anti-TMCC3 mAb

[0135] like Figure 9A As shown in Figure 5, mAb8D6 can bind to the BCSC population (CD44 + 、H2k d-, 7AAD - ) and enriched BCSC populations from BC0145 PDX tumors, as determined by 8D6 - BCSC compared to 8D6 + This is reflected by the greater in vitro mammosphere-forming capacity of BCSCs. + / TMCC3 - / H2k d- / 7AAD - Compared with cells, CD44 + / TMCC3 + / H2k d- / 7AAD - cells produce faster-growing and larger tumors ( Figure 9B ). Similarly, mAb8D6 can enrich CSC populations from ovarian cancer PDX tumors OC057 and OC085. Figure 9C and Figure 9D As shown in 8D6 + Cells have 8D6 than OC057 and OC085 PDX - These findings suggest that mAb8D6 can enrich breast and ovarian CSCs from PDX tumors.

[0136] Example 8 Anticancer Efficacy of Prototype Anti-TMCC3 Antibody-Drug Conjugate (ADC)

[0137] In vitro, mAb 8D6 was found to be internalized into TMCC3 after binding to surface TMCC3. + In cells ( Figure 10A This finding suggests that mAb 8D6 may be suitable for designing antibodies targeting TMCC3. + Thus, chimeric mAb 8D6 was generated and combined with the anti-tubulin agent MMAF at a drug to antibody ratio (DAR) of 3.8 ( Figure 10B ADC-8D6-bound MMAF was activated in vitro at 10 μM (IC 50 =102.6 nM) caused 60% cell death in BC0145 PDX tumor cells ( Figure 10C We have evaluated the anti-tumor efficacy of this first generation ADC-8D6 in two breast cancer PDXs. 3 ADC-8D6 (4 mg / kg) conjugated to MMAF was administered to BC0145 or BC0634 PDX tumor-bearing mice twice weekly starting at 4 hr. Figure 10D and Figure 10EAs shown in Figure 2, ADC-8D6 significantly inhibited tumor growth compared to mice treated with human IgG (hIgG) (P < 0.0001). In addition, treatment with ADC-8D6 reduced metastasis to lymph nodes and vital organs compared to the hIgG control group (Table 2). These findings show that ADC-8D6 combined with MMAF can inhibit tumor growth and metastasis in vivo in BC0145 and BC0634 breast cancer PDX. However, DAR is heterogeneous in this prototype ADC, which is an inherent challenge in conventional chemical combinations used to develop first-generation ADCs.

[0138] Table 2: Inhibition of tumor metastasis by ADC-MMAF (8D6) in vivo

[0139]

[0140] Example 9 Preparation of trimannosyl anti-TMCC3 8D6 mAb

[0141] In order to produce ADCs with homogeneous DARs and specific drug binding sites, we implemented a high-efficiency glycoengineering technology platform to combine trimannosyl core antibodies for novel ADCs. This platform converts trimannosyl antibodies into trimannosyl-4GlcNAz antibodies and then attaches the payload to the terminal GlcNAz group at the specific site of the antibody through a strain-promoted azide alkyne click chemistry (SPAAC) reaction. Anti-TMCC3 8D6 mAb was treated with β1,4-galactosidase and α2-3,6,8 neuraminidase in 1× sugar buffer at 37°C for 24 hours to remove galactose and sialic acid portions of N-glycans from anti-TMCC3 8D6 mAb. β1,4-galactosidase was further added to the reaction and the reaction was allowed to proceed at 37°C for another 24 hours to obtain G0F / G0 antibody samples. The antibody samples were purified and subjected to reduced mass chromatography analysis. As Figure 11 As shown in , the major amount of antibody in the sample is G0F (with a heavy chain of molecular weight of 50,019 Da) and only a small amount is G0 (no fucose; with a heavy chain of molecular weight of 49,875 Da).

[0142] Example 10 Preparation of anti-TMCC3 8D6 mAb-4Az

[0143] Trimannosyl anti-TMCC3 8D6 mAb and UDP-GlcNAz in 1× buffer SP were incubated at 37°C for 16 hours in the presence of rabbit MGAT-1 and rat MGAT-2. After incubation, reduced mass chromatography and intact mass chromatography of anti-TMCC3 8D6 mAb-4Az were performed. Figure 12AAs shown in , a trimannosyl-anti-TMCC3 8D6 mAb-4GlcNAz antibody product was obtained, wherein the heavy chain contained two GlcNAz molecules (molecular weight of 219 Da × 2 = 438) and each heavy chain had a molecular weight of 50,457 Da. This result indicates that GlcNAz is bound 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 chromatography. Figure 12B As shown in , a G0F trimannosyl anti-TMCC38D6 mAb-4GlcNAz antibody product was obtained, which contained four GlcNAz molecules (molecular weight of 244 Da×4=976 Da) and had a molecular weight of 148,331 Da, compared to the intact trimannosyl core anti-TMCC3 8D6 mAb with a molecular weight of 147,355 Da.

[0144] Example 11 Preparation of anti-TMCC3 8D6 mAb-4 (DBCO-vc-MMAE)

[0145] DBCO-vc-MMAE (10 mM in DMSO) was slowly added to a solution of trimannosyl anti-TMCC3 8D6 mAb-4GlcNAz antibody product in MES buffer (pH 6.5) at 37° C. for 18 hours. 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-TMCC38D6 mAb-4 (DBCO-vc-MMAE). Figure 13 As shown in Figure 1, a trimannosyl anti-TMCC3 8D6 mAb-4 (GlcNAc-triazole-DBCO-(PEG)4-MMAE) ADC product containing four DBCO-(PEG)4-MMAE molecules (molecular weight 1,614 Da x 4 = 6,456 Da) (Formula I) was obtained. The ADC had a molecular weight of 154,788 Da and a drug-to-antibody ratio (DAR) of 3.78.

[0146]

[0147] Example 12 Anticancer Efficacy of Trimannosyl-MMAE 8D6-ADC

[0148] To evaluate the efficacy of trimannosyl ADC-8D6 in ovarian cancer, we conducted four animal studies using the OC085 PDX tumor model of ovarian cancer. The clinical information and drug treatment history of the OC085 patient are summarized in Figure 14In brief, after being diagnosed with stage 4 ovarian cancer, she underwent debulking surgery followed by first-line chemotherapy with carboplatin + paclitaxel. The patient had a very short period of disease stabilization (2.5M) and moved to second-line chemotherapy (carboplatin + lipodox), followed by the addition of nivolumab and concurrent DC-CIK cell therapy, both of which failed to prevent disease progression. We found that TMCC3 was highly expressed in the patient's primary tumor and PDX tumors, as shown in Figure 2. Figure 14 Therefore, the PDX OC085, derived from a patient with advanced ovarian cancer who had an inadequate response to first-line / second-line therapy, is an ideal model for our target patient population.

[0149] In one study, 4 mg / kg of trimannosyl ADC-8D6 conjugated to MMAE was administered twice weekly starting on the same day after tumor inoculation. ADC treatment initiated on the same day after tumor inoculation mimics minimal residual disease (MRD) status in high-risk ovarian cancer patients, such as complete response after chemotherapy and / or surgical resection. Figure 15B In a second study, 4 mg / kg of trimannosyl ADC-8D6-conjugated MMAE was administered to mice at 400 mm Tg after tumors reached 100 mm Tg. 3 Tumor growth in mice treated with ADC was slower than that in control mice treated with human IgG ( Figure 15D )(p<0.001). Figure 15C and Figure 15E As shown in , the body weight of tumor-bearing mice was not affected by ADC treatment at 4 mg / kg / dose × 2 / week. In addition, we analyzed Ki67 expression and CSC frequency in ADC-treated tumors after sacrifice. Figure 15F As shown in Figure 2, it was noted that the expression of Ki67 was lower in ADC-treated tumors than in hIgG-treated tumors. As shown in Figure 2, the spheroid formation ability was lower in ADC-treated tumors compared to the hIgG control group ( Figure 15G In the third experiment, we increased the dose of trimannosyl ADC-8D6-conjugated MMAE to 8 mg / kg / dose (iv) weekly for 4 weeks in mice bearing OC085 ovarian PDX tumors, starting the day after tumor inoculation. Figure 16A As shown in Figure 2, tumor growth was completely abolished 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 sacrifice on day 128 ( Figure 16A). Weight loss was noted in the human IgG-treated group but not in the ADC-treated mice ( Figure 16B ).

[0150] To explore the anticancer ability of trimannosyl ADC-8D6 as a single agent or in combination with niraparib (PARP inhibitor), we tested the effect of niraparib on the tumors when they reached ∼100 mm 3 Mice bearing OC085 PDX tumors were treated with trimannosyl ADC-8D6 alone at 8 mg / kg / dose per week (iv injection), niraparib at 25 mg / kg / day (oral gavage, 5 days / week) alone or in combination. Figure 17A As shown in , the tumor growth of mice treated with trimannosyl ADC-8D6 was negligible compared to the robust tumor growth in mice treated with human IgG (p < 0.001). The dose of 25 mpk niraparib was selected based on reports that this is a therapeutic dose in mice with ovarian tumors and bile duct carcinomas (Bezrookove, V., et al. Cancers 13, 4405 (2021); Meng, J., et al. Journal of Translational Medicine 19 (2021)). However, in mice with OC085 PDX, treatment with 25 mpk niraparib only slightly reduced tumor growth, but did not reach statistical significance. The combination of ADC-8D6 and niraparib abolished tumor growth, similar to 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 Higher doses of niraparib were further tested. As shown in Figure 18 , administration of 50 mg / kg / day of niraparib did not suppress tumor growth in mice bearing OC085 PDX tumors, but did cause weakness and significant weight loss. These results demonstrate that trimannosyl ADC-8D6 has higher in vivo anticancer activity and fewer side effects than niraparib.

[0151] TMCC3 has been shown to be crucial for CSCs in breast and ovarian cancers, and mAb8D6 has been shown to be an anti-TMCC3-specific monoclonal antibody with therapeutic diagnostic potential. In the six animal experiments described above, we demonstrated that ADC-8D6 not only exhibited anti-cancer activity in breast cancer PDX (BC0145 and BC0634) and ovarian PDX (OC085) tumors, but also demonstrated CSC-targeting capabilities in vivo. In a comparative experiment comparing ADC-8D6 with therapeutic doses of niraparib, ADC-8D6 clearly demonstrated superior anti-tumor efficacy compared to niraparib in ovarian PDX (OC085) tumors. These results provide momentum for further development of anti-TMCC3 / ADCs as anticancer agents targeting ovarian cancer stem cells.

[0152] Although the present invention has been described in conjunction with the specific embodiments illustrated, many alternatives and modifications and variations thereof will be apparent to those skilled in the art. All such alternatives, modifications and variations are deemed to fall within the scope of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof that is specific for an antigenic determinant 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, wherein the CDRs of the heavy chain variable region comprise: CDRH1 of the amino acid sequence of SEQ ID NO:3, CDRH2 of the amino acid sequence of SEQ ID NO:4, and CDRH3 of the amino acid sequence of SEQ ID NO:5; and wherein the CDRs of the light chain variable region comprise: CDRL1 of the amino acid sequence of SEQ ID NO:6, CDRL2 of the amino acid sequence of SEQ ID NO:7, and CDRL3 of the amino acid sequence of SEQ ID NO:

8.

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

3. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody 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.

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

5. The antibody or antigen-binding fragment thereof of claim 4, which is linked to a second antibody or antigen-binding fragment thereof that is specific for a second antigenic determinant. The antibody or antigen-binding fragment thereof according to claim 1 , which is conjugated to a therapeutic agent.

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

8. The antibody or antigen-binding fragment thereof according to claim 7, wherein the therapeutic agent is selected from DM1, DM3, DM4, monomethyl auristatin E (MMAE) and monomethyl auristatin F (MMAF). The antibody or antigen-binding fragment thereof according to claim 1 , which is expressed on the surface of a cell.

10. The antibody or antigen-binding fragment thereof according to claim 9, wherein the cell is an immune cell, a cancer stem cell or a stem cell. The antibody or antigen-binding fragment thereof according to claim 10 , wherein the cell is a T cell. A vector encoding the antibody or antigen-binding fragment thereof according to claim 1 .

13. A genetically engineered cell expressing the antibody or antigen-binding fragment thereof according to claim 1 or containing the vector according to claim 12.

14. A method for producing an antibody or antigen-binding fragment thereof according to 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 conducive to expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment from the host cell and / or the culture medium in which the host cell is grown. 15 . A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to claim 1 or the genetically engineered cell according to claim 13 and a pharmaceutically acceptable carrier. 16 . A container or injection device comprising the antibody or antigen-binding fragment thereof according to claim 1 or the genetically engineered cell according to claim 13 .

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

18. The pharmaceutical composition according to claim 17, wherein the disease is cancer. The pharmaceutical composition according to claim 18 , wherein the cancer is a solid cancer.

20. The pharmaceutical composition according to 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. The pharmaceutical composition according to claim 17, wherein the pharmaceutical composition is in a form suitable for injection.

22. The pharmaceutical composition according to 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 the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

24. The method of claim 23, further comprising: The antibody or antigen-binding fragment thereof is used to assess the expression level of TMCC3 in a sample, wherein an increase in the expression level of TMCC3 compared to a standard indicates the presence of cancer stem cells in the sample.

25. The method of claim 24, wherein the cancer stem cell is a cancer stem cell of hematopoietic, epidermal, breast, ovarian, lung, pancreatic, prostate, brain, colorectal, bone marrow, or lymphoma. 26 . A kit for detecting TMCC3 or cancer in a sample, wherein the kit comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11.

27. A method for detecting a predisposition to cancer in an individual or predicting the likelihood, treatment response, prognosis or recurrence of said cancer, comprising The expression level of TMCC3 in a sample is assessed using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein an increase in the expression level of TMCC3 compared to a standard indicates the presence of cancer stem cells in the sample.

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