TEM8-targeted antibody or its antigen-binding fragment and its use

Abstract

The present invention belongs to the field of antibodies, and more specifically relates to TEM8-binding molecules, in particular antibodies that specifically recognize TEM8, and fragments thereof. Furthermore, the present invention also relates to nucleic acids or host cells containing such antibodies or fragments thereof, drugs containing such antibodies or fragments thereof, and treatment and diagnostic methods using such antibodies and fragments thereof, or the use thereof.

Description

[Technical Field]

[0001] This application claims priority to Chinese Patent Application No. 202310636114.4, filed on 31 May 2023, which is incorporated herein by reference in its entirety.

[0002] The present invention belongs to the field of antibodies, and more specifically relates to TEM8-binding molecules, in particular antibodies that specifically recognize TEM8, and fragments thereof. Furthermore, the present invention also relates to nucleic acids or host cells containing such antibodies or fragments thereof, drugs containing such antibodies or fragments thereof, and treatment and diagnostic methods using such antibodies and fragments thereof, or the use thereof. [Background technology]

[0003] Angiogenesis is the process by which new blood vessels develop and form from existing capillaries or postcapillary veins, and is important for various physiological processes, including organ growth, embryonic development, and wound healing. Angiogenesis also plays a crucial role in many diseases, particularly tumor formation and growth, as tumors require a sufficient blood supply to sustain their growth. As described in Croix et al. (Croix BS, Rago C., Velculescu V., Traverso G., Romans KE, Montegomery E., Lal A., Riggins GJ, Lengauer C., Vogelstein B., et al. Genes expressed in human tumor endothelium. Science. 2000;289:1197-1202. doi:10.1126 / science.289.5482.1197.), by comparing the expression profiles of vascular endothelial cell genes in normal tissue and tumor tissue, 46 genes were found to be highly expressed in tumor vascular endothelial cells. These were named tumor endothelial marker (TEM) molecules, and among them, TEM8 exhibits particularly excellent tumor specificity. Subsequently, TEM8, also known as anthrax toxin receptor 1 (ANTXR1), was found to be an anthrax toxin receptor, sharing 58% homology with another anthrax toxin receptor, CMG2 (ANTXR2).

[0004] TEM8 has five splice variants: V1, V2, V3, V4, and V5. These variants share the same N-terminal sequence and differ mainly in the length of their C-terminal splicing (Vargas M., Karamsetty R., Leppla SH, Chaudry G. J.Broad Expression Analysis of Human ANTXR1 / TEM8 Transcripts Reveals Differential Expression and Novel Splizce Variants. PLoS ONE. 2012;7:e43174.doi:10.1371 / annotation / cebf633f-19e7-496b-b370-c0f1b1aea888). The major splice variant, V1, is a TEM8 with a total length of 564 amino acids, of which amino acids 322-342 belong to the transmembrane domain. V2, V3, V4, and V5 have 368, 333, 528, and 358 amino acids, respectively. Therefore, V2 and V4 have transmembrane domains and are membrane proteins like V1, while V3 and V5 lack a TM domain and are soluble proteins. Furthermore, TEM8 is evolutionarily highly conserved, having identical amino acid sequences between humans and cynomolgus monkeys, and being completely identical between mice and rats. Human and mouse TEM8 share 98.9% homology.

[0005] TEM8 is significantly overexpressed in various tumor tissues, including those of breast, colorectal, lung, esophageal, gastric, pancreatic, and ovarian cancers. Further studies have revealed that TEM8 is primarily expressed in tumor-associated endothelial cells, pericytes, and tumor-associated fibroblasts (Szot C, et al. Tumor stroma-targeted antibody-drug conjugate triggers localized anticancer drug release. J Clin Invest. 2018;128:2927-2943. doi:10.1172 / JCI120481). TEM8 has also been reported to be expressed by a subset of tumor cells, and its abundance in tumor-associated fibroblasts is further increased under nutrient deficiencies in the tumor microenvironment. Furthermore, these solid tumors frequently generate a pronounced stromal response known as "fibrogenic stroma" or "reactive stroma." This component accounts for 20-60% of the entire tumor mass and is characterized by the presence of abundant stromal cells and a dense extracellular matrix (ECM).

[0006] TEM8 binds to type I and type VI collagen, allowing tumor-associated fibroblasts to migrate along collagen fibers in the tumor stroma (Nanda A., Carson-Walter EB, Seaman S., Barber TD, Stampfl J., Singh S., Vogelstein B., Kinzler KW, St Croix B. TEM8 interacts with the cleaved C5 domain of collagen alpha 3(VI) Cancer Res. 2004;64:817-820.doi:10.1158 / 0008-5472.can-03-2408). More recently, it has been reported that in the nutrient-deficient tumor microenvironment, TEM8 mediates the uptake of collagen fibers in tumor-associated fibroblasts, and these cells promote tumor cell growth by processing collagen fibers with glutamine (Hsu K.-S., Dunleavey JM, Szot C., Yang L., Hilton MB, Morris K., Seaman S., Feng Y., Lutz EM, Koogle R., et al. Cancer cell survival depends on collagen uptake into tumor-associated stroma. Nat.Commun.2022;13:7078.doi:10.1038 / s41467-022-34643-5). In TEM8 knockout mice, tumor cells grow remarkably slowly (Nanda A., Carson-Walter EB, Seaman S., Barber TD, Stampfl J., Singh S., Vogelstein B., Kinzler KW, St Croix B. TEM8 interacts with the cleaved C5 domain of collagen alpha 3(VI) Cancer Res. 2004;64:817-820.doi:10.1158 / 0008-5472.can-03-2408). Furthermore, inhibition of TEM8-to-collagen fiber binding by TEM8-specific antibodies shows antitumor effects in various tumor models, including melanoma, lung cancer, and colorectal cancer.

[0007] Unlike VEGF, VEGFR, and other angiogenic factors, TEM8 does not affect physiological angiogenesis. TEM8 knockout mice show no significant abnormalities in vascular development or wound healing. (Cullen M, Seaman S, Chaudhary A, Yang MY, Hilton MB, Logsdon D, et al. Host-Derived Tumor Endothelial Marker 8 (TEM8) Promotes the Growth of Melanoma. Cancer Res (2009) 69:6021-6. doi:10.1158 / 0008-5472.CAN-09-1086). The above research suggests that TEM8 has the potential to be a novel target for tumor therapy, and that antibodies with high affinity and specificity for TEM8 have great potential for clinical application. According to the present invention, TEM8-specific antibodies are discovered and identified using a complete human phage library, and the applicability of these antibodies is investigated.

[0008] Although several anti-TEM8 antibodies have already been developed, there is still a need to further develop TEM8 antibodies based on prior art, particularly to develop anti-human TEM8 antibodies that possess high specificity, high biological activity, high endocytosis capability, and / or can be used in the production of ADCs and CAR-T cells. [Overview of the project]

[0009] This invention discloses a TEM8-targeted antibody or its antigen-binding fragment, and its use.

[0010] Therefore, the present invention provides a novel TEM8-conjugated antibody and its antigen-conjugated fragment.

[0011] In some embodiments, the anti-TEM8 antibody of the present invention has the following characteristics: (1) It can bind to TEM8, for example, human, cynomolgus monkey, rat, or mouse TEM8 with high affinity. (2) It can bind with high affinity to TEM8 expressed on the surface of the cell membrane, for example, to human, cynomolgus monkey, rat, or mouse TEM8. (3) It is possible to induce endocytosis of an antibody or a fragment thereof, or a molecule containing an antibody or a fragment thereof (e.g., ADC), by TEM8-positive cells (e.g., TEM8-overexpressing CHO cells). (4) It is possible to target and kill TEM8-positive cells, such as TEM8-positive tumor cells. (5) The ability to treat tumors such as cancer, (6) Suitable for constructing antibody-drug conjugates (ADCs) for targeting and killing TEM8-positive cells, such as TEM8-positive tumor cells, or for treating tumors such as cancer.

[0012] In one embodiment, the present invention provides a nucleic acid encoding an antibody or a fragment thereof as described herein, a vector containing the nucleic acid, and a host cell containing the vector.

[0013] In some embodiments, the present invention provides a method for producing an antibody or a fragment thereof as described herein.

[0014] In some embodiments, the present invention provides molecules comprising antibodies described herein, such as antibody-drug conjugates, pharmaceutical compositions, and compound products.

[0015] The present invention also provides a method for preventing or treating TEM8-related diseases in a subject, such as tumors, preferably TEM8-positive tumors, such as TEM8-positive cancer, using the antibodies or antibody-drug conjugates described herein.

[0016] The present invention also relates to a method for detecting TEM8 in a sample. [Brief explanation of the drawing]

[0017] [Figure 1]Construction of CHO stable cell lines of human and mouse TEM8 is shown.

[0018] [Figure 2A] EC50 of five candidate molecules binding to human and mouse TEM8 overexpressing cells detected by FACS is shown. [Figure 2B] EC50 of five candidate molecules binding to human and mouse TEM8 overexpressing cells detected by FACS is shown.

[0019] [Figure 3A] IC50 of endocytosis of five antibodies by human and mouse TEM8 overexpressing cells detected by DT3C method is shown. [Figure 3B] IC50 of endocytosis of five antibodies by human and mouse TEM8 overexpressing cells detected by DT3C method is shown.

[0020] [Figure 4] IC50 of in vitro killing of five MMAE conjugate candidate molecules is shown.

[0021] [Figure 5] In vivo activity of five MMAE conjugate candidate molecules in HCT116 CDX model is shown.

[0022] [Figure 6] In vivo activity of naked antibody 6A6Q in HCT116 CDX model is shown.

Mode for Carrying Out the Invention

[0023] For the purposes of interpreting this specification, the following definitions are used, and where appropriate, a singular term may also include a plural form, and vice versa. It should be understood that the present invention is not limited to the specific methodologies, protocols, and reagents described herein, and these may vary. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit the scope of the present invention, which is limited only by the appended claims. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains.

[0024] When used with a number, the term "approximately" is intended to encompass numbers within a range that has a lower limit of 5% lower than the specified number and an upper limit of 5% higher than the specified number.

[0025] As used herein, the term "and / or" means one or more of the options.

[0026] As used herein, the terms “comprising” or “including” mean that the listed elements, integers, or steps are included, but not excluded from any other elements, integers, or steps. Unless otherwise indicated, as used herein, the terms “comprising” or “including” also encompass situations involving combinations of the listed elements, integers, or steps. For example, a reference to an antibody variable region “comprising” a particular sequence is intended to also encompass the antibody variable region consisting of that particular sequence.

[0027] As used herein, the terms “anti,” “binding,” or “specific binding” mean that binding to a target or antigen is selective and can be distinguished from undesirable or nonspecific interactions. The ability of a binding site to a particular target or antigen can be measured by flow cytometry or enzyme-linked immunosorbent assay (ELISA), or by conventional binding assays known in the art, such as radioimmunoassay (RIA), biolayer interferometry, MSD assay, or surface plasmon resonance (SPR).

[0028] As used herein, the terms “anti-TEM8 antibody,” “anti-TEM8,” “TEM8 antibody,” or “antibody that binds to TEM8” refer to an antibody or fragment thereof that can bind to TEM8 (of primates, e.g., humans or cynomolgus monkeys, or mice or rats) with sufficient affinity.

[0029] An “isolated” antibody or molecule is an antibody or molecule that has been separated from its natural environment. In some embodiments, the antibody or molecule is purified to a purity of 95% or greater than 99%, as measured by electrophoresis (e.g., SDS-PAGE, IEF, capillary electrophoresis) or chromatography (e.g., ion exchange or reverse-phase HPLC).

[0030] The terms "whole antibody" or "full-length antibody" are used interchangeably herein and refer to antibody molecules having the structure of a natural immunoglobulin molecule. In the case of a conventional quadruple-chain IgG antibody, the full-length antibody contains two heavy chains (H) and two light chains (L) interconnected by disulfide bonds. In the case of a heavy-chain antibody that has only a heavy chain and no light chain, the full-length antibody contains two heavy chains (H) interconnected by disulfide bonds. For a conventional quadruple-chain IgG antibody, the heavy chain of the full-length antibody typically consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, the heavy chain constant region containing at least three domains: CH1, CH2, and CH3. The light chain of the full-length antibody consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region, the light chain constant region consisting of one domain CL. Each heavy chain variable region (VH) and each light chain variable region consists of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. In some embodiments, the antibody according to the present invention is a full-length antibody, for example, a conventional four-chain IgG full-length antibody.

[0031] The term “antibody fragment” encompasses a portion of an intact antibody. In preferred embodiments, the antibody fragment is an antigen-binding fragment. The term “antigen-binding fragment” of an antibody is a molecule distinct from the full-length antibody, containing a portion of the full-length antibody but binding to the antigen of the full-length antibody, or competing with the full-length antibody (i.e., the full-length antibody from which the antigen-binding fragment originates) for binding to the antigen. Antigen-binding fragments can be produced by recombinant DNA technology or by enzymatic or chemical cleavage of an intact antibody. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, dAb (domain antibodies), linear antibodies, single-chain antibodies (e.g., scFv), single-domain antibodies such as VHH, bivalent antibodies or their fragments, or camel antibodies, diabodies, single-domain antibodies (sdAb), and nanobodies. For example, Fab fragments can be obtained by papain digestion of a full-length antibody. Furthermore, digestion of a complete antibody with pepsin below the disulfide bond in the hinge region produces F(ab')2, which is a dimer of Fab' and a bivalent antibody fragment. F(ab)'2 can be reduced by cleaving the disulfide bond in the hinge region under neutral conditions, thereby converting the (Fab')2 dimer back to the Fab' monomer. The Fv fragment consists of the VL and VH domains of a single arm of the antibody. The two domains of the Fv fragment, VL and VH, can be encoded by separate genes, but they can also be linked by recombination using synthetic linker peptides, thereby producing them as a single protein chain in which the VL and VH regions pair to form a single-chain Fv(scFv).

[0032] A "complementarity-determining region," "CDR region," or "CDR" is a region of the antibody variable domain whose sequence is highly variable, forms a structurally defined loop ("hypervariable loop"), and / or contains antigen contact residues ("antigen contact sites"). CDRs are primarily responsible for binding to antigenic epitopes. Heavy chain and light chain CDRs are generally numbered sequentially from the N-terminus and referred to as CDR1, CDR2, and CDR3. CDRs located within the variable domain of the antibody's heavy chain are called HCDR1, HCDR2, and HCDR3, and CDRs located within the variable domain of the antibody's light chain are called LCDR1, LCDR2, and LCDR3. In a given amino acid sequence of the light chain variable region or heavy chain variable region, the precise boundaries of the amino acid sequence of each CDR can be determined using one or a combination of many well-known antibody CDR assignment schemes.For example, the Chothia scheme based on the three-dimensional structure of antibodies and the topology of the CDR loop (Chothia et al., (1989) Nature 342:877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)); the Kabat scheme based on the variability of antibody sequences (Computer access to the Kabat antibody sequence database; Martin AC; Proteins, 1996 May; 25(1):130-3; doi:10.1002 / (SICI)1097-0134(199605)25:1<130:AID-PROT11>3.0.CO;2-L.PMID:8727325); the AbM scheme (University of Bath); the Contact scheme (University College London); the International ImMunoGeneTics Database (IMGT) scheme (IMGT, the international ImMunoGeneTics information system, http: / / imgt.cines.fr; Lefranc MP; Novartis Found Symp. 2003; 254: 126-36; discussion 136-42, 216-22, 250-2. PMID: 14712935 Review); and the North CDR definition scheme based on affinity propagation clustering using numerous crystal structures.

[0033] In some embodiments, the CDR of the heavy chain variable region of the antibody of the present invention is determined according to the Kabat or IMGT scheme. In some embodiments, the CDR of the light chain variable region of the antibody of the present invention is determined according to the Kabat or IMGT scheme.

[0034] Unless otherwise indicated herein, amino acid residues in the Fc or heavy chain constant region are numbered according to the EU numbering system (also known as the EU index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991, NIH Publication 91-3242, and amino acid residues in the light chain variable region and heavy chain variable region are numbered according to the Kabat numbering system (also known as the Kabat index) as described in Kabat et al., Sequences of Proteins of Immunological Interes, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991, NIH Publication 91-3242.

[0035] The terms “amino acid substitution” or “amino acid replacement” are used interchangeably herein and refer to the replacement of at least one amino acid residue in a given parent amino acid sequence with a different “substituted” amino acid residue. The substituted residue may be a “naturally occurring amino acid residue” (i.e., encoded by the genetic code) and is selected from the group consisting of alanine (Ala), arginine (Arg), asparagine (Asn), aspartic acid (Asp), cysteine ​​(Cys), glutamine (Gln), glutamic acid (Glu), glycine (Gly), histidine (His), isoleucine (Ile), leucine (Leu), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val). Substitutions by one or more naturally occurring amino acid residues are also included in the definition of amino acid substitution herein. "Naturally unexisting amino acid residues" refer to amino acid residues that can covalently bond to adjacent amino acid residues in a polypeptide chain, in addition to the naturally existing amino acid residues listed above. Examples of naturally unexisting amino acid residues include norleucine, ornithine, norvaline, homoserine, Aib, and other amino acid residue analogs.

[0036] As described herein, “conservative modification” includes substitutions, deletions, or additions to a polypeptide sequence that do not substantially alter the desired functional activity of the polypeptide sequence. In some embodiments, a conservative modification is a conservative substitution. A “conservative substitution” means substituting one amino acid with another amino acid within the same class, for example, substituting one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid. For example, conservative substitutions often involve the substitution of amino acids with chemically similar amino acids. Tables of conservative substitutions that provide functionally similar amino acids are well known in the art. The following are eight groups of amino acids that contain conserved substitutions with each other: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). In some embodiments, the term “conservative modification” applied to the amino acid sequence of an antibody molecule is used to refer to amino acid modifications that do not significantly affect, or alter, the binding properties of the antibody molecule of the present invention, including its amino acid sequence, to the target antigen. For example, a conservatively modified variant retains at least 80%, 85%, 90%, 95%, 98%, 99%, or more, for example, 100-110% or more, binding affinity to the target antigen compared to the parent antibody.

[0037] As used herein, the term “therapeutic agent” includes any substance effective in preventing or treating tumors such as cancer, such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

[0038] "Chemotherapy agents" include chemical compounds useful for treating cancer or diseases of the immune system.

[0039] The term "small molecule drug" refers to organic compounds with low molecular weight that can modulate biological processes. A "small molecule" is defined as a molecule with a molecular weight of less than 10 kD, typically less than 2 kD, and preferably less than 1 kD. Small molecules include, but are not limited to, inorganic molecules, organic molecules, organic molecules containing inorganic components, molecules containing radioactive atoms, synthetic molecules, peptide mimetic compounds, and antibody mimetic compounds. As therapeutic agents, small molecules may be more permeable to cells, less susceptible to degradation, and less likely to induce an immune response than large molecules.

[0040] As used herein, the term “immunomodulator” refers to a natural or synthetic activator or drug that inhibits or modulates an immune response. An immune response may be a humoral or cellular response. Immunomodulators include immunosuppressants. In some embodiments, the immunomodulators of the present invention include immune checkpoint inhibitors or immune checkpoint agonists.

[0041] "An isolated nucleic acid encoding an anti-TEM8 antibody or a fragment thereof" means one or more nucleic acid molecules encoding the heavy chain or light chain (or a fragment thereof, e.g., a heavy chain variable region or a light chain variable region) of an antibody, and includes such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[0042] The “percentage of amino acid sequence identity (%)” refers to the percentage of amino acid residues in a candidate sequence that are identical to the amino acid residues of the specific amino acid sequence shown herein, without considering any conservative substitutions as part of the sequence identity, after aligning the candidate sequence with a specific amino acid sequence shown herein to achieve the highest possible percentage of sequence identity and introducing gaps as necessary. In some embodiments, the present invention intends to provide variants of the antibody molecule of the present invention that have a substantial degree of identity, e.g., at least 80%, 85%, 90%, 95%, 97%, 98%, or 99% or more, compared to the antibody molecule and its sequence specifically disclosed herein. Variants may include conservative modifications.

[0043] The term "pharmaceutically acceptable adjuvants" refers to diluents, adjuvants (e.g., Freund's adjuvants (complete and incomplete)), excipients, carriers, or stabilizers administered together with the active substance.

[0044] As used herein, the term “labeling” refers to a compound or composition that is directly or indirectly conjugated or fused to an active substance, such as a polynucleotide probe or antibody, to facilitate the detection of the conjugated or fused active substance. The labeling may be detectable on its own (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzymatic labeling, may catalyze a chemical change in a detectable substrate compound or composition. This term is intended to encompass both direct labeling of a probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, and indirect labeling of a probe or antibody by reaction with another directly labeled active substance.

[0045] The term “combination therapy” refers to the administration of two or more therapeutic agents or modes of treatment for treating the diseases described herein. Such administrations include co-administration of therapeutic agents substantially simultaneously, for example, in a single capsule having a fixed ratio of active ingredients. Alternatively, such administrations include co-administration of each active ingredient in multiple or separate containers (e.g., tablets, capsules, powders, and liquids). Powders and / or liquids may be reconstituted or diluted to the desired dose before administration. Furthermore, such administrations also include using each type of therapeutic agent approximately simultaneously or sequentially at different times. In any case, the treatment regimen brings about the beneficial effect of the combined pharmaceutical agent in the treatment of the disorders or conditions described herein.

[0046] As used herein, the term “to treat” (or “to treat” or “treatment”) means to delay, interrupt, prevent, alleviate, stop, reduce or reverse the onset of symptoms, complications or biochemical indicators of a disease, to relieve symptoms, or to prevent or inhibit the further progression of a disease, condition or disorder.

[0047] As used herein, “prevention” (or “prevention” or “prevention”) includes the suppression of the onset or progression of a disease or disorder, or the symptoms of a particular disease or disorder.

[0048] "Subject / patient / individual sample" refers to a collection of cells or fluids obtained from a patient or subject. Sources of tissue or cell samples may include solid tissues, such as fresh, frozen, and / or preserved organ or tissue samples, biopsy samples, or puncture samples; blood or any blood component; body fluids, such as tears, vitreous fluid, cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; or cells derived from the subject at any point in pregnancy or development. Tissue samples may contain compounds that do not naturally coexist with tissues in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, and antibiotics.

[0049] In a first aspect of the present invention, an antibody or a fragment thereof (e.g., an antigen-binding fragment) that specifically binds to TEM8 is provided.

[0050] In some embodiments, the anti-TEM8 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, derived from the heavy chain variable region. In some embodiments, the anti-TEM8 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, derived from the light chain variable region. In some embodiments, the anti-TEM8 antibody or its antigen-binding fragment of the present invention comprises three complementarity-determining regions (HCDRs) derived from the heavy chain variable region and three complementarity-determining regions (LCDRs) derived from the light chain variable region.

[0051] In some embodiments, the anti-TEM8 antibody or its antigen-binding fragment of the present invention includes a heavy chain variable region (VH). In some embodiments, the anti-TEM8 antibody or its antigen-binding fragment of the present invention includes a light chain variable region (VL). In some embodiments, the anti-TEM8 antibody or its antigen-binding fragment of the present invention includes both a heavy chain variable region (VH) and a light chain variable region (VL). In some embodiments, the heavy chain variable region includes three complementarity-determining regions (HCDRs), HCDR1, HCDR2, and HCDR3, derived from the heavy chain variable region. In some embodiments, the light chain variable region includes three complementarity-determining regions (LCDRs), LCDR1, LCDR2, and LCDR3, derived from the light chain variable region.

[0052] In some embodiments, the heavy chain variable region of the present invention is (i) Consists of or includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 14, 27, 42, or 55, or (ii) SEQ ID NO: Contains or consists of an amino acid sequence selected from the group consisting of 1, 14, 27, 42, or 55, (iii) Compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 14, 27, 42, or 55, it includes or comprises an amino acid sequence having one or more (preferably 10 or less, more preferably 5, 4, 3, 2, or 1 or less) amino acid modifications (e.g., conservative modifications such as amino acid substitutions, more preferably conservative amino acid substitutions), preferably the amino acid modifications are not present in the CDR region.

[0053] In some embodiments, the light chain variable region of the present invention is (i) Consists of or comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 15, 28, 40, 43, or 56, or (ii) SEQ ID NO: Contains or consists of an amino acid sequence selected from the group consisting of 2, 15, 28, 40, 43, or 56, (iii) Compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 15, 28, 40, 43, or 56, the amino acid sequence includes or comprises one or more (preferably 10 or less, more preferably 5, 4, 3, 2, or 1 or less) amino acid modifications (e.g., conservative modifications such as amino acid substitutions, more preferably conservative amino acid substitutions), preferably the amino acid modifications are not present in the CDR region.

[0054] In some embodiments, the three complementarity-determining regions (HCDRs), HCDR1, HCDR2, and HCDR3 derived from the heavy chain variable region of the present invention are: (i) Three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in the VH described in SEQ ID NO: 1, 14, 27, 42, or 55 or (ii) A sequence selected from any one of the sequences of (i) that contains, in total, at least one amino acid modification (preferably an amino acid substitution, preferably a conservative substitution) in any one of the three HCDR regions, 5, 4, 3, 2, or 1 or fewer. The HCDR may be determined according to any CDR allocation scheme, for example, by the Kabat, AbM, Chothia, or IMGT allocation schemes, or a combination thereof, for example, the HCDR may be determined according to the IMGT scheme or the Kabat scheme, respectively.

[0055] In some embodiments, the three complementarity determination regions (LCDRs), LCDR1, LCDR2, and LCDR3 derived from the light chain variable region of the present invention are: (i) Three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in the VL described in SEQ ID NO: 2, 15, 28, 40, 43, or 56 or (ii) A sequence selected from any one of the sequences of (i) that contains, in total, at least one amino acid modification (preferably an amino acid substitution, preferably a conservative substitution) in any one of the three LCDR regions, 5, 4, 3, 2, or 1 or fewer. The LCDR may be determined according to any CDR designation scheme, for example, by the designation schemes of Kabat, AbM, Chothia, or IMGT, or a combination thereof. For example, the LCDR is determined according to either the IMGT scheme or the Kabat scheme, respectively.

[0056] In some embodiments, the HCDR1 determined based on the IMGT scheme includes or consists of an amino acid sequence described in any one of SEQ ID NO: 3, 16, 29, 44, or 57, or the HCDR1 determined based on the IMGT scheme includes an amino acid sequence having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence described in any one of SEQ ID NO: 3, 16, 29, 44, or 57.

[0057] In some embodiments, HCDR1 determined based on the Kabat scheme includes or consists of an amino acid sequence described in any one of SEQ ID NO: 9, 22, 35, 50, or 63, or HCDR1 determined based on the Kabat scheme includes an amino acid sequence having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence described in any one of SEQ ID NO: 9, 22, 35, 50, or 63.

[0058] In some embodiments, the HCDR2 determined based on the IMGT scheme includes or consists of an amino acid sequence described in any one of SEQ ID NO: 4, 17, 30, 45, or 58, or the HCDR2 determined based on the IMGT scheme includes an amino acid sequence having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence described in any one of SEQ ID NO: 4, 17, 30, 45, or 58.

[0059] In some embodiments, the HCDR2 determined based on the Kabat scheme includes or consists of an amino acid sequence described in any one of SEQ ID NO: 10, 23, 36, 51, or 64, or the HCDR2 determined based on the Kabat scheme includes an amino acid sequence having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence described in any one of SEQ ID NO: 10, 23, 36, 51, or 64.

[0060] In some embodiments, the HCDR3 determined based on the IMGT scheme includes or consists of an amino acid sequence described in any one of SEQ ID NO: 5, 18, 31, 46, or 59, or the HCDR3 determined based on the IMGT scheme includes an amino acid sequence having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence described in any one of SEQ ID NO: 5, 18, 31, 46, or 59.

[0061] In some embodiments, the HCDR3 determined based on the Kabat scheme includes or consists of an amino acid sequence described in any one of SEQ ID NO: 11, 24, 37, 52, or 65, or the HCDR3 determined based on the Kabat scheme includes an amino acid sequence having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence described in any one of SEQ ID NO: 11, 24, 37, 52, or 65.

[0062] In some embodiments, LCDR1 determined based on the IMGT scheme includes or consists of an amino acid sequence described in any one of SEQ ID NO: 6, 19, 32, 47, or 60, or LCDR1 determined based on the IMGT scheme includes an amino acid sequence having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence described in any one of SEQ ID NO: 6, 19, 32, 47, or 60.

[0063] In some embodiments, LCDR1 determined based on the Kabat scheme includes or consists of an amino acid sequence described in any one of SEQ ID NO: 12, 25, 38, 53, or 66, or LCDR1 determined based on the Kabat scheme includes an amino acid sequence having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to an amino acid sequence described in any one of SEQ ID NO: 12, 25, 38, 53, or 66.

[0064] In some embodiments, the LCDR2 determined based on the IMGT scheme includes or consists of an amino acid sequence described in any one of SEQ ID NO: 7, 20, 33, 48, or 61, or the LCDR2 determined based on the IMGT scheme includes an amino acid sequence having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence described in any one of SEQ ID NO: 7, 20, 33, 48, or 61.

[0065] In some embodiments, the LCDR2 determined based on the Kabat scheme includes or consists of an amino acid sequence described in any one of SEQ ID NO: 13, 26, 39, 54, or 67, or the LCDR2 determined based on the Kabat scheme includes an amino acid sequence having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence described in any one of SEQ ID NO: 13, 26, 39, 54, or 67.

[0066] In some embodiments, the LCDR3 determined based on the IMGT scheme includes or consists of an amino acid sequence described in any one of SEQ ID NO: 8, 21, 34, 41, 49, or 62, or the LCDR3 determined based on the IMGT scheme includes an amino acid sequence having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence described in any one of SEQ ID NO: 8, 21, 34, 41, 49, or 62.

[0067] In some embodiments, the LCDR3 determined based on the Kabat scheme includes or consists of an amino acid sequence described in any one of SEQ ID NO: 8, 21, 34, 41, 49, or 62, or the LCDR3 determined based on the Kabat scheme includes an amino acid sequence having one, two, or three modifications (preferably amino acid substitutions, preferably conservative substitutions) compared to the amino acid sequence described in any one of SEQ ID NO: 8, 21, 34, 41, 49, or 62.

[0068] In some specific embodiments of the present invention, the anti-TEM8 antibody or its antigen-binding fragment is: 1) The three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in VH as described in SEQ ID NO:1, and the three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in VL as described in SEQ ID NO:2; 2) The three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in VH as described in SEQ ID NO:14, and the three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in VL as described in SEQ ID NO:15; 3) The three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in VH described in SEQ ID NO:27, and the three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in VL described in SEQ ID NO:28 or 40; 4) The three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in VH as described in SEQ ID NO:42, and the three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in VL as described in SEQ ID NO:43; or 5) Including the three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in VH described in SEQ ID NO: 55, and the three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in VL described in SEQ ID NO: 56, For example, the HCDR and LCDR are determined according to the IMGT or Kabat scheme.

[0069] In some specific embodiments of the present invention, the anti-TEM8 antibody or antigen-binding fragment of the present invention comprises three complementarity-determining regions HCDR1, HCDR2, and HCDR3 in the heavy chain variable region, and three complementarity-determining regions LCDR1, LCDR2, and LCDR3 in the light chain variable region, wherein each of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 contains or consists of the amino acid sequences listed in the SEQ ID NO in the table below, based on the IMGT scheme. [Table 1] Alternatively, HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain or consist of the amino acid sequences listed in the SEQ ID NO in the table below, based on the Kabat scheme. [Table 2]

[0070] In some specific embodiments of the present invention, the anti-TEM8 antibody or its antigen-binding fragment comprises VH and VL, each comprising or consisting of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the [Table 3]

[0071] In a preferred embodiment, the present invention provides an anti-TEM8 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region each contain or consist of an amino acid sequence described in the SEQ ID NO shown in the table below. [Table 4]

[0072] In some embodiments, the anti-TEM8 antibody or its antigen-binding fragment of the present invention further comprises an antibody heavy chain constant region. In some embodiments, the anti-TEM8 antibody or its antigen-binding fragment of the present invention further comprises an antibody light chain constant region. In some embodiments, the anti-TEM8 antibody or its antigen-binding fragment of the present invention further comprises a heavy chain constant region and a light chain constant region.

[0073] In some embodiments, the antibody heavy chain constant region of the present invention is the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, and preferably the heavy chain constant region of IgG1. In some embodiments, the antibody light chain constant region of the present invention is the lambda or kappa light chain constant region.

[0074] In some preferred embodiments, the antibody heavy chain constant region of the present invention is (i) Consists of or comprises an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence selected from SEQ ID NO:71. (ii) containing or consisting of an amino acid sequence selected from SEQ ID NO:71, (iii) The amino acid sequence comprises or consists of one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, or 1 or less) amino acid modifications (for example, conservative modifications such as amino acid substitutions, more preferably conservative amino acid substitutions) compared to the amino acid sequence selected from SEQ ID NO:71.

[0075] In some embodiments, the constant region of the antibody light chain of the present invention is (i) Containing or consisting of an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 72 or 73, (ii) containing or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 72 or 73, (iii) Compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 72 or 73, it comprises or consists of an amino acid sequence having one or more (preferably 20 or 10 or less, more preferably 5, 4, 3, 2, or 1 or less) amino acid modifications (for example, conservative modifications such as amino acid substitutions, more preferably conservative amino acid substitutions).

[0076] In some embodiments, the antibody or fragment thereof of the present invention comprises a heavy chain constant region comprising or consisting of the amino acid sequence described in SEQ ID NO:71 and a light chain constant region comprising or consisting of the amino acid sequence described in SEQ ID NO:72 or 73.

[0077] In some embodiments, the anti-TEM8 antibody of the present invention or its antigen-binding fragment comprises an antibody heavy chain (HC). In some embodiments, the anti-TEM8 antibody of the present invention or its antigen-binding fragment further comprises an antibody light chain (LC). In some embodiments, the anti-TEM8 antibody of the present invention or its antigen-binding fragment comprises a heavy chain and a light chain. In some embodiments, the antibody heavy chain of the present invention comprises or consists of an antibody heavy chain variable region and an antibody heavy chain constant region. In some embodiments, the antibody light chain of the present invention comprises or consists of an antibody light chain variable region and an antibody light chain constant region. In some embodiments, the antibody of the present invention comprises or consists of two heavy chains and two light chains, for example, two heavy chains and two light chains that are identical.

[0078] In some embodiments, the antibody of the present invention comprises a heavy chain including a heavy chain variable region and a heavy chain constant region, and a light chain including a light chain variable region and a light chain constant region. (i) The heavy chain variable region includes or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO:1, the light chain variable region includes or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO:2, the heavy chain constant region includes or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO:71, the light chain constant region is SEQ ID It includes or comprises an amino acid sequence that is identical to or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in NO:72. (ii) The heavy chain variable region includes or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO:14, the light chain variable region includes or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO:15, the heavy chain constant region includes or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO:71, the light chain constant region is SEQ ID It includes or comprises an amino acid sequence that is identical to or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in NO:72. (iii) The heavy chain variable region includes or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO:27, the light chain variable region includes or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO:28 or 40, the heavy chain constant region includes or consists of an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO:71, the light chain constant region is SEQ ID It includes or comprises an amino acid sequence that is identical to or at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the amino acid sequence described in NO:73. (iv) The heavy chain variable region includes or comprises the amino acid sequence described in SEQ ID NO:42, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. The light chain variable region contains or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO:43. The heavy chain constant region includes or comprises the amino acid sequence described in SEQ ID NO:71, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. The light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:73, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, or (v) The heavy chain variable region includes or comprises the amino acid sequence described in SEQ ID NO:55, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. The light chain variable region contains or comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence described in SEQ ID NO:56. The heavy chain constant region includes or consists of the amino acid sequence described in SEQ ID NO:71, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto, and the light chain constant region includes or consists of the amino acid sequence described in SEQ ID NO:73, or an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0079] In some embodiments, the antibody of the present invention comprises a heavy chain including a heavy chain variable region and a heavy chain constant region, and a light chain including a light chain variable region and a light chain constant region. (vi) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:1, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:2, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:72. (vii) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:14, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:15, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:72. (viii) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:27, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:28 or 40, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:73. (ix) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:42, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:43, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:71, the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:73, or (x) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 55, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 56, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO: 71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO: 73.

[0080] In one embodiment of the present invention, the amino acid modification described herein is a conservative modification. In one embodiment of the present invention, the amino acid modification described herein includes the substitution, insertion, or deletion of amino acids. Preferably, the amino acid modification described herein is an amino acid substitution, preferably a conservative substitution.

[0081] In preferred embodiments, the amino acid modifications described herein occur in the region outside the CDR (e.g., FR). More preferably, the amino acid modifications described herein occur in the region outside the heavy chain variable region and / or the light chain variable region.

[0082] In some embodiments, the substitution is a conservative substitution. A "conservative substitution" refers to substituting one amino acid with another amino acid within the same class, for example, substituting one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid with another neutral amino acid.

[0083] In certain embodiments, the substitution occurs in the CDR region of the antibody. Typically, the resulting variant has a modification (e.g., improvement) of a certain biological property (e.g., increased affinity) compared to the parent antibody, and / or substantially retains a certain biological property of the parent antibody.

[0084] In some embodiments, the antibodies provided herein are modified to remove a glycosylation site. In some embodiments, the glycosylation site NRS of the antibody or fragment thereof is removed, preferably by mutation, for example, by mutating NRS to QRS.

[0085] In certain embodiments, it may be desirable to produce cysteine-modified antibodies, such as "thioMAb," in which one or more residues of the antibody are substituted with cysteine ​​residues.

[0086] In certain embodiments, the antibodies provided herein may be further modified to include other non-protein moieties known and readily available in the art. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymer, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymer or random copolymer), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, poly(propylene oxide / ethylene oxide) copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.

[0087] In some embodiments, the anti-TEM8 antibody or its antigen-binding fragment of the present invention has the following characteristics: (1) To TEM8, it exhibits the same or similar binding affinity and / or specificity as any of the antibodies listed in the present invention. (2) Inhibiting the binding of any of the antibodies listed in the present invention to TEM8, (3) To bind to an epitope that is the same as or overlaps with any of the antibodies shown in the present invention, (4) Regarding binding to TEM8, it competes with any of the antibodies shown in the present invention. The present invention further comprises an anti-TEM8 antibody or antigen-binding fragment having one or more of the following:

[0088] As defined herein, a “reference antibody” and an “antibody that binds to the same or overlapping epitopes” mean an antibody that, in a competitive assay, blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to that antigen; conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of that antibody to that antigen in a competitive assay.

[0089] As defined herein, a reference antibody and an antibody that competes for binding to its antigen refers to an antibody that, in a competitive assay, blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to that antigen. Conversely, a reference antibody blocks 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of that antibody to that antigen in a competitive assay. Numerous types of competitive binding assays, such as direct or indirect solid-phase radioimmunoassays (RIAs), direct or indirect solid-phase enzyme immunoassays (EIAs), sandwich competitive assays, bio-optical interference (e.g., Fortebio), or surface plasmon resonance (Biacore), can be used to determine whether one antibody competes with another.

[0090] As defined herein, an antibody that inhibits (e.g., competitively inhibits) the binding of a reference antibody to its antigen refers to an antibody that inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the reference antibody to its antigen. Conversely, a reference antibody inhibits 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding of the antibody to its antigen. The binding of an antibody to its antigen can be measured by affinity (e.g., equilibrium dissociation constant). Methods for determining affinity are known in the art.

[0091] An antibody exhibiting the same or similar binding affinity and / or specificity as a reference antibody refers to an antibody that can have at least 50%, 60%, 70%, 80%, 90%, or 95% or more of the binding affinity and / or specificity of the reference antibody. This can be determined by any method known in the art for determining binding affinity and / or specificity.

[0092] In some embodiments, the anti-TEM8 antibody is a monoclonal antibody.

[0093] In some embodiments, the anti-TEM8 antibody is a human antibody.

[0094] In some embodiments, the anti-TEM8 antibody is a full-length antibody.

[0095] In one embodiment, the anti-TEM8 antibody of the present invention also comprises its antibody fragments, such as its antigen-binding fragment. In some embodiments, the antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, Fv, single-chain Fv, diabody, single-domain antibody (sdAb), nanobody, or sc(Fv)2.

[0096] In certain embodiments, the anti-TEM8 antibody molecule is in the form of a bispecific or multispecific antibody molecule. In one embodiment, the bispecific antibody may bind to two different epitopes of the TEM8 protein. In one embodiment, the bispecific antibody may bind to TEM8 and another protein. Therefore, the anti-TEM8 antibody of the present invention also includes multispecific antibodies, such as bispecific antibodies, that specifically bind to TEM8 and other antigens.

[0097] Therefore, in some embodiments, the present invention relates to bispecific or multispecific antibodies that specifically bind to TEM8 and one or more other antigens.

[0098] In a second aspect of the present invention, an antibody-drug conjugate comprising the anti-TEM8 antibody of the present invention or its antigen-binding fragment is provided.

[0099] The antibody or fragment thereof of the present invention can induce endocytosis of the antibody or fragment thereof, or a molecule containing the antibody or fragment thereof, by TEM8-positive cells, and therefore it is suitable for use in the construction of antibody-drug conjugates.

[0100] In some embodiments, the present invention provides an antibody-drug conjugate comprising one of the anti-TEM8 antibodies provided herein and an additional substance such as a therapeutic agent or label. In some embodiments, the therapeutic agent is a cytotoxic agent. In some embodiments, the therapeutic agent is an MMAE.

[0101] In some embodiments, the antibody or antigen-binding fragment of the present invention is linked to a cytotoxic agent (e.g., MMAE) via a linker to form an antibody-drug conjugate. In some embodiments, the linker is a Vc linker. In some embodiments, the antibody or antigen-binding fragment of the present invention is linked to a VcMMAE to form an antibody-drug conjugate.

[0102] A third aspect of the present invention provides a nucleic acid, such as an isolated nucleic acid molecule, that encodes either the anti-TEM8 antibody of the present invention, a fragment thereof, or a chain thereof.

[0103] For example, the nucleic acids of the present invention include nucleic acids encoding an amino acid sequence selected from any one of SEQ ID NO: 1, 2, 14, 15, 27, 28, 40, 42, 43, 55, or 56, or an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from any one of SEQ ID NO: 1, 2, 14, 15, 27, 28, 40, 42, 43, 55, or 56. As will be understood by those skilled in the art, each antibody or polypeptide amino acid sequence can be encoded by various nucleic acid sequences by codon degeneracy. The nucleic acid sequences encoding the molecules of the present invention can be generated using methods well known in the art, for example, by de novo solid-phase DNA synthesis or PCR amplification.

[0104] When expressed from a suitable expression vector, the polypeptide encoded by the nucleic acid can exhibit human (and / or mouse, rat, dog, or cynomolgus monkey) TEM8 antigen-binding ability. For example, in some embodiments, the nucleic acid encoding the variable regions of the heavy and / or light chains is operably linked in a reading frame to the nucleic acid encoding the constant regions of the heavy and / or light chains, resulting in the nucleic acid encoding the antibody heavy and / or light chains when expressed from a suitable expression vector.

[0105] To facilitate production and purification, a secretion signal peptide and / or a tag peptide for facilitating purification, such as a his tag, may be fused to the N-terminus of the antibody's heavy and / or light chains.

[0106] A fourth aspect of the present invention provides one or more vectors comprising the nucleic acid of the present invention.

[0107] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. This term includes vectors that are self-replicating nucleic acid structures, and vectors that are incorporated into the genome of a host cell into which they are introduced. The term “expression vector” refers to a vector containing recombinant polynucleotides that include an expression regulatory sequence operably linked to the nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression, and other elements for expression may be provided by the host cell or in an in vitro expression system. Examples of expression vectors include all known in the art into which recombinant polynucleotides are incorporated, e.g., cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0108] In one embodiment, the vector is an expression vector such as a eukaryotic expression vector. Examples of vectors include, but are not limited to, viruses, plasmids, cosmids, lambda phages, or yeast artificial chromosomes (YACs). In one embodiment, the vector is a pCDNA vector, for example, pCDNA3.4.

[0109] A fifth aspect of the present invention provides a host cell comprising a nucleic acid encoding the antibody of the present invention or an antigen-binding fragment thereof, or a vector comprising a nucleic acid.

[0110] The term “host cell” refers to a cell into which an exogenous polynucleotide has been introduced, and includes the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their offspring, regardless of the number of passages. Offspring may not be exactly identical to the parent cells in nucleic acid content and may contain mutations. Mutant offspring having the same function or biological activity as those screened or selected in the initially transformed cells are included herein. Host cells are any type of cell line that can be used to produce the antibody molecules of the present invention, and include eukaryotic cells, e.g., mammalian cells, insect cells, yeast cells; and prokaryotic cells, e.g., Escherichia coli cells. Host cells include cultured cells, as well as cells in transgenic animals, transgenic plants, or cultured plant or animal tissues.

[0111] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from the group consisting of yeast cells, mammalian cells (e.g., CHO cells, e.g., CHO-S, CHO-K, or CHO-K1, or 293 cells, e.g., 293T, 293F, or HEK293 cells), or other cells suitable for producing antibodies or fragments thereof. In one embodiment, the host cell is prokaryotic, such as bacteria like Escherichia coli.

[0112] In one embodiment, the host cell is eukaryotic. In another embodiment, the host cell is selected from yeast cells, mammalian cells, or other cells suitable for producing antibodies or fragments thereof. For example, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Vertebrate cells can also be used as hosts. For example, mammalian cell lines engineered to be suitable for growth in suspension can be used. Other examples of useful mammalian host cell lines include the SV40-transformed monkey kidney CV1 cell line (COS-7); and human fetal kidney cells (HEK293, 293F, or 293T cells). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, e.g., CHO-S, CHO-K, or CHO-K1, and myeloma cell lines, e.g., Y0, NS0, and Sp2 / 0. Mammalian host cell lines suitable for antibody production are known in the art.

[0113] In a sixth aspect of the present invention, the present invention provides a method for producing an anti-TEM8 antibody or a fragment thereof (preferably an antigen-binding fragment), comprising: culturing host cells under conditions suitable for the expression of the antibody or a fragment thereof (preferably an antigen-binding fragment) or a nucleic acid encoding any one or two strands thereof; and optionally isolating the antibody or a fragment thereof (preferably an antigen-binding fragment). In certain embodiments, the method further comprises recovering the anti-TEM8 antibody or a fragment thereof (preferably an antigen-binding fragment) from host cells.

[0114] The polynucleotides encoding the polypeptide chain of the antibody or its antigen-binding fragment of the present invention can be inserted into one or more vectors for further cloning and / or expression in host cells. Expression vectors can be constructed using methods well known to those skilled in the art. Once an expression vector containing one or more nucleic acid molecules of the present invention is prepared for expression, the expression vector can be transfected or introduced into a suitable host cell. To achieve this objective, various techniques, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, bioristic, liposome-based transfection, or other prior arts can be used.

[0115] Antibodies prepared as described herein can be purified by known existing techniques, such as high-performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography (e.g., protein A), and size exclusion chromatography. The actual conditions used to purify a particular protein also depend on factors such as net charge, hydrophobicity, and hydrophilicity, which will be apparent to those skilled in the art.

[0116] The purity of the antibody molecule of the present invention can be determined by any of the various well-known analytical methods, including size exclusion chromatography, gel electrophoresis, and high-performance liquid chromatography.

[0117] A seventh aspect of the present invention provides a method or assay for identifying, screening, or characterizing the physical / chemical properties and / or biological activity of anti-TEM8 antibodies provided herein.

[0118] In one embodiment, the antigen-binding activity of the antibody in the present invention is tested by known methods such as biolayer interference assay or ELISA. Binding to TEM8 can be measured using methods known in the art, for example, by radioimmunoassay (RIA), biolayer interference assay (BLI), electrochemiluminescence (ECL) assay, surface plasmon resonance (SPR), or flow cytometry (FCM, also known as fluorescence-activated cell sorting (FACS)).

[0119] The present invention also provides an assay for identifying biologically active anti-TEM8 antibodies. The biological activity is selected from the properties of the antibody of the present invention and may include, for example, binding to TEM8 (e.g., binding to human, cynomolgus monkey, mouse, and / or rat TEM8), cellular endocytosis activity, or killing activity against TEM8-positive cells.

[0120] For example, the binding activity of the antibody molecule of the present invention to TEM8 or TEM8-expressing cells can be measured by methods known in the art, such as Fortebio, flow cytometry, Octet, or plasmon resonance (Biacore), or by exemplary methods disclosed in Example 3 of this specification.

[0121] For example, the endocytosis activity of the antibody molecule of the present invention against cells (e.g., TEM8-positive cells, e.g., TEM8-overexpressing cells) can be measured by methods known in the art, such as DT3C, or by exemplary methods disclosed in Example 4 of this specification.

[0122] For example, the killing or antitumor activity of the antibody molecule of the present invention or the antibody-drug conjugate containing it against cells (e.g., TEM8-positive cells, e.g., tumor cells) can be measured by methods known in the art, such as cell killing assays including in vitro cell killing assays, or in vivo animal model experiments, or by exemplary methods disclosed in Examples 7, 8, or 9 herein.

[0123] The cells used in any of the in vitro assays described above may include cell lines that naturally express TEM8 or cell lines that have been engineered to express TEM8. Such cells may include cell lines that express TEM8, as well as cell lines that do not normally express TEM8, such as the HCT116 cell line, which have been transfected with DNA encoding TEM8.

[0124] It will be understood that all of the above assays may be performed using the antibody-drug conjugate of the present invention, either in place of or in addition to the anti-TEM8 antibody.

[0125] It should be understood that all of the above assays can be performed using a combination of anti-TEM8 antibody and other activators.

[0126] In an eighth aspect of the present invention, a composition such as a pharmaceutical composition or pharmaceutical preparation is provided, comprising the antibody of the present invention, a fragment of its antigen-binding region, or its antibody-drug conjugate.

[0127] As used herein, the term “pharmaceutical composition” refers to a composition in which the biological activity of the active ingredient contained herein is present and which does not contain any additional ingredients that are unacceptably toxic to the subject to which the composition is administered.

[0128] In some embodiments, the pharmaceutical composition or pharmaceutical preparation further comprises a pharmaceutically acceptable carrier, a pharmaceutically acceptable excipient, and pharmaceutically acceptable auxiliary materials such as buffers, as are known in the art. As used herein, “pharmaceutically acceptable carrier” includes any physiologically compatible solvent, dispersion medium, isotonic agent, and absorption retarder.

[0129] For information on pharmaceutically acceptable adjuvants and their use, see also "Handbook of Pharmaceutical Excipients," 8th Edition, RCRowe, PJSeskey, and SCOwen, Pharmaceutical Press, London, Chicago.

[0130] The compositions or formulations of the present invention may be in various forms. Such forms include, for example, liquid solutions (e.g., solutions for injection and infusion), powders or suspensions, liposomal formulations, and liquid, semi-solid, and solid dosage forms such as suppositories. The preferred form depends on the intended mode of administration and therapeutic use.

[0131] Pharmaceutical formulations comprising the antibody of the present invention as described herein can be prepared, preferably in the form of a lyophilized formulation or an aqueous solution, by mixing the antibody of the present invention having a desired purity with one or more pharmaceutically acceptable auxiliary materials.

[0132] The pharmaceutical composition or formulation of the present invention may contain two or more active ingredients as necessary for the specific indication being treated, preferably having complementary activities that do not adversely affect each other. For example, it is desirable to also provide additional therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants). The active ingredients are appropriately present in combination in amounts effective for the intended use.

[0133] A ninth aspect of the present invention provides a pharmaceutical combination or pharmaceutical combination product comprising the anti-TEM8 antibody of the present invention or a fragment thereof (preferably an antigen-binding fragment), or its antibody-drug conjugate, and one or more additional therapeutic agents (e.g., chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, vaccines, small molecule drugs, or immunomodulators (e.g., immunosuppressants)).

[0134] As used herein, the term “pharmaceutical combination or combination product” refers to an unfixed or fixed combination product, which includes, but is not limited to, kits of parts / kits. “Unfixed combination” means that the active ingredients (e.g., (i) the antibody of the present invention or its antibody-drug conjugate, and (ii) additional therapeutic agents) are administered to a patient as separate entities, simultaneously or sequentially at the same or different time intervals, without specific time constraints, such administration providing a prophylactic or therapeutic level of efficacy of two or more activators in the patient. “Fixed combination” means that two or more activators are administered to a patient simultaneously in the form of a single entity. Preferably, the doses and / or time intervals of the two or more activators are selected such that the combined use of these components produces an effect that exceeds the effect that could be achieved by using any one component alone in the treatment of a disease or disorder. The components may be in separate formulations, and these formulations may be the same or different.

[0135] In a tenth aspect of the present invention, a kit of parts is provided comprising the pharmaceutical formulation of the present invention, preferably in the form of pharmaceutical dosing units. Thereafter, the dosing units may be provided according to a dosing regimen or drug dosing interval.

[0136] In one embodiment, the kit of parts according to the present invention is contained in the same package. - A first container for containing a pharmaceutical composition comprising an anti-TEM8 antibody or its antibody-drug conjugate, - A second container for containing a pharmaceutical composition comprising additional therapeutic agents, such as chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, vaccines, small molecule drugs, or immunomodulators (e.g., immunosuppressants). Includes.

[0137] An eleventh aspect of the present invention provides a method for preventing or treating TEM8-related disease in a subject, comprising administering an effective amount (e.g., a prophylactic or therapeutic effective amount) of the anti-TEM8 antibody of the present invention or its antigen-binding fragment, antibody-drug conjugate, pharmaceutical composition, or combination product to the subject.

[0138] As used herein, the term “to treat” (or “to treat” or “treatment”) means to delay, interrupt, prevent, alleviate, stop, reduce or reverse the onset of symptoms, complications or biochemical indicators of a disease, to relieve symptoms, or to prevent or inhibit the further progression of a disease, condition or disorder.

[0139] As used herein, “prevention” (or “prevention” or “prevention”) includes the suppression of the onset or progression of a disease or disorder, or the symptoms of a particular disease or disorder.

[0140] As used herein, the term “effective dose” refers to the amount or dose of the antibody, fragment, composition, or formulation of the present invention that, when administered to a patient in a single or multiple dose, produces the desired effect in a patient requiring treatment or prevention.

[0141] As used herein, “therapeutic dose” refers to the amount effective in the required dose for the required duration to achieve the desired therapeutic outcome. The therapeutic dose is also the amount in which any toxicity or adverse effect of the antibody or antibody fragment or composition or combination is less than the therapeutically beneficial effect. The “therapeutic dose” preferably suppresses a measurable parameter (e.g., tumor volume) or improves a measurable parameter by at least about 40%, more preferably at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or even 100% compared to an untreated subject.

[0142] As used herein, "prophylactic effective dose" refers to the amount effective at the required dose for the required duration to achieve the desired prophylactic outcome. Generally, since prophylactic doses are administered to subjects before or at an early stage of the disease, the prophylactic effective dose is less than the therapeutic effective dose.

[0143] "Individual" or "Subject" includes mammals. Mammals include, but are not limited to, livestock (e.g., cattle, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, e.g., monkeys), rabbits, and rodents (e.g., mice and rats). In some embodiments, the individual or subject is a human.

[0144] In some embodiments, the TEM8-related diseases described herein are diseases related to the abnormal proliferation and / or dysfunction of tumor stromal cells, e.g., tumors, e.g., cancer cells.

[0145] In some embodiments, the TEM8-related diseases described herein include tumors, such as cancer. The cancer may be early, mid, or advanced, or it may be metastatic. In some embodiments, the cancer may be a solid tumor or a hematological malignancy. In some embodiments, the cancer may be selected from colorectal cancer, rectal cancer, and colon cancer.

[0146] In one embodiment, a tumor refers to a tumor in tumor tissue or tumor cells of an individual, which expresses TEM8, has TEM8 nucleic acid, or has elevated levels of TEM8 protein (e.g., expression) or TEM8 nucleic acid levels compared to adjacent normal tissue or normal cells of the same individual (e.g., normal cells in the tissue) or the same tissue or cells within it of a healthy individual.

[0147] In some embodiments, the antibodies or antibody fragments or antibody-drug conjugates or compositions or formulations or combination products of the present invention delay the onset of disorders and / or symptoms associated with disorders.

[0148] In some embodiments, the preventive or therapeutic methods described herein further include administering to a subject or individual a combination of an antibody molecule or pharmaceutical composition or antibody-drug conjugate disclosed herein and one or more additional treatments, such as a treatment mode and / or additional therapeutic agents.

[0149] In some embodiments, the treatment method includes surgical procedures, radiotherapy (e.g., external particle beam therapy, including three-dimensional conformal radiotherapy in which the irradiation area is designed), local irradiation (e.g., irradiation directed at a pre-selected target or organ), or focused irradiation.

[0150] In some embodiments, the therapeutic agent is selected from chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, vaccines, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressants).

[0151] In some embodiments, the antibodies described herein can be administered in combination with other antibodies, either separately, for example, as individual antibodies administered independently, or conjugated to each other (for example, as bispecific or multispecific antibody molecules).

[0152] The combination therapy of the present invention encompasses both combined administration (e.g., two or more therapeutic agents contained in the same or separate formulations) and separate administration, in the latter case, administration of the antibody of the present invention, and antibody-drug conjugates, compositions, pharmaceutical compositions, formulations, etc. containing the same, can be performed before, simultaneously with, and / or after the administration of additional therapeutic agents and / or active substances.

[0153] The anti-TEM8 antibody of the present invention (and antibody-drug conjugates, compositions, pharmaceutical compositions, formulations, combination products, etc., containing the same) may be administered by any suitable method, including parenteral administration and, if local treatment is required, intra-focal administration. Parenteral injection or infusion includes intramuscular, intravenous, intra-arterial, intraperitoneal, or subcutaneous injection or infusion. Administration may be by any suitable route, e.g., by injection, e.g., intravenous or subcutaneous, depending to some extent whether it is short-term or long-term. Various administration schedules, including but not limited to single doses, multiple doses at multiple time points, bolus doses, and pulse infusions, are incorporated herein.

[0154] In other embodiments, the present invention provides the use of the anti-TEM8 antibody or fragment thereof, or an antibody-drug conjugate or composition containing thereof, in the manufacture or preparation of a medicament for use as described herein, for example, to prevent or treat a related disease or disorder as referred to herein.

[0155] In a twelfth aspect of the present invention, the present invention also relates to a method for diagnosing and detecting an antibody or an antigen-binding fragment thereof, and a composition for diagnosis and detection containing the same.

[0156] In certain embodiments, the presence of TEM8 in a biological sample can be detected using any anti-TEM8 antibody or its antigen-binding fragment provided herein.

[0157] As used herein, the term “detect” includes quantitative or qualitative detection, and exemplary detection methods may include immunohistochemistry, immunocytochemistry, flow cytometry (e.g., FACS), magnetic beads combined with antibody molecules, ELISA assays, and PCR techniques (e.g., RT-PCR). In certain embodiments, the biological sample is blood, serum, or other liquid sample of biological origin. In certain embodiments, the biological sample includes cells or tissue. In certain embodiments, the biological sample is derived from tumor or cancerous tissue.

[0158] In one embodiment, an anti-TEM8 antibody or its antigen-binding fragment is provided for diagnostic and detection methods.

[0159] In another embodiment, a method for detecting the presence of TEM8 in a biological sample is provided. In a particular embodiment, the method includes detecting the presence of TEM8 in a biological sample. In a particular embodiment, TEM8 is human TEM8, or mouse, rat, dog, or cynomolgus monkey TEM8. In a particular embodiment, the method includes contacting a biological sample with an anti-TEM8 antibody described herein under conditions that allow the binding of the anti-TEM8 antibody to TEM8, and detecting whether a complex has been formed between the anti-TEM8 antibody and TEM8. The formation of a complex indicates the presence of TEM8. The method may be in vitro or in vivo. In one embodiment, the anti-TEM8 antibody is used to select a subject suitable for treatment with the anti-TEM8 antibody, for example, in which case TEM8 is a biomarker used for subject selection.

[0160] In certain embodiments, labeled antibodies or fragments thereof are provided. The labeling includes, but is not limited to, directly detectable labels or parts (e.g., fluorescent labels, chromogenic labels, high electron density labels, chemiluminescent labels, and radioactive labels) as well as parts that are indirectly detectable, for example, by enzymatic reactions or intermolecular interactions (e.g., enzymes or ligands).

[0161] In some embodiments provided herein, the sample is obtained before treatment with the antibody or fragment thereof of the present invention. In some embodiments, the sample is obtained before treatment with additional therapeutic agents. In some embodiments, the sample is obtained during or after treatment with additional therapeutic agents.

[0162] In some embodiments, TEM8 is detected before a procedure, for example, before the start of a procedure or before a particular procedure after a procedure interval.

[0163] In some embodiments, the present invention provides a method for treating a TEM8-related disease, the method comprising: testing a subject (e.g., a sample) (e.g., a control sample) for the presence of TEM8 and thereby determining a TEM8 value; comparing the TEM8 value to a control value (e.g., a value in the same tissue or cells of a healthy individual, or a value in adjacent normal tissue or cells of the patient being tested); and, if the TEM8 value is greater than the control value, administering a therapeutically effective amount of an antibody or fragment thereof, or an antibody-drug conjugate, composition, pharmaceutical composition, formulation, or combination product containing thereof, in combination with one or more optional additional treatments, to the subject, thereby treating the disease.

[0164] In the thirteenth aspect, the present invention relates to the following specific embodiments. 1. An antibody or antigen-binding fragment thereof that specifically binds to TEM8, wherein the antibody is 1) The three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in VH as described in SEQ ID NO:1, and the three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in VL as described in SEQ ID NO:2; 2) The three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in VH as described in SEQ ID NO:14, and the three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in VL as described in SEQ ID NO:15; 3) The three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in VH described in SEQ ID NO:27, and the three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in VL described in SEQ ID NO:28 or 40; 4) The three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in VH as described in SEQ ID NO:42, and the three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in VL as described in SEQ ID NO:43; or 5) Including the three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in VH described in SEQ ID NO: 55, and the three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in VL described in SEQ ID NO: 56, Preferably, an antibody or its antigen-binding fragment, in which the HCDR and LCDR are determined according to the IMGT or Kabat scheme.

[0165] 2. An antibody or antigen-binding fragment thereof that specifically binds to TEM8, wherein the antibody comprises three complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region, and three complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region, and each of these HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 contains or consists of the amino acid sequences listed in the SEQ ID NO in the table below, based on the IMGT scheme. [Table 5] Alternatively, an antibody or antigen-binding fragment thereof, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain or consist of the amino acid sequences listed in the SEQ ID NO in the table below, based on the Kabat scheme. [Table 6]

[0166] 3. The antibody includes a heavy chain variable region, and the heavy chain variable region includes or consists of an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 14, 27, 42, or 55, or the heavy chain variable region includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 14, 27, 42, or 55, and / or The TEM8-conjugated antibody or antigen-conjugated fragment thereof according to Embodiment 1 or 2, wherein the antibody includes a light chain variable region, and the light chain variable region includes or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 15, 28, 40, 43, or 56, or the light chain variable region includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 15, 28, 40, 43, or 56.

[0167] 4. An anti-TEM8 antibody or an antigen-binding fragment thereof, wherein the antibody comprises a heavy chain variable region and a light chain variable region. (i) The heavy chain variable region contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:1, and the light chain variable region contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:2. (ii) The heavy chain variable region contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:14, and the light chain variable region contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:15. (iii) The heavy chain variable region includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:27, and the light chain variable region includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:28 or 40. (iv) The heavy chain variable region contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:42, and the light chain variable region contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:43. (v) An anti-TEM8 antibody or its antigen-binding fragment, wherein the heavy chain variable region contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:55, and the light chain variable region contains an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:56.

[0168] 5. The TEM8-conjugated antibody or antigen-conjugated fragment thereof according to Embodiment 1 or 2, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region each contain or consist of the amino acid sequence described in the SEQ ID NO in the table below. [Table 7]

[0169] 6. The antibody includes a heavy chain constant region, for example, the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, for example, the heavy chain constant region of IgG1, and the heavy chain constant region is (i) It contains an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO:71, or (ii) Consists of or comprises an amino acid sequence selected from the group consisting of SEQ ID NO:71 A TEM8-conjugated antibody or its antigen-conjugated fragment according to any one of Embodiments 1 to 5.

[0170] 7. The antibody comprises a light chain constant region, for example, a lambda or kappa light chain constant region, for example, the light chain constant region is (i) It contains or consists of an amino acid sequence that has at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 72 or 73, or (ii) containing or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 72 or 73 A TEM8-conjugated antibody or its antigen-conjugated fragment according to any one of Embodiments 1 to 6.

[0171] 8. The antibody comprises a heavy chain constant region and a light chain constant region, The heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:72 or 73. A TEM8-conjugated antibody or its antigen-conjugated fragment according to any one of Embodiments 1 to 7.

[0172] 9. The antibody comprises a heavy chain and a light chain, the heavy chain comprising a heavy chain variable region and a heavy chain constant region, the light chain comprising a light chain variable region and a light chain constant region, (i) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:1, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:2, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:72. (ii) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:14, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:15, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:72. (iii) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:27, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:28 or 40, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:73. (iv) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:42, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO:43, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:71, the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:73, or (v) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 55, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 56, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO: 71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO: 73. A TEM8-conjugated antibody or its antigen-conjugated fragment according to any one of Embodiments 1 to 8.

[0173] 10. The following characteristics: (1) It can bind to TEM8, for example, human, cynomolgus monkey, rat, or mouse TEM8 with high affinity. (2) It can bind with high affinity to TEM8 expressed on the surface of the cell membrane, for example, to human, cynomolgus monkey, rat, or mouse TEM8. (3) It is possible to induce endocytosis of an antibody or a fragment thereof, or a molecule containing an antibody or a fragment thereof (e.g., ADC), by TEM8-positive cells (e.g., TEM8-overexpressing CHO cells). (4) It is possible to target and kill TEM8-positive cells, such as TEM8-positive tumor cells. (5) The ability to treat tumors such as cancer, (6) Suitable for constructing antibody-drug conjugates (ADCs) for targeted killing of TEM8-positive cells, such as TEM8-positive tumor cells, or for the treatment of tumors such as cancer. (7) Inhibiting the binding of any of the antibodies listed in Embodiment 9 to TEM8, (8) To bind to the same or overlapping epitope as any of the antibodies described in Embodiment 9, or (9) Regarding binding to TEM8, it competes with any of the antibodies described in Embodiment 9. A TEM8-conjugated antibody or antigen-conjugated fragment thereof according to any one of embodiments 1 to 8, having one or more of the above.

[0174] 11. The antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 10, wherein the antibody is a monoclonal antibody.

[0175] 12. The antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 11, wherein the antibody is a human antibody.

[0176] 13. The antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 12, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, a single-chain antibody, e.g., scfv, a (Fab')2 fragment, a single-domain antibody, a diabody, or a linear antibody.

[0177] 14. The antibody or antigen-binding fragment thereof according to Embodiment 13, wherein the single-chain antibody is scFv.

[0178] 15. The antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 14, wherein the antibody is a bispecific antibody or a multispecific antibody.

[0179] 16. An isolated nucleic acid encoding an anti-TEM8 antibody or an antigen-binding fragment thereof as described in any one of Embodiments 1 to 15.

[0180] 17. An expression vector comprising the nucleic acid described in Embodiment 16.

[0181] 18. An expression vector according to Embodiment 17, which is a pCDNA vector, for example, a pCDNA3.4 vector.

[0182] 19. A host cell comprising the nucleic acid described in Embodiment 16 or the expression vector described in Embodiment 17 or 18.

[0183] 20. A host cell according to Embodiment 19, which is prokaryotic or eukaryotic.

[0184] 21. A host cell according to Embodiment 20, selected from the group consisting of Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for producing antibodies or antigen-binding fragments thereof.

[0185] The host cell according to Embodiment 21, which is a 22.293 cell or a CHO cell.

[0186] 23. A method for producing an anti-TEM8 antibody or an antigen-binding fragment thereof, comprising culturing a host cell described in any one of Embodiments 19 to 22 under conditions suitable for the expression of a nucleic acid encoding the anti-TEM8 antibody or an antigen-binding fragment described in any one of Embodiments 1 to 15, and optionally isolating the antibody or an antigen-binding fragment thereof, further optionally recovering the anti-TEM8 antibody or an antigen-binding fragment thereof from the host cell.

[0187] 24. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof as described in any one of Embodiments 1 to 15, and an additional therapeutic agent.

[0188] 25. The antibody-drug conjugate according to Embodiment 24, wherein the additional therapeutic agent is a cytotoxic agent, for example, an MMAE, and optionally the MMAE is conjugated to an antibody or its antigen-binding fragment via a Vc linker, for example, the antibody or its antigen-binding fragment is linked to a VcMMAE to form an antibody-drug conjugate.

[0189] 26. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of Embodiments 1 to 15, or an antibody-drug conjugate as described in Embodiment 24 or 25, and optionally a pharmaceutically acceptable auxiliary material.

[0190] 27. A pharmaceutical composition comprising an antibody or its antigen-binding fragment according to any one of Embodiments 1 to 15, or an antibody-drug conjugate according to Embodiment 24 or 25, and an additional therapeutic agent.

[0191] 28. A pharmaceutical combination comprising an antibody or its antigen-binding fragment as described in any one of Embodiments 1 to 15, or an antibody-drug conjugate as described in Embodiment 24 or 25, and an additional therapeutic agent.

[0192] 29. The pharmaceutical composition according to Embodiment 27 or the pharmaceutical combination according to Embodiment 28, wherein the additional therapeutic agent is selected from the group consisting of chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressants).

[0193] 30. Use in the preparation of a pharmaceutical for the prevention or treatment of tumors in a subject or individual of an effective amount of an anti-TEM8 antibody or its antigen-binding fragment according to any one of Embodiments 1 to 15, or an antibody-drug conjugate according to Embodiment 24 or 25, or a pharmaceutical composition according to Embodiment 26.

[0194] 31. The use according to Embodiment 30, wherein the tumor is a solid tumor, for example, the tumor is cancer, for example, the cancer is selected from the group consisting of colon cancer, rectal cancer, or colorectal cancer.

[0195] 32. The use according to embodiment 30 or 31, further comprising administering one or more additional treatments in combination to the subject.

[0196] 33. The use according to Embodiment 32, wherein the treatment comprises a treatment mode and / or additional therapeutic agents.

[0197] 34. The use according to Embodiment 33, wherein the additional therapeutic agent is selected from the group consisting of chemotherapeutic agents, cytokines, other antibodies, cytotoxic agents, vaccines, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressants), and / or the mode of treatment includes surgical procedures and / or radiotherapy.

[0198] 35. A method for detecting TEM8 in a sample, (a) Contacting the sample with the anti-TEM8 antibody or its antigen-binding fragment described in any of Embodiments 1 to 15, (b) A method comprising detecting the formation of a complex between an anti-TEM8 antibody or an antigen-binding fragment thereof and TEM8, wherein the anti-TEM8 antibody is optionally labeled for detection.

[0199] All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. Any or all of the features described above and throughout this application may be combined in various embodiments of the present invention. Furthermore, the materials, methods, and examples described herein are illustrative and not intended to limit the invention. Other features, purposes, and advantages of the present invention will become apparent from this specification, the drawings, and the appended claims. [Examples]

[0200] Example 1. Interspecific sequence homology of TEM8 antigen and construction of a stable high-expression cell line.

[0201] Homology analysis was performed on TEM8 sequences from different species (human TEM8 (hTEM8, SEQ ID NO: 68), cynomolgus monkey TEM8 (CynoTEM8), mouse TEM8 (mTEM8, SEQ ID NO: 69), rat TEM8 (ratTEM8), and dog TEM8 (dogTEM8, SEQ ID NO: 70)), and it was found that TEM8 exhibits high homology in humans, cynomolgus monkeys, mice, and rats. The extracellular region sequences of human TEM8 and cynomolgus monkey TEM8 are identical. The sequences of mouse TEM8 and rat TEM8 are identical. The sequence homology between human TEM8 and mouse TEM8 is 98.9% (see Table 1).

[0202] [Table 8]

[0203] The nucleic acids encoding human TEM8 (SEQ ID NO: 68) and mouse TEM8 (SEQ ID NO: 69) were cloned into the expression vector pCDNA3.1(+) (constructed by Beijing Tsingke Biotech Co., Ltd.), and then transfected into CHO-K1 cells (Cobioer Biosciences Co., Ltd., catalog number: CBP60296) using the transfection reagent Lipofectamine™ 3000 (ThermoFisher, L3000075). On day 14 after transfection, these cells were subjected to flow cell sorting using L2 (67nM) (L2 antibody heavy chain: SEQ ID NO: 74; L2 antibody light chain: SEQ ID NO: 75) as the primary antibody and goat anti-human IgG H&L (FITC) (abcam, 6854) as the secondary antibody. Cells with a positive rate of 2.5% were selected and cultured, and after flow cytometry analysis, the culture of stable cell lines was completed. Among these, CHO stable cells that highly express human TEM8 were named CHO-hTEM8, and CHO stable cells that highly express mouse TEM8 were named CHO-mTEM8. CHO-hTEM8 and CHO-mTEM8 were detected by flow cytometry analysis using L2 (67nM) (L2 antibody heavy chain: SEQ ID NO: 74; L2 antibody light chain: SEQ ID NO: 75) as the primary antibody and goat anti-human IgG H&L (FITC) (abcam, ab6854) as the secondary antibody. The results are shown in Figure 1. In Figure 1, the left panel shows the flow cytometry results for CHO-hTEM8, and the right panel shows the flow cytometry results for CHO-mTEM. These results indicate that CHO-hTEM8 cells and CHO-mTEM8 cells highly express hTEM8 and mTEM8, respectively.

[0204] Example 2. Screening of TEM8-specific antibodies using a complete human scFv phage library.

[0205] The process for screening fully human scFv phage antibodies was as follows: A phage library of 2 × 10^12 cells (CRO, human naive scFv library) was added to 1 × 10^7 CHO-K1 negative cells (Cobioer Biosciences Co., Ltd., catalog number: CBP60296) and incubated at 4°C for 2 hours. The phage suspension after negative panning was added to 1 × 10^7 CHO-hTem8 cells and incubated at 4°C for 2 hours. The incubated mixture was washed 8-11 times with PBS, and unbound phages were removed by centrifugation. Cell-bound phages were eluted with elution buffer (glycine-hydrochloride buffer, 0.2 M, pH 2.5). After repeating the above panning three times, specific binding between the phage supernatant and hTEM8-his antigen (hTEM8 containing 6 His tags) was detected to obtain positive clones, and the results are shown in Table 2.

[0206] [Table 9] Five positive phage clones were sequenced and identified, and the sequence results were obtained. The original light chain of 6A6 contained the glycosylation site NRS, but this was modified to QRS by mutation, and the clone was named 6A6Q. The sequences of the light chain variable region and heavy chain variable region of the scFv obtained by screening are shown in the sequence listing.

[0207] Example 3. Construction of IgG antibody and FACS detection of the binding activity of IgG antibody to TEM8-overexpressing cells.

[0208] Production of IgG antibodies Nucleic acids encoding the variable regions of the antibody's light and heavy chains were cloned into expression vector pCDNA 3.4 containing nucleic acids encoding either the heavy chain constant region (SEQ ID NO: 71) or the light chain constant region (SEQ ID NO: 72; SEQ ID NO: 73) (combinations are shown in the table below). These vectors were co-introduced into CHO suspension cells for expression, the cell supernatant was collected, purified using a Protein A column, and the purified antibodies were detected for size and purity by SDS-PAGE and HPLC-SEC. [Table 10]

[0209] Detection of IgG antibody binding activity to cells The TEM8-overexpressing cells (CHO-hTEM8 and CHO-mTEM8) prepared in Example 1 were treated with trypsin and 1 × 10⁶ cells were placed in 100 μL of a U-bottom 96-well plate. 6 The antibodies were added in a / well manner. Then, each of the antibodies (3A3, 6A6Q, 7E3, 11B7, 3B3, L2 (L2 antibody heavy chain: SEQ ID NO: 74; L2 antibody light chain: SEQ ID NO: 75)) and anti-HEL antibodies (anti-HEL-human IgG1, Biointron, B117901) were subjected to 3-fold serial dilutions using PBS, starting at 26.67 nM, to obtain eight concentration gradients. 100 μL of diluted antibody was added to the cell plate to resuspend the cells, the plate was incubated at 4°C for 1 hour, and then washed three times with PBS. Secondary antibody: anti-human IgG H&L (FITC, abcam, ab6854) was diluted 1:2000 in PBS, and 100 μL of the diluted secondary antibody was added to a cell plate to resuspend the cells. The plate was incubated at 4°C for 1 hour, and then washed three times with PBS. The cells were resuspended in 100 μL of PBS, binding signals were detected by FACS, and the data were analyzed using Graphpad.

[0210] The results are shown in Figure 2. All five antibodies, 3A3, 6A6Q, 7E3, 11B7, and 3B3, cross-bound to human and mouse TEM8-expressing cells. The EC50 values ​​for binding to CHO-hTEM8 were 0.71 nM, 0.63 nM, 0.75 nM, 0.85 nM, and 1.51 nM, respectively. The EC50 values ​​for binding to CHO-mTEM8 were 1.48 nM, 1.16 nM, 1.41 nM, 1.21 nM, and 2.14 nM, respectively. The control antibody L2 bound to CHO-hTEM8 and CHO-mTEM8 with EC50 values ​​of 1.72 nM and 2.84 nM, respectively.

[0211] Example 4. Antibody Endocytosis Assay The antibody endocytosis assay employed the classic DT3C method and included the following steps.

[0212] Logarithmically growing TEM8-overexpressing cells (CHO-hTEM8 & CHO-mTEM8) were treated with trypsin and seeded at a rate of 5000 cells / well in 100 μL of TEM8 in 96-well plates. The plates were incubated at 37°C in a 5% CO2 constant temperature incubator for 24 hours. Recombinant DT3C protein (CUSABIO, CSB-EP360556CQR1) was diluted to 587 nM in cell culture medium (Ham's F-12K, 10% FBS). The antibody was diluted to 267 nM in cell culture medium. The diluted recombinant DT3C protein was mixed with an equal volume of antibody to obtain a mixture, which was incubated at room temperature for 30 minutes. The mixture was subjected to 3-fold serial dilution in cell culture medium (Ham's F-12K, 10% FBS) to obtain 10 concentration gradients. 100 μL of the diluted sample was added to a cell plate and mixed thoroughly, and the plate was incubated in a 37°C, 5% CO2 incubator for 48 hours. 20 μL of Cell Counting Kit-8 (CellorLab, CX001) was added to the plate, and the plate was incubated in a 37°C, 5% CO2 incubator for 3 hours. Absorbance values ​​at 450 nm were measured using a microplate reader, and the data were then analyzed using Graphpad.

[0213] As shown in Figure 3, each mixture of DT3C with five types of antibodies and the control antibody L2 killed cells, indicating that all antibodies were endocytized by the cells.

[0214] Example 5. Identification of antibody epitopes The antibody epitopes were identified by a competitive ELISA including the following steps.

[0215] The antibodies were biotinylated using the EZ-Linker NHS kit (Thermo Scientific, catalog number: 20217). 2 nM labeled antibody was mixed with 240 nM unlabeled antibody in a molar ratio of 1:120, and the mixture was incubated with CHO-hTEM8 at 4°C for 1 hour. After incubation, the cells were washed three times with PBS, and the supernatant was removed by centrifugation. SA-bio secondary antibody (FITC-streptavidin conjugate (AAT Bioquest, catalog number: 16910)) was added to the sample, the mixture was incubated at 4°C for 1.5 hours, and washed three times with PBS. The mixture was centrifuged to remove the supernatant, and the cells were resuspended in 120 μL of PBS and detected by FACS for epitope binding. As shown in Table 3, the results indicated that 3A3, 6A6Q, 7E3, 3B3, and L2 competed for binding and belonged to the same antigenic determinant cluster Bin#1, while 11B7 did not compete for binding with any of the above antibodies and belonged to a different antigenic determinant cluster Bin#2.

[0216] [Table 11]

[0217] Example 6. Preparation of TEM8 antibody-drug conjugate

[0218] Antibodies (L2, 7E3, 3A3, 6A6Q, 3B3, and 11B7) were reduced by adding an appropriate amount of reducing agent to expose the sulfhydryl groups between antibody chains, and VcMMAE (MCE, catalog number: 646502-53-6) was dissolved in DMSO to prepare a solution.

[0219] Dissolved VcMMAE was added according to an antibody-to-linker-payload molar ratio of 10:1, and the antibody and VcMMAE were conjugated together via a Michael addition reaction between the sulfhydryl group and the maleimide group on the linker.

[0220] The conjugated mixture was subjected to buffer replacement to remove residual VcMMAE, and the post-reaction purity and DAR (drug / antibody ratio) values ​​were determined by SEC (size exclusion chromatography) and HIC (hydrophobic chromatography), respectively. The conjugation results for different candidate molecules are shown in Table 4.

[0221] [Table 12]

[0222] Example 7. In vitro cell killing assay of ADCs

[0223] HCT116 (catalog number: CBP60028, Cobioer Biosciences Co., Ltd.) in the logarithmic growth phase was trypsin-treated and seeded at 3000 cells / well in 100 μL of water into a 96-well plate. The plate was incubated at 37°C in a 5% CO2 incubator for 24 hours. ADC was subjected to 5-fold serial dilutions starting at 30000 nM using cell culture medium (Ham's F-12K, 10% FBS, AusGenex, catalog number: FBS500-S) to obtain a concentration gradient of 10. 10 μL of the diluted sample was added to the cell plate and mixed thoroughly. The plate was incubated at 37°C in a 5% CO2 incubator for 72 hours. 10 μL of Cell Counting Kit-8 (CellorLab, CX001) was added to a plate, and the plate was incubated in a 37°C, 5% CO2 constant temperature incubator for 3 hours. Absorbance values ​​at 450 nm were measured using a microplate reader. The data were analyzed using Graphpad. Table 5 shows the in vitro killing activity of the TEM8 antibody-drug conjugate, and Figure 4 shows the IC50 curve.

[0224] [Table 13]

[0225] Example 8. In vivo efficacy of the HCT116 CDX model

[0226] In Balb / c nude mice (obtained from GemPharmatech Co.,Ltd.) (Jiangsu Provincial Laboratory Animal Quality Certificate: 202223503), a subcutaneous xenograft tumor model was constructed using HCT116 human colon cancer cells (catalog number: CBP60028, Cobioer Biosciences Co.,Ltd.), and the in vivo efficacy of each TEM8 antibody-drug conjugate was evaluated.

[0227] Specifically, HCT116 cells in the logarithmic growth phase were collected, the culture medium was removed, and the cells were washed twice with PBS. These cells were then inoculated into the right dorsal side of Balb / c nude mice at a dose of 7.5*10^6 / 200 ul / mouse (without Matrigel). Twelve days after inoculation, the tumors had grown to an appropriate size, and the tumor volume reached a standard level (tumor volume > 100 mm). 3 Mice were randomly divided into seven groups (5 mice per group): a blank control group (PBS), a positive control L2-MMAE group, a 3A3-MMAE group, a 6A6-MMAE group, a 7E3-MMAE group, a 3B3-MMAE group, and an 11B7-MMAE group. DPBS was used as the solvent for all related drugs.

[0228] Administration began on the day of group assignment (day 0), and the administration regimen consisted of intraperitoneal (ip) administration of 5 mg / kg twice weekly (BIW) for 28 days. The body weight and tumor volume of the mice were measured three times a week after group assignment for administration. The size of the mouse tumors was measured each time using calipers, and the volume was calculated using the formula V = (L × W^2) / 2 (where L is the longest diameter of the tumor and W is the shortest diameter perpendicular to the longest diameter). The experiment was terminated 28 days after group assignment for administration, i.e., after the 9th dose, and all mice were euthanized.

[0229] As shown in Figure 5, all treatment groups (i.e., the 3A3-MMAE group, 6A6-MMAE group, 7E3-MMAE group, 3B3-MMAE group, and 11B7-MMAE group) showed a significant inhibitory effect on tumor growth, and statistically significant differences were observed compared to the blank control group.

[0230] Based on data from the entire administration cycle, the 6A6-MMAE group and the 7E3-MMAE group showed a more significant advantage, with both groups demonstrating statistically significant differences compared to the positive control L2-MMAE group (p=0.0107 and p=0.0163).

[0231] In the final data, the tumor volume in the blank control group was 2478.9 ± 637.3 mm². 3 The tumor volume in the positive control L2-MMAE group was 437.6 ± 163.5 mm².3 and the tumor volumes in the 3A3-MMAE, 6A6-MMAE, 7E3-MMAE, 3B3-MMAE, and 11B7-MMAE groups were 487.9 ± 192.3 mm 3 , 123.1 ± 17.7 mm 3 , 141.9 ± 51.9 mm 3 , 383.8 ± 147.2 mm 3 , and 361.4 ± 146.2 mm 3 respectively. It was shown that all of the ADC drugs had strong tumor suppression activity. From the above results, it was shown that all of the ADC drugs had strong tumor suppression activity.

[0232] Example 9. In Vivo Efficacy of HCT116 CDX Model

[0233] In Balb / c nude mice (obtained from Shanghai Lingchang Biotechnology Co., Ltd., license number: SCXK(Shanghai)2018 - 0003), a subcutaneous xenograft tumor model was constructed using HCT116 human colon cancer cells (Cobioer Biosciences Co., Ltd., catalog number: CBP60028), and the in vivo efficacy of the TEM8 antibody drug 6A6Q naked antibody was evaluated.

[0234] Specifically, HCT116 cells in the logarithmic growth phase were collected, the culture medium was removed, and the cells were washed twice with PBS and then inoculated into the right dorsal side of Balb / c nude mice at an inoculation dose of 1*10^7 / 200ul / mouse (without Matrigel). Seven days after inoculation, the tumors grew to an appropriate size, and when the tumor volume reached the standard (tumor volume > 100 mm 3 ), the mice were randomly divided into two groups (6 mice per group): the blank control group (PBS) and the 6A6Q naked antibody group. For all related drugs, DPBS was used as the solvent.

[0235] Administration started on the day of grouping (day 0), and the administration regimen was to perform intraperitoneal (i.p.) administration at a dose of 20 mg / kg once a week (QW) for 23 days.

[0236] The body weight and tumor volume of the mice were measured three times a week for one week after grouping for administration. The size of the mouse tumor was measured each time using calipers, and the volume was calculated using the formula V = (L × W^2) / 2 (where L is the longest diameter of the tumor and W is the short diameter perpendicular to the longest diameter). 23 days after grouping for administration, that is, after the fourth administration, the experiment was terminated and all the mice were euthanized.

[0237] As shown in Figure 6, based on the data for the entire administration cycle, the 6A6Q naked antibody group showed a significant inhibitory effect on tumor growth, and a statistically significant difference was observed compared with the blank control group (p < 0.0001).

[0238] In the final data, the tumor volume of the blank control group was 1447.5 ± 190.8 mm 3 and that of the 6A6Q naked antibody group was 776.3 ± 52.4 mm 3 These results demonstrated that the TEM8 antibody drug 6A6Q naked antibody has stronger tumor inhibitory activity.

[0239]

Table 14-1

Table 14-2

Table 14-3

Table 14-4

Table 14-5

Table 14-6

Table 14-7

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to TEM8, wherein the antibody is 1) The three complementarity-determining regions HCDR1, HCDR2, and HCDR3 included in VH as described in SEQ ID NO: 1, and the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 included in VL as described in SEQ ID NO: 2; 2) The three complementarity-determining regions HCDR1, HCDR2, and HCDR3 included in VH as described in SEQ ID NO: 14, and the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 included in VL as described in SEQ ID NO: 15; 3) The three complementarity-determining regions HCDR1, HCDR2, and HCDR3 included in VH as described in SEQ ID NO: 27, and the three complementarity-determining regions LCDR1, LCDR2, and LCDR3 included in VL as described in SEQ ID NO: 28 or 40; 4) The three complementarity-determining regions HCDR1, HCDR2, and HCDR3 included in VH as described in SEQ ID NO: 42, and the three complementarity-determining regions LCDR1, LCDR2, and HCDR3 included in VL as described in SEQ ID NO: 43; or 5) Including the three complementarity determination regions HCDR1, HCDR2, and HCDR3 included in VH as described in SEQ ID NO: 55, and the three complementarity determination regions LCDR1, LCDR2, and LCDR3 included in VL as described in SEQ ID NO: 56, Preferably, the HCDR and LCDR are determined according to an IMGT or Kabat scheme, and the antibody or antigen-binding fragment thereof.

2. An antibody or antigen-binding fragment thereof that specifically binds to TEM8, wherein the antibody comprises three complementarity-determining regions HCDR1, HCDR2, and HCDR3 of the heavy chain variable region, and three complementarity-determining regions LCDR1, LCDR2, and LCDR3 of the light chain variable region, and each of the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 contains or consists of the amino acid sequences listed in the SEQ ID NO in the table below, based on the IMGT scheme. Table 1 Alternatively, an antibody or antigen-binding fragment thereof, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each contain or consist of the amino acid sequences described in the SEQ ID NO in the table below, based on the Kabat scheme. Table 2

3. The antibody includes a heavy chain variable region, and the heavy chain variable region includes or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 14, 27, 42, or 55, or the heavy chain variable region includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 14, 27, 42, or 55, and / or The TEM8-conjugated antibody or antigen-conjugated fragment according to claim 1 or 2, wherein the antibody includes a light chain variable region, and the light chain variable region includes or consists of an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 15, 28, 40, 43, or 56, or the light chain variable region includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 2, 15, 28, 40, 43, or 56.

4. An anti-TEM8 antibody or its antigen-binding fragment, wherein the antibody comprises a heavy chain variable region and a light chain variable region. (i) The heavy chain variable region includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 1, and the light chain variable region includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 2, (ii) The heavy chain variable region includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 14, and the light chain variable region includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:

15. (iii) The heavy chain variable region includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 27, and the light chain variable region includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 28 or 40, (iv) The heavy chain variable region includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 42, and the light chain variable region includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:

43. (v) An anti-TEM8 antibody or its antigen-binding fragment, wherein the heavy chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO: 55, and the light chain variable region comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence described in SEQ ID NO:

56.

5. The TEM8-conjugated antibody or antigen-conjugated fragment thereof according to claim 1 or 2, wherein the antibody comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region and the light chain variable region each contain or consist of an amino acid sequence described in the SEQ ID NO in the table below. Table 3

6. The antibody includes a heavy chain constant region, for example, the heavy chain constant region of IgG1, IgG2, IgG3, or IgG4, for example, the heavy chain constant region of IgG1, and the heavy chain constant region is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 71, or (ii) Containing or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 71, A TEM8-conjugated antibody or its antigen-conjugated fragment according to any one of claims 1 to 5.

7. The antibody comprises a light chain constant region, for example, a lambda or kappa light chain constant region, and for example, the light chain constant region is (i) an amino acid sequence having at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 72 or 73, or (ii) SEQ ID NO: Containing or consisting of an amino acid sequence selected from the group consisting of 72 or 73, A TEM8-conjugated antibody or its antigen-conjugated fragment according to any one of claims 1 to 6.

8. The antibody comprises a heavy chain constant region and a light chain constant region, The heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO: 71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO: 72 or 73. A TEM8-conjugated antibody or its antigen-conjugated fragment according to any one of claims 1 to 7.

9. The antibody comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain variable region and a heavy chain constant region, and the light chain comprises a light chain variable region and a light chain constant region. (i) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 1, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 2, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO: 71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:

72. (ii) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 14, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 15, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO: 71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:

72. (iii) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 27, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 28 or 40, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO: 71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:

73. (iv) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 42, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 43, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO: 71, the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO: 73, or (v) The heavy chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 55, the light chain variable region contains or consists of the amino acid sequence described in SEQ ID NO: 56, the heavy chain constant region contains or consists of the amino acid sequence described in SEQ ID NO: 71, and the light chain constant region contains or consists of the amino acid sequence described in SEQ ID NO:

73. A TEM8-conjugated antibody or its antigen-conjugated fragment according to any one of claims 1 to 8.

10. The following characteristics: (1) It can bind with high affinity to TEM8, for example, human, cynomolgus monkey, rat, or mouse TEM8. (2) It can bind with high affinity to TEM8 expressed on the surface of the cell membrane, for example, to human, cynomolgus monkey, rat, or mouse TEM8. (3) The ability to induce endocytosis of the antibody or its fragment or a molecule containing the antibody or its fragment (e.g., ADC) by TEM8-positive cells (e.g., TEM8-overexpressing CHO cells), (4) It is possible to target and kill TEM8-positive cells, such as TEM8-positive tumor cells. (5) The ability to treat tumors such as cancer, (6) Suitable for constructing antibody-drug conjugates (ADCs) for targeting and killing TEM8-positive cells, such as TEM8-positive tumor cells, or for treating tumors such as cancer. (7) Inhibiting the binding of any of the antibodies listed in claim 9 to TEM8, (8) To bind to an epitope that is the same as or overlaps with any one of the antibodies described in claim 9, or (9) With respect to binding to TEM8, it competes with any one of the antibodies described in claim 9. A TEM8-conjugated antibody or antigen-conjugated fragment thereof according to any one of claims 1 to 8, having one or more of the above.

11. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 10, wherein the antibody is a monoclonal antibody.

12. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 11, wherein the antibody is a human antibody.

13. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 12, wherein the antigen-binding fragment is an antibody fragment selected from the group consisting of Fab, Fab', Fab'-SH, Fv, a single-chain antibody (e.g., scfv), a (Fab')2 fragment, a single-domain antibody, a diabody, or a linear antibody.

14. The antibody or antigen-binding fragment thereof according to claim 13, wherein the single-chain antibody is scFv.

15. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 14, wherein the antibody is a bispecific antibody or a multispecific antibody.

16. An isolated nucleic acid encoding an anti-TEM8 antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15.

17. An expression vector comprising the nucleic acid described in claim 16.

18. The expression vector according to claim 17, which is a pCDNA vector, for example, a pCDNA 3.4 vector.

19. A host cell comprising the nucleic acid described in claim 16 or the expression vector described in claim 17 or 18.

20. The host cell according to claim 19, which is prokaryotic or eukaryotic.

21. The host cell according to claim 20, selected from the group consisting of Escherichia coli cells, yeast cells, mammalian cells, or other cells suitable for producing antibodies or antigen-binding fragments thereof.

22. The host cell according to claim 21, which is a 293 cell or a CHO cell.

23. A method for producing an anti-TEM8 antibody or an antigen-binding fragment thereof, comprising: culturing a host cell according to any one of claims 19 to 22 under conditions suitable for the expression of a nucleic acid encoding the anti-TEM8 antibody or antigen-binding fragment according to any one of claims 1 to 15; optionally isolating the antibody or antigen-binding fragment thereof; and optionally further comprising recovering the anti-TEM8 antibody or antigen-binding fragment thereof from the host cell.

24. An antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 15, and an additional therapeutic agent.

25. The antibody-drug conjugate according to claim 24, wherein the additional therapeutic agent is a cytotoxic agent, for example, an MMAE, and optionally the MMAE is conjugated to an antibody or its antigen-binding fragment via a Vc linker, for example, the antibody or its antigen-binding fragment is linked to VcMMAE to form an antibody-drug conjugate.

26. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or an antibody-drug conjugate according to claim 24 or 25, and optionally a pharmaceutically acceptable auxiliary material.

27. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or an antibody-drug conjugate according to claim 24 or 25, and an additional therapeutic agent.

28. A pharmaceutical combination comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 15, or an antibody-drug conjugate according to claim 24 or 25, and an additional therapeutic agent.

29. The pharmaceutical composition according to claim 27 or the pharmaceutical combination according to claim 28, wherein the additional therapeutic agent is selected from the group consisting of chemotherapeutic agents, cytokines, cytotoxic agents, other antibodies, small molecule drugs, or immunomodulators (e.g., immunosuppressants).

30. Use of an effective amount of an anti-TEM8 antibody or its antigen-binding fragment according to any one of claims 1 to 15, or an antibody-drug conjugate according to claim 24 or 25, or a pharmaceutical composition according to claim 26, in the preparation of a pharmaceutical for the prevention or treatment of tumors in a subject or individual.

31. The use according to claim 30, wherein the tumor is a solid tumor, for example, the tumor is cancer, for example, the cancer is selected from the group consisting of colon cancer, rectal cancer, or colorectal cancer.

32. The use according to claim 30 or 31, further comprising administering one or more additional treatments in combination to the subject.

33. The use according to claim 32, wherein the treatment comprises a treatment method and / or additional therapeutic agents.

34. The use according to claim 33, wherein the additional therapeutic agent is selected from the group consisting of chemotherapeutic agents, cytokines, other antibodies, cytotoxic agents, vaccines, small molecule drugs, or immunomodulatory agents (e.g., immunosuppressants), and / or the mode of treatment includes surgical procedures and / or radiotherapy.

35. A method for detecting TEM8 in a sample, (a) Contacting the sample with the anti-TEM8 antibody or its antigen-binding fragment according to any one of claims 1 to 15, (b) A method comprising detecting the formation of a complex between an anti-TEM8 antibody or an antigen-binding fragment thereof and TEM8, wherein the anti-TEM8 antibody is optionally labeled for detection.

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