WO2019015696A2 - New target for treating cancer - Google Patents

Abstract

The present invention relates to a target for treating cancer. Specifically, the present invention relates to the use of the extracellular loop 1 (ECL 1) of TM4SF1 as a target for cancer treatment. More specifically, the present invention relates to specificity binding in respect of antibodies of the ECL 1 of TM4SF1 and polypeptides of said ECL 1.

Description

New targets for the treatment of cancer

This application claims the priority of a Chinese patent application filed on July 17, 2017 by the Chinese Patent Office, application number 201710581764.8, the invention titled "New Target for Cancer Treatment", and the Chinese patent filed on November 17, 2017. The priority of the Chinese Patent Application No. 201711146247.4, entitled "New Target for the Treatment of Cancer", the entire contents of which is hereby incorporated by reference.

Technical field

The present invention relates to a target for the treatment of cancer, in particular, the invention relates to the use of extracellular loop 1 (ECL1) of TM4SF1 as a target for cancer therapy. More specifically, the present invention relates to an antibody that specifically binds to extracellular loop 1 (ECL1) of TM4SF1 and an extracellular loop 1 (ECL1) polypeptide of TM4SF1.

Background technique

Angiogenesis involves the formation of new blood vessels from existing blood vessel networks by vascular endothelial cell (EC) proliferation, migration, and morphogenesis. There is convincing evidence that the establishment of a vascular network supply through angiogenesis is essential for the development of normal tissues and organs during embryonic development and the process of tissue proliferation under pathological conditions. The vascular network provides a pathway for the delivery of oxygen and nutrients as well as the removal of catabolic products, representing the rate-limiting step in most growth processes occurring in multicellular organisms. Therefore, it has been generally accepted that vascular compartments are necessary not only for the development and differentiation of organs during embryogenesis, but also for the healing and regeneration functions of adult wounds.

Angiogenesis is also implicated in the pathogenesis of a variety of conditions, including but not limited to: cancer, obesity, proliferative retinopathy, age-related macular degeneration, tumors, rosacea, atherosclerosis, rheumatoid arthritis (RA) , cellular immunity and psoriasis. Angiogenesis is a cascade of processes that degrade the extracellular matrix at a localized location after protease release, proliferation of vascular endothelial cells, and migration of capillaries to angiogenic stimuli. Given the significant physiological and pathological importance of angiogenesis, much work has been devoted to elucidating the factors that can modulate this process.

TM4SF1 (Transmembrane-4L Six Family member 1) was discovered as a tumor cell antigen in 1986. It is a member of the L6 protein superfamily and is a four-transmembrane membrane protein with two Extracellular loop loop structure (Hellstrom et al. Cancer Res. 46: 3917-3923, 1986). It has been found that TM4SF1 is abundantly expressed on many cancer cells. Studies have also shown that TM4SF1 has only a small expression on vascular endothelial cells of normal tissues (DeNardo et al. Int J Rad Appl Instrum B. 18:621-631, 1991; Wright et al. Protein Sci. 9:1594-1600, 2000; Richman et al. Cancer Res. 5916s-5920s, 1995; O'Donnell et al. Prostate. 37: 91-97, 1998), and vascular endothelial cells in tumor tissues (Shih et al. Cancer Res. 69: 3272-3277, 2009; Zukauskas et al. Angiogenesis. 14 :345-354,2011), retinal neovascular endothelial cells (English, J Biomed Inform. 42:287-295, 2009), and VEGF-A-induced endothelial cells of neovascularization (Shih et al. Cancer Res. 69:3272- Highly expressed in 3277, 2009) (Shih et al. Cancer Res. 69: 3272-3277, 2009; Zukauskas et al. Angiogenesis. 14:345-354, 2011).

Although studies have shown that TM4SF1 can be used as a marker of neovascularization to inhibit pathological angiogenesis and thus be used to treat pathological angiogenesis-related disorders, such as cancer, but whether TM4SF1 can pass signal transduction Lack of understanding of tumor growth and metastasis. In particular, little is known about how TM4SF1 affects tumor cells at the molecular level. Therefore, it is necessary to explore a therapeutic method that directly targets tumor cells on the surface of tumor cell surface TM4SF1, for example, to study a novel anti-TM4SF1 antibody for anti-tumor.

Summary of the invention

The present inventors have unexpectedly discovered in the study that the extracellular loop 1 (ECL1) of TM4SF1 has antibody binding potential and can be a new therapeutic target. In particular, the inventors have found that an antibody capable of specifically binding to extracellular loop 1 (ECL1) of TM4SF1 can be a potential anticancer drug.

Based on the above findings, the present invention provides the use of an antibody that specifically binds to extracellular loop 1 (ECL1) of TM4SF1 in the preparation of a medicament for treating cancer.

In some embodiments, an antibody of the invention is a chimeric, humanized or human antibody.

In some embodiments, an antibody of the invention is a monoclonal antibody.

In some embodiments, an antibody of the invention is a bispecific antibody.

In some embodiments, an antibody of the invention is an antibody-drug conjugate.

In some embodiments, an antibody of the invention specifically binds to an extracellular loop 1 (ECL1) polypeptide of TM4SF1, wherein the sequence is a contiguous fragment of SEQ ID NO: 2 (full length sequence of TM4SF1) The starting point is at position 27, 28, 29, 30, 31, 32 or 33 of SEQ ID NO: 2, and the end point is at position 42, 43 , 44, 45, 46 of SEQ ID NO: 2. Bit, 47 or 48 bits.

In some embodiments, an antibody of the invention specifically binds to an extracellular loop 1 (ECL1) polypeptide having the sequence SEQ ID NO: 4 (TM4SF1).

In some embodiments, the antibodies of the invention comprise heavy and light chains, wherein

(i) the heavy chain comprises three CDR regions having the sequences shown in SEQ ID No: 7, SEQ ID No: 8, and SEQ ID No: 9, respectively;

(ii) The light chain comprises three CDR regions having the sequences shown in SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12, respectively.

In some embodiments, the cancer treated by the method of the invention is selected from the group consisting of: ovarian cancer; lung cancer; gastric cancer; breast cancer; liver cancer; pancreatic cancer; skin cancer; malignant melanoma; head and neck cancer; sarcoma; cholangiocarcinoma; Kidney cancer; colon cancer; small intestine cancer; testicular cancer; placental choriocarcinoma; cervical cancer; testicular cancer; uterine cancer; prostate cancer; leukemia; multiple myeloma; malignant lymphoma;

In some embodiments, the cancer cells of the cancer treated by the methods of the invention express TM4SF1.

In another aspect, the invention provides a method of treating cancer comprising administering to a subject in need thereof a therapeutically effective amount of an antibody that specifically binds to extracellular loop 1 (ECL1) of TM4SF1.

In yet another aspect, the invention provides the use of an extracellular loop 1 (ECL1) polypeptide of TM4SF1 for the manufacture of a medicament for the treatment of cancer. One useful protocol is to use the extracellular loop 1 polypeptide of TM4SF1 or the TM4SF1 recombinant protein containing this region for immunization of animals to prepare specific antibodies for the treatment of tumors and other diseases. In some embodiments, the extracellular loop 1 polypeptide of TM4SF1 or the TM4SF1 recombinant protein comprising the region can be used for screening for anti-TM4SF1-specific antibodies, for example, for screening hybridoma monoclonal antibodies, screening phage display antibody libraries. The affinity of the monoclonal antibody can also be determined by using the extracellular loop 1 polypeptide of TM4SF1 or the TM4SF1 recombinant protein containing the region, screening for anti-TM4SF1 antibody with neutralizing ability, and performing various cell tests and animal tests for screening, Confirm and validate antibodies or other molecular forms of the drug.

In some embodiments, the sequence of the extracellular loop 1 (ECL1) polypeptide of TM4SF1 of the invention is a contiguous fragment of SEQ ID NO: 2 (full length sequence of TM4SF1) and the origin is at position 27 of SEQ ID NO: 2, 28, 29, 30, 31, 32 or 33, the endpoint is at position 42, 43 , 44, 45, 46, 47 or 48 of SEQ ID NO: 2.

In some embodiments, the sequence of the extracellular loop 1 (ECL1) polypeptide of TM4SF1 of the invention is SEQ ID NO:4.

In yet another aspect, the invention provides an isolated TM4SF1 extracellular loop 1 (ECL1) polypeptide, the sequence of which is a contiguous fragment of SEQ ID NO: 2 (full-length sequence of TM4SF1) and whose origin is at 27 of SEQ ID NO: Bits, 28, 29, 30, 31, 32 or 33, the end point is at position 42, 43 , 44, 45, 46, 47 or 48 of SEQ ID NO: 2.

In some embodiments, the polypeptides of the invention have tumor suppressor activity.

In some embodiments, the sequence of the polypeptide of the invention is SEQ ID NO:4.

In yet another aspect, the invention provides an antibody that specifically binds to extracellular loop 1 (ECL1) of TM4SF1.

In some embodiments, the antibody of the invention is a chimeric, humanized or human antibody.

In some embodiments, the antibodies of the invention are monoclonal antibodies.

In some embodiments, an antibody of the invention is a bispecific antibody.

In still other embodiments, the antibodies of the invention have tumor suppressor activity, preferably have inhibitory activity against tumor metastasis, such as liver cancer, prostate cancer, lung cancer, colon cancer or metastasis thereof.

In still other embodiments, the antibodies of the invention are used to inhibit tumors, preferably for inhibiting tumor metastasis, such as liver cancer, prostate cancer, lung cancer, colon cancer, or metastasis thereof.

In some embodiments, an antibody of the invention specifically binds to an extracellular loop 1 (ECL1) polypeptide of TM4SF1, wherein the sequence is a contiguous fragment of SEQ ID NO: 2 (full length sequence of TM4SF1) The starting point is at position 27, 28, 29, 30, 31, 32 or 33 of SEQ ID NO: 2, and the endpoint is at position 42, 43 , 44, 45, 46 of SEQ ID NO: 2. Bit, 47 or 48 bits.

In some embodiments, an antibody of the invention specifically binds to an extracellular loop 1 (ECL1) polypeptide having the sequence TM4SF1 set forth in SEQ ID NO:4.

In some embodiments, the antibodies of the invention comprise heavy and light chains, wherein

(i) the heavy chain comprises three CDR regions having the sequences shown in SEQ ID No: 7, SEQ ID No: 8, and SEQ ID No: 9, respectively;

(ii) The light chain comprises three CDR regions having the sequences shown in SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12, respectively.

In a further aspect, the invention provides an antibody-drug conjugate comprising an antibody of the invention and a therapeutic agent, preferably the therapeutic agent is selected from the group consisting of a cytotoxic drug, an immunopotentiator, and a radioisotope, more preferably said The therapeutic agent is selected from the group consisting of a dolastatin peptide and derivatives thereof, and most preferably the therapeutic agent is selected from the group consisting of MMAE and MMAF.

In still another aspect, the invention provides a pharmaceutical composition comprising an ECL1 polypeptide of TM4SF1 of the invention, an antibody of the invention and/or a conjugate of the invention, and a pharmaceutically acceptable carrier.

In another aspect, the invention provides a conjugate comprising an antibody of the invention or a functional fragment thereof, conjugated to one or more therapeutic agents, preferably the therapeutic agent is a cytotoxic drug (eg An antimetabolite, an antitumor antibiotic, an alkaloid), an immunopotentiator or a radioisotope, more preferably the therapeutic agent is selected from maytansinoids (eg, Ansamitocin or Mertansine) )), dolastatin and derivatives thereof, most preferably the therapeutic agent is selected from the group consisting of MMAE (Monomethyl auristatin E, monomethyl auristatin peptide E) and MMAF (Monomethyl auristatin F, monomethyl ear) Inhibin peptide F). In other embodiments, the therapeutic agent can also be selected from those listed in Table 1 below.

Table 1 List of therapeutic agents available in the conjugates of the invention

In some embodiments, the therapeutic agent is coupled to the antibody or a functional fragment thereof via a linker. The linkers used in the present invention can be attached to the antibody by any means known in the art, preferably via a thiol group and/or an amino group. In a particularly preferred embodiment, the antibodies of the invention are linked to the linker via a thiol group. The joint used in the present invention may be a cleavable joint (i.e., a joint that can be broken in an in vivo environment) or a non-cleavable joint. In some embodiments, the linker of the invention is selected from a cleavable linker, preferably selected from the group consisting of a peptide, a guanidine, and a disulfide linker, such as a maleimidocaproyl-valine-citrulline-p-amino group. Benzyloxycarbonyl (hereinafter abbreviated as mc-vc-pAB or vc, ie, maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl). In other embodiments, the linkers of the invention are selected from non-cleavable linkers, such as maleimidocaproyl (hereinafter abbreviated as mc, ie, maleimidocaproyl). In other embodiments, the linker can also be selected from those listed in Table 2 below.

Table 2 List of available linkers in the conjugates of the invention

In another aspect, the invention provides a conjugate having the general formula Ab-(LU) _n , wherein Ab represents an antibody or functional fragment thereof according to the invention, and L represents a linker (eg mc-vc-pAB or mc) U represents a therapeutic agent (preferably the therapeutic agent is selected from the group consisting of a cytotoxic drug, an immunopotentiator, and a radioisotope, and more preferably the therapeutic agent is selected from the group consisting of maytansinoids, dolastatin peptides and derivatives thereof Most preferably the therapeutic agent is selected from the group consisting of MMAE and MMAF), and n is an integer from 1 to 8 ( eg 1, 2, 3, 4, 5, 6, 7, or 8). The joint used in the present invention may be a cleavable joint (i.e., a joint that can be broken in an in vivo environment) or a non-cleavable joint.

DRAWINGS

Figure 1 is a graph showing the affinity of the monoclonal antibody FC17-7 of the present invention and TM4SF1;

Figure 2 is a graph showing that monoclonal antibody FC17-7 blocks the interaction of TM4SF1 with DDR1 in an immunoprecipitation experiment;

Figure 3 shows that P-STAT3 is down-regulated when cells are treated with monoclonal antibody FC17-7 in the Western Blot assay;

Figure 4 shows that monoclonal antibodies attenuate tumor suspension sphere formation ability after treatment of cells with monoclonal antibody FC17-7;

Figures 5-8 are graphs showing that monoclonal antibodies significantly inhibit brain metastasis, bone metastasis, and prolonged survival in breast cancer;

Figure 9 is a diagram showing that the TM4SF1 extracellular loop 1 small peptide disrupts the interaction of TM4SF1 with DDR1;

Figure 10 is a graph showing that TM4SF1 extracellular loop 1 small peptide inhibits downstream signal transduction;

Figure 11 to Figure 12 are graphs showing that TM4SF1 extracellular loop 1 small peptide significantly inhibits metastasis of breast cancer;

Figure 13 is a graph showing that TM4SF1 extracellular loop 1 small peptide prolongs mouse survival.

Detailed ways

definition:

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art. For specific definitions and terminology in the art, the professional can refer to Current Protocols in Molecular Biology (Ausubel). Abbreviations for amino acid residues are standard 3-letter and/or 1-letter codes used in the art to refer to one of the 20 commonly used L-amino acids. In particular, the meaning of the terms used herein can also be found in Chinese patent application 201110131029.X.

Although numerical ranges and parameter approximations are shown in the broad scope of the invention, the numerical values shown in the specific embodiments are described as being as accurate as possible. However, any numerical value inherently contains certain errors due to the standard deviations present in their respective measurements. In addition, all ranges disclosed herein are to be understood as encompassing any and all sub-ranges. For example, the stated range of "1 to 10" should be considered to encompass any and all subranges between the minimum 1 and the maximum 10 (including the endpoints); that is, all subranges starting with a minimum of 1 or greater For example, 1 to 6.1, and a subrange ending at a maximum of 10 or less, such as 5.5 to 10. In addition, any reference that is referred to as "incorporated herein" is understood to be incorporated in its entirety.

In addition, it should be noted that, as used in the specification, the singular The term "or" can be used interchangeably with the term "and/or" unless the context clearly dictates otherwise.

The term "soluble" protein as used herein refers to a protein that is soluble in aqueous solution at biologically relevant temperatures, pH levels, and osmotic pressures. In certain embodiments, the fusion proteins of the invention are soluble proteins. As used herein, "soluble fusion protein" means that the fusion protein does not comprise a transmembrane region and an intracellular region.

As used herein, the term "isolated" refers to the following substances and/or entities that (1) are separated from at least some of the components associated with and at the time of initial production (in the natural environment and/or in the experimental setting) and / or (2) by manual production, preparation and / or manufacturing. The isolated material and/or entity can be at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, About 98%, about 99%, substantially 100% or 100% of the other components whose initial association are separated. In certain embodiments, the fusion proteins of the invention are isolated fusion proteins.

The terms "portion" and "fragment" are used interchangeably to refer to a portion of a polypeptide, nucleic acid or other molecular construct.

The term "subject" as used herein refers to a mammal, such as a human, but may also be other animals, such as domestic animals (such as dogs, cats, etc.), livestock (such as cattle, sheep, pigs, horses, etc.) or experimental animals ( Such as monkeys, rats, mice, rabbits, guinea pigs, etc.).

The terms "identity," "percent identity," "homology," or "identity" as used herein, refer to sequence identity between two amino acid sequences or between nucleic acid sequences. Percent identity can be determined by aligning two sequences, which refers to the number of identical residues (ie, amino acids or nucleotides) that are shared by the sequences being shared. Standard algorithms in the art can be used (e.g., Smith and Waterman, 1981, Adv. Appl. Math. 2: 482; Needleman and Wunsch, 1970, J. MoI. Biol. 48: 443; Pearson and Lipman, 1988, Proc. Natl .Acad.Sci., USA, 85: 2444) or for sequence alignment and comparison by a computerized version of these algorithms (Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI) The versions are publicly available as BLAST and FASTA. In addition, ENTREZ available through the National Institutes of Health (Bethesda MD) can be used for sequence comparisons. When using the BLAST and Gapped BLAST programs, default parameters for various programs (e.g., BLASTN, available on the Internet site of the National Center for Biotechnology Information) can be used. In one embodiment, a GGC with a gap weight of 1 can be used to determine the percent identity of the two sequences such that each amino acid gap is given a weight as if it were a single amino acid mismatch between the two sequences. Alternatively, the ALIGN program (version 2.0), which is part of the GCG (Accelrys, San Diego, CA) sequence alignment software package, can be used.

In some embodiments, the subject methods of the present disclosure can be used separately. Alternatively, the subject methods can be used in combination with other conventional anti-cancer therapies for the treatment or prevention of proliferative diseases such as tumors. For example, these methods can be used to prevent cancer, prevent cancer recurrence and post-operative metastasis, and as an adjunct to other cancer treatments. The present disclosure demonstrates that the effectiveness of conventional cancer treatments (eg, chemotherapy, radiation therapy, phototherapy, immunotherapy, and surgery) can be enhanced by the use of therapeutic polypeptides of interest.

The terms "pharmaceutical composition," "combination drug," and "drug combination," as used herein, are used interchangeably and mean at least one drug, and optionally a pharmaceutically acceptable carrier, that are combined together to achieve a particular purpose or A combination of excipients. In certain embodiments, the pharmaceutical compositions include combinations that are separated in time and/or space, as long as they are capable of acting together to achieve the objectives of the present invention. For example, the components contained in the pharmaceutical composition (eg, antibodies, nucleic acid molecules, nucleic acid molecule combinations, and/or conjugates according to the invention) can be administered to the subject as a whole or separately to the subject. When the components contained in the pharmaceutical composition are separately administered to a subject, the components may be administered to the subject simultaneously or sequentially. Preferably, the pharmaceutically acceptable carrier is water, a buffered aqueous solution, an isotonic saline solution such as PBS (phosphate buffer), dextrose, mannitol, dextrose, lactose, starch, magnesium stearate, cellulose, carbonic acid. Magnesium, 0.3% glycerol, hyaluronic acid, ethanol or polyalkylene glycols such as polypropylene glycol, triglycerides and the like. The type of pharmaceutically acceptable carrier employed depends inter alia on whether the composition according to the invention is formulated for oral, nasal, intradermal, subcutaneous, intramuscular or intravenous administration. The composition according to the invention may comprise a wetting agent, an emulsifier or a buffer substance as an additive.

In some embodiments, cancers treatable with the antibodies disclosed herein include, for example but are not limited to, primary mesenchymal tumors (sarcoma); fibrosarcoma; mucinous sarcoma; liposarcoma; chondrosarcoma; osteogenic sarcoma; angiosarcoma Endothelial sarcoma; lymphangiosarcoma; synovial sarcoma; mesothelioma; Ewing's tumor; granulocytic leukemia; monocytic leukemia; malignant leukemia; lymphocytic leukemia; plasmacytoma; leiomyosarcoma; and rhabdomyosarcoma; Epithelial cancer (cancer); squamous cell or epidermal carcinoma; basal cell carcinoma; sweat adenocarcinoma; sebaceous gland cancer; adenocarcinoma; papillary carcinoma; papillary adenocarcinoma; cystadenocarcinoma; medullary carcinoma; Bronchial carcinoma; bronchial carcinoma; melanoma; renal cell carcinoma; hepatocellular carcinoma; cholangiocarcinoma; transitional cell carcinoma; squamous cell carcinoma; choriocarcinoma; seminoma; embryonal carcinoma; malignant teratoma; Carcinoma; leukemia; acute lymphocytic leukemia and acute myeloid leukemia (myeloid, promyelocytic, myelomonocytic; monocyte and erythroid leukemia); slow Leukemia; chronic myeloid (granulocytic) leukemia; chronic lymphocytic leukemia; polycythemia vera; lymphoma; Hodgkin's disease; non-Hodgkin's disease; multiple myeloma; primary macroglobulin Hypertension; heavy chain disease. In some preferred embodiments, the cancer is selected from the group consisting of ovarian cancer; lung cancer; gastric cancer; breast cancer; liver cancer; pancreatic cancer; skin cancer; malignant melanoma; head and neck cancer; sarcoma; cholangiocarcinoma; bladder cancer; Cancer; small bowel cancer; testicular cancer; placental choriocarcinoma; cervical cancer; testicular cancer; uterine cancer; prostate cancer; ovarian cancer;

The pharmaceutical composition, vaccine or pharmaceutical preparation according to the invention may be administered by any suitable route, for example, orally, nasally, intradermally, subcutaneously, intramuscularly or intravenously.

The term "therapeutic agent" as used herein denotes any substance or entity capable of exerting a therapeutic effect (eg, treating, preventing, ameliorating or inhibiting any disease and/or condition), including but not limited to: chemotherapeutic agents, radiation A therapeutic agent, an immunotherapeutic agent, a thermally therapeutic agent, and the like.

As used herein, "CDR region" or "CDR" refers to the hypervariable region of the heavy and light chains of an immunoglobulin, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed. , USDepartment of Health and Human Services, NIH, 1991, and later). There are three heavy chain CDRs and three light chain CDRs. The term CDR or CDRs as used herein is used to indicate one of these regions, or a few or even all of these regions, which contain a majority of the amino acid residues responsible for binding by the affinity of the antibody for the antigen or its recognition epitope. base.

For the purposes of the present invention, "identity", "identity" or "similarity" between two nucleic acid or amino acid sequences refers to the two to be compared after the optimal alignment (optimal alignment). The percentage of identical nucleotides or identical amino acid residues between the sequences, which are purely statistical and the differences between the two sequences are randomly distributed and cover the full length thereof. Sequence comparisons between two nucleic acid or amino acid sequences are typically performed by comparing the sequences after they have been matched in an optimal manner, and the comparison can be performed by a segment or by a "comparison window". In addition to being able to be manually implemented, the optimal alignment for comparing sequences can also be achieved by the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2:482], by Neddleman and Wunsch (1970). [J. MoI. Biol. 48: 443] local homology algorithm, by Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85: 2444) similarity search method, by using these algorithms Computer software implementation (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by BLAST N or BLAST P comparison software).

As used herein, "therapeutically effective amount" or "effective amount" refers to a dose sufficient to demonstrate its benefit to the subject to which it is administered. The actual amount administered, as well as the rate and time course of administration, will depend on the condition and severity of the subject being treated. The prescription for treatment (eg, the determination of the dose, etc.) is ultimately the responsibility of the GP and other physicians and depends on their decision, usually considering the disease being treated, the condition of the individual patient, the site of delivery, the method of administration, and the Other factors known.

The term "subject" as used herein refers to a mammal, such as a human, but may also be other animals, such as wild animals (such as herons, donkeys, cranes, etc.), livestock (such as ducks, geese, etc.) or experimental animals (such as Orangutans, monkeys, rats, mice, rabbits, guinea pigs, woodchucks, ground squirrels, etc.).

The term "antibody" refers to an intact antibody and any antigen-binding fragment thereof ("antigen-binding portion") or single strand thereof. "Full length antibody" refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by a disulfide bond. Each heavy chain comprises a heavy chain variable region (abbreviated as VH) and a heavy chain constant region. The heavy chain constant region comprises three domains, CH1, CH2 and CH3. Each light chain comprises a light chain variable region (abbreviated as VL) and a light chain constant region. The light chain constant region contains a domain, CL. The VH and VL regions can also be subdivided into multiple regions of high variability, referred to as complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FR). Each VH and VL consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. These variable regions of the heavy and light chains comprise a binding domain that interacts with the antigen. The constant region of the antibody mediates binding of the immunoglobulin to the host's tissues or factors, including various cells of the immune system (such as effector cells) and the first component (Clq) of the classical complement system. Chimeric or humanized antibodies are also encompassed in the antibodies according to the invention.

The term "humanized antibody" refers to an antibody comprising a CDR region derived from a non-human antibody, and the other portion of the antibody molecule is derived from one (or several) human antibodies. Moreover, in order to retain binding affinity, some residues of the backbone (referred to as FR) segment can be modified (Jones et al., Nature, 321:522-525, 1986; Verhoeyen et al., Science, 239: 1534-1536, 1988; Riechmann et al., Nature, 332: 323-327, 1988). Humanized antibodies or fragments thereof according to the invention can be prepared by techniques known to those skilled in the art (for example, as described in the document Singer et al., J. Immun. 150: 2844-2857, 1992; Mountain et al., Biotechnol. Genet. Eng. Rev., 10: 1-142, 1992; or Bebbington et al., Bio/Technology, 10: 169-175, 1992).

The term "chimeric antibody" refers to an antibody wherein the variable region sequence is from one species and the constant region sequence is from another species, eg, the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. A chimeric antibody or fragment thereof according to the invention can be prepared by using genetic recombination techniques. For example, the chimeric antibody can be produced by cloning recombinant DNA comprising a promoter and a sequence encoding a variable region of a non-human, in particular murine, monoclonal antibody according to the invention, and a sequence encoding a constant region of a human antibody . The chimeric antibody of the present invention encoded by such a recombinant gene will be, for example, a murine-human chimera whose specificity is determined by a variable region derived from murine DNA, and whose isotype is derived from human DNA. Constant zone to determine. For methods of preparing chimeric antibodies, for example, reference can be made to the document Verhoeyn et al. (BioEssays, 8: 74, 1988).

The term "monoclonal antibody" refers to a preparation of an antibody molecule having a single molecular composition. Monoclonal antibody compositions display a single binding specificity and affinity for a particular epitope.

The term "derivative" refers to a (chemical) modification of an amino acid/amino acid chain at the N-terminus, C-terminus, backbone, peptide bond and/or side chain residue. The term is not intended to mean any addition, substitution or deletion of an amino acid in an amino acid chain. (Chemical) derivatives derived from such L-amino acid or L-enantiomer amino acids typically include any natural or non-naturally occurring derivative of these amino acids - including but not limited to: amino acids as defined above contain post-translational modifications Or synthetic modifications, including acetylation (at the N-terminus of the (poly)peptide sequence, at the lysine residue, etc.), deacetylation, alkylation such as methylation, ethylation, etc. (preferably at (multiple) a lysine or arginine residue in the peptide sequence), dealkylation such as demethylation, deethylation, etc., amidation (preferably at the C-terminus of the (poly)peptide sequence), A Acylation, γ-carboxylation, glutamylation, glycosylation (preferably asparagine, lysine, hydroxylysine, serine or threonine residues in the (poly)peptide sequence) Increase in heme or heme fraction, hydroxylation, iodization, prenylation, increase of isoprenoid moieties such as farnesyl or geranylgeraniol, etc. ), lipoylation (lipidation of lipoic acid functional groups) such as prenylation, formation of GPI anchors, including myristoylation, methods Alkalization, geranyl-based geranylgernaylation, oxidation, phosphorylation (eg, serine, tyrosine, threonine or histidine moiety in the (poly)peptide sequence), sulfuric acid Chemolysis (for example, sulfation of tyrosine), selenylation, sulfation, and the like.

Application

The fusion protein of the present invention may be administered alone, but is preferably administered as a pharmaceutical composition, which typically includes a suitable pharmaceutical excipient, diluent or carrier selected according to the intended mode of administration. The fusion protein can be applied to a patient in need of treatment by any suitable means. Accurate metering will depend on a number of factors, including the precise nature of the fusion protein.

Some suitable modes of use include, but are not limited to, oral, rectal, nasal, topical (including buccal and sublingual), subcutaneous, vaginal or parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intrathecal and hard) Administration outside the brain.

For intravenous injection and injection at the site of the disease, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is free of heat and has suitable pH, isotonicity and stability.

Those skilled in the art can formulate fusion proteins using suitable solvents or formulations, for example, isotonic vehicles such as sodium chloride injection, Ringer's injection, lactated Ringer's injection. Preservatives, stabilizers, buffers, antioxidants, and/or other additives may be added as desired. The pharmaceutical composition for oral administration may be in the form of a tablet, a capsule, a powder or an oral solution. Tablets may include a solid carrier such as gelatin or an adjuvant. Liquid pharmaceutical compositions typically include a liquid carrier such as water, petroleum, animal or vegetable oil, mineral oil or synthetic oil. Physiological saline solutions, dextrose or other sugar solutions or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may also be included.

Examples of the techniques and protocols mentioned above, as well as other techniques and protocols used in accordance with the present invention, can be found in Remington's Pharmaceutical Sciences, 16th edition, Oslo, A. (ed), 1980.

Example

Example 1. Preparation of anti-TM4SF1 monoclonal antibody

To prepare a monoclonal antibody with high affinity for TM4SF1, we designed a synthetic polypeptide based on the amino acid sequence of extracellular loop 1 of TM4SF1, the sequence of which is FPNGETKYASENHLSC. BALB/c mice (Shanghai Slack Laboratory Animal Center) were immunized with TM4SF1 polypeptide, spleen lymphocytes of immunized mice were taken, and hybridomas were prepared by fusion with SP2/0 myeloma cells (ATCC), and the fused cells were 96-well cells. The culture plate was subjected to cloning culture, and the anti-TM4SF1 antibody-positive clone was detected by ELISA using the ELISA plate coated with TM4SF1 polypeptide to obtain a plurality of hybridoma cell lines resistant to TM4SF1 antibody. The positive cloned hybridoma cells were subjected to expansion culture and subcloned culture. Thus, several anti-TM4SF1 antibody hybridoma cell lines were obtained, and the antibody was confirmed to have high affinity for TM4SF1 by ELISA and flow cytometry. It was finally determined that several monoclonal antibodies, such as clone FC17-7, have a high affinity for the target. The FC17-7 hybridoma cell clone has been deposited in the China CENTER FOR TYPE CULTURE COLLECTION (CCTCC), material name: anti-TM4SF1 antibody (FC17-7), accession number CCTCC NO: C2017110, preservation date: 2017 July 13th). In order to obtain the sequence of the FC17-7 monoclonal antibody, we extracted total RNA from the FC17-7 monoclonal antibody hybridoma cell line, synthesized cDNA by reverse transcription, and amplified the heavy and light chain variable region DNA of anti-TM4SF1 by PCR amplification. Sequence, and further obtain the variable region amino acid sequences of the heavy and light chains, and the six CDR region sequences of the heavy and light chains of the antibody were determined by sequence analysis (amino acid sequence: SEQ ID No: 7, SEQ ID No: 8 And SEQ ID No: 9; SEQ ID No: 10, SEQ ID No: 11 and SEQ ID No: 12).

Example 2. Monoclonal antibody binding to TM4SF1

A549 lung cancer cell line with high expression of TM4SF1 was cultured in DMEM-HG medium. After the cell density reached 90% or more, discard the medium, wash it once with PBS, add 0.25% trypsin, 37 degrees, digest in the incubator for 5 minutes, then terminate the digestion with the same volume of medium, 1000 rpm, 5 min, remove Clear, resuspended in PBS containing 10% FBS, 1% sodium azide. The cells were counted, tubed, and the number of cells per tube was 1×10 ⁶ , centrifuged, 1000 rpm, 5 minutes, and the supernatant was removed; 100 μl of PBS containing 3% BSA was added to each sample, and the corresponding concentration of the sample prepared in Example 1 was added. Cloning antibody (primary antibody), incubating for 30 min; washing: each sample was resuspended in PBS, centrifuged, 1000 rpm, 5 min, repeated three times; each sample was added with 100 μl of PBS containing 3% BSA, and then diluted 1:500. APC-conjugated anti-mouse secondary antibody was added and incubated for 30 min; each sample was resuspended in PBS containing 3% BSA, 1% sodium azide, centrifuged, 1000 rpm, 5 min, repeated three times, and finally resuspended in 200 μl PBS. Upstream cytometry for detection. The results showed that the antibody strain FC17-7 specifically binds to TM4SF1. Live cell FACS experiments demonstrated that the monoclonal antibody FC17-7 can efficiently bind to TM4SF1 on the cell membrane surface with high affinity (Fig. 1).

Example 3. Preparation of TM4SF1 polypeptide

To inhibit the TM4SF1 signal, we also designed soluble peptides based on the sequence of extracellular loop 1 of TM4SF1. The sequence of this polypeptide is FPNGETKYASENHLSRC. The peptide contains 17 amino acids and is synthesized by Shanghai Jill Biochemical Co., Ltd. by chemical means. The synthesized peptides are qualified for quality testing and are used in cell and animal experiments.

Example 4. Monoclonal antibody blocking TM4SF1 interaction with DDR1

To demonstrate that monoclonal antibodies can be used to block the interaction of TM4SF1 with DDR1, we used 293FT cells to simultaneously overexpress TM4SF1 and DDR1. 293FT cells were cultured in DMEM-HG medium, and the cells were trypsinized, counted, and 1 × 10 ⁶ cells were cultured in a 6 cm culture dish. The next day, 700 μl of serum-free DMEM-HG medium was added to each of the two centrifuge tubes. 10 μl of P-Pei transfection reagent was added to the first centrifuge tube. 1 μg of PQCXIP-hTM4SF1 plasmid and 1 μg of PQCXIN-hDDR1 plasmid were added to the second centrifuge tube and mixed. The liquid from the second centrifuge tube was then added to the first centrifuge tube and incubated for 20 min, which was the transfection mixture. The 293FT cells inoculated the previous day were taken out, the medium was removed, and the above transfection mixture was added to the culture dish, and after incubation for 6 hours, fresh medium was added to continue the culture. After 48 hours of transfection, the medium was removed and 2 ml of serum-free DMEM-HG medium containing a certain concentration of the FC17-7 antibody prepared in Example 1 or IgG as a negative control was added. After incubating at 37 ° C for 5 min, Collagen I was added to the culture dish to a final concentration of 10 μg/ml, and the cells were collected after further culture for 6 hours. The cells were lysed with IP-RIPA buffer, lysed in a refrigerator at 4 ° C for 1 h, centrifuged at 12000 rpm for 30 min, and the supernatant was taken to determine the protein concentration, and the protein concentration of each sample was adjusted to be uniform. Anti-flag M2 affinity gel beads were added to each sample, and after incubation at 4 ° C for 2 h, the supernatant was discarded by centrifugation at 3000 rpm for 2 min, and washed with IP-RIPA, and repeated 3 times. To the Anti-flag M2 affinity gel beads, the eluate was added to contain 3 μl of the Plasmid (5 μg/ml), incubated on ice, and eluted for 1 h. After the end of the elution, the supernatant was centrifuged at 3000 rpm for 2 min, and protein expression was detected by Western Blot. The results showed that because TM4SF1 and DDR1 interaction sites are their extracellular regions, co-immunoprecipitation experiments have shown that FC17-7 antibody can not only bind TM4SF1 extracellular loop loop well, but also block the interaction between the two (Fig. 2 ).

Example 5. Monoclonal antibodies block the non-classical DDR1 signaling pathway involved in TM4SF1

To demonstrate that a monoclonal antibody can be used to block the signaling pathway of TM4SF1, we have prepared TM4SF1 and DDR1 overexpressing cells. For the transfection method, the method in Example 4 was followed. After 48 h, the medium was removed, and 2 ml of serum-free DMEM-HG medium containing a certain concentration of the FC17-7 antibody prepared in Example 1 or as a negative was added. Control IgG. The culture dish was cultured at 37 ° C, incubated for 5 min, and Collagen I was added to the culture dish to a final concentration of 10 μg/ml, and the cells were collected after further incubation for 12 hours. The cells were lysed with RIPA buffer, lysed in a refrigerator at 4 ° C for 1 h, sonicated twice (15 s 30% power), centrifuged at 12000 rpm for 30 min, and the supernatant was taken to determine the protein concentration, and the protein concentration of each sample was adjusted to be uniform. The protein was subjected to SDS-PAGE electrophoresis, and the protein was transferred to a membrane by a conventional Western Blot method, and the antibody was stained. Experiments show that when TM4SF1 is involved in the non-classical DDR1 signaling pathway, the downstream P-STAT3 level will increase, affecting downstream functions. Western Blot experiments demonstrated that when cells were treated with the FC17-7 antibody prepared in Example 1, they inhibited the downstream signal transduction of TM4SF1 involved in the non-canonical DDR1 signaling pathway, and P-STAT3 was down-regulated (Fig. 3).

Example 6. Monoclonal antibody inhibits tumor cell suspension sphere formation experiment

MDAMB231-BoM-1833-TGL cells were prepared as single cell suspensions, seeded in low adhesion 24-well plates in an amount of 1000 cells per well, medium was added to MEBM medium, and 1:50 B27, 5 mg/ml was added thereto. Insulin, 0.5 mg/ml hydrocortisone, 20 ng/ml bFGF, 20 ng/ml EGF, 4 mg/ml heparin, 30 μg/ml Collagen I and 10 μg/ml FC17-7 antibody prepared in Example 1 or as a negative control IgG. After 7 days of cell culture, the number of tumor-forming suspension spheres was counted by microscopic observation. The experimental results show that TM4SF1 interacts with DDR1 to activate the downstream signaling pathway and enhance the expression of downstream soluble transcription factors such as Sox2, Oct4 and Nanog, thus promoting the dryness of tumor cells. The tumor cell suspension sphere formation experiment can reflect the dryness of tumor cells. When the cells were treated with FC17-7 antibody, the formation of tumor cell suspension spheres was significantly reduced, indicating that monoclonal antibodies can inhibit the dryness of tumor cells (Fig. 4).

Example 7. In vivo experiments in mice in which monoclonal antibodies or polypeptides inhibit tumor cell metastasis

The day before the injection of the tumor cells into the mice was recorded as day -1 of the experiment, on which the mice were fasted for 4 hours, and then the mice were injected with the corresponding concentrations of the FC17-7 antibody prepared in Example 1 by intravenous injection or As a negative control IgG, the mice were given food after 2 hours, after which the mice were injected with antibodies every two days. On day 0 of the experiment, MDAMB231-BoM-1833-TGL cells were blocked with antibodies according to the method of Example 4. After 6 hours, the cells were digested, the medium was removed, washed once with PBS, and resuspended in PBS to a concentration of 1 × 10 ⁶ cells/ml. The mice were then injected with 1×10 ⁵ /100 μl of tumor cells by ventricular injection to construct a mouse metastasis model. After every other week, the mice were subjected to in vivo imaging and the mouse fluorescence signal data was recorded. By constructing a breast cancer metastasis model, we used the monoclonal antibody FC17-7 prepared in Example 1 to periodically administer the mice, and found that FC17-7 can significantly inhibit the metastasis of multiple organs of the breast, including the brain and bone (Fig. 5 to Figure 7); at the same time, the monoclonal antibody can prolong the survival time of mice (Fig. 8).

Example 8. Effect of TM4SF1 extracellular loop 1 small peptide on tumor metastasis - blocking TM4SF1 and DDR1 interaction

First, we used 293FT cells to overexpress TM4SF1 extracellular loop 1 small peptide or TM4SF1 extracellular loop 2 small peptide or DDR1 extracellular peptide, respectively. The cells were lysed with IP-RIPA buffer, lysed in a refrigerator at 4 ° C for 1 h, centrifuged at 12000 rpm for 30 min, and the supernatant was taken to determine the protein concentration, and the protein concentration of each sample was adjusted to be uniform. Anti-HA affinity gel beads were added to each sample, and after incubation at 4 ° C for 2 h, the supernatant was discarded by centrifugation at 3000 rpm for 2 min, and washed with IP-RIPA, and repeated 3 times. To the Anti-HA affinity gel beads, the eluate was added and 3 μl of the HA peptide (5 μg/ml) was incubated on ice and eluted for 1 h. After the end of the elution, the supernatant was taken by centrifugation at 3000 rpm for 2 min. Then, 293FT cells were used to simultaneously overexpress TM4SF1 and DDR1. For the transfection method, the method in Example 4 was followed. After 48 hours, the medium was removed, and 2 ml of serum-free DMEM-HG medium was prepared, which was prepared in the same manner as in Example 8. TM4SF1 extracellular loop 1 small peptide or TM4SF1 extracellular loop 2 small peptide or DDR1 extracellular peptide or PBS as a negative control. The culture dish was cultured at 37 ° C, incubated for 5 min, and Collagen I was added to the culture dish to a final concentration of 10 μg/ml. After further culture for 6 hours, the cells were collected for co-immunoprecipitation. The method of Example 4 is shown in the immunoprecipitation method and the subsequent Western Blot method. The results showed that we treated the cells with TM4SF1 extracellular loop 1 small peptide, extracellular loop 2 small peptide, and DDR1 extracellular small peptide, and found that TM4SF1 extracellular loop 1 small peptide can significantly disrupt the interaction between TM4SF1 and DDR1 (Fig. 9 ).

Example 9. Effect of TM4SF1 extracellular loop 1 small peptide on tumor metastasis - inhibition of TM4SF1 involvement Code DDR1 signal path

To show that the TM4SF1 signal can be blocked with the TM4SF1 extracellular loop 1 small peptide, we prepared TM4SF1 and DDR1 overexpressing cells. For the transfection method, the method in Example 4 was carried out. After 48 hours, the medium was removed, and 2 ml of serum-free DMEM-HG medium containing a certain concentration of TM4SF1 extracellular loop 1 small peptide prepared in Example 3 was added. Or as a negative control for PBS. The culture dish was cultured at 37 ° C, incubated for 5 min, and Collagen I was added to the culture dish to a final concentration of 10 μg/ml, and the cells were collected after further incubation for 12 hours. The cells were lysed with RIPA buffer, lysed in a refrigerator at 4 ° C for 1 h, sonicated twice (15 s 30% power), centrifuged at 12000 rpm for 30 min, and the supernatant was taken to determine the protein concentration, and the protein concentration of each sample was adjusted to be uniform. The protein was subjected to SDS-PAGE electrophoresis, and the protein was transferred to a membrane by a conventional Western Blot method, and the antibody was stained. The results showed that we treated cells with different concentrations of TM4SF1 extracellular loop 1 small peptide, and the results showed that extracellular loop 1 small peptide began to inhibit TM4SF1 involvement in P-STAT3 downstream of non-canonical DDR1 signaling pathway at a concentration of 9 μg/ml. At the same time, it has concentration dependence (Fig. 10).

Example 10. Effect of TM4SF1 extracellular loop 1 small peptide on tumor metastasis - inhibition of breast cancer metastasis

The day before the injection of the tumor cells into the mice was recorded as day -1 of the experiment, on which the mice were fasted for 4 hours, and then the mice were injected with the corresponding concentration of TM4SF1 extracellular ring 1 obtained in Example 3 by intravenous injection. The peptide or IgG as a negative control was administered to the mice 2 hours later, after which the small peptide was injected once every other day. On day 0 of the experiment, MDAMB231-BoM-1833-TGL cells were blocked with a small peptide according to the method of Example 9. After 6 hours, the cells were digested, the medium was removed, washed once with PBS, and resuspended in PBS to a concentration of 1 × 10 ⁶ cells/ml. For the method of constructing a mouse breast cancer metastasis model, see the method of Example 7. The experimental results show that by constructing a breast cancer metastasis model, the mice were treated with the TM4SF1 extracellular loop 1 small peptide, and we found that the small peptide can significantly inhibit the metastasis of breast cancer (Figures 11 and 12). Mouse survival can be extended (Figure 13).

The invention has been illustrated by way of specific embodiments. However, those skilled in the art can understand that the present invention is not limited to the specific embodiments, and various modifications and changes can be made by those skilled in the art within the scope of the invention, and various technical features mentioned throughout the specification. The invention can be combined with one another without departing from the spirit and scope of the invention. Such modifications and variations are within the scope of the invention.

Claims (29)

1. Use of an antibody that specifically binds to extracellular loop 1 (ECL1) of TM4SF1 in the manufacture of a medicament for the treatment of cancer.
2. The use according to claim 1, wherein the antibody is a chimeric antibody, a humanized antibody or a human antibody.
3. The use according to claim 1 wherein the antibody is a monoclonal antibody.
4. The use according to claim 1 wherein the antibody is a bispecific antibody.
5. The use according to claim 1 wherein the antibody is an antibody-drug conjugate.
6. The use according to claim 1, wherein the antibody specifically binds to an extracellular loop 1 (ECL1) polypeptide of TM4SF1 having the sequence, wherein the sequence is a contiguous fragment of SEQ ID NO: 2 and the origin is at SEQ ID NO: 2, 27, 28, 29, 30, 31, 32, or 33, the endpoint is located at 42, 43, 44, 45, 46, 47 of SEQ ID NO: 2. Bit, or 48 bits.
7. The use according to claim 1, wherein the antibody specifically binds to an extracellular loop 1 (ECL1) polypeptide having the sequence TM4SF1 of SEQ ID NO:4.
8. The use according to claim 1 wherein the antibody comprises a heavy chain and a light chain, wherein

   (i) the heavy chain comprises three CDR regions having the sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively;

   (ii) The light chain comprises three CDR regions having the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.
9. The use according to claim 1, wherein the cancer is selected from the group consisting of: ovarian cancer; lung cancer; gastric cancer; breast cancer; liver cancer; pancreatic cancer; skin cancer; malignant melanoma; head and neck cancer; sarcoma; cholangiocarcinoma; bladder cancer; Cancer; colon cancer; small intestine cancer; testicular cancer; placental choriocarcinoma; cervical cancer; testicular cancer; uterine cancer; prostate cancer; multiple myeloma; malignant lymphoma;
10. The use according to claim 1, wherein the cancer cells of the cancer express TM4SF1.
11. Use of the extracellular loop 1 (ECL1) polypeptide of TM4SF1 in the manufacture of a medicament for the treatment of cancer.
12. The use according to claim 11, wherein the sequence of the polypeptide is a contiguous fragment of SEQ ID NO: 2 and the origin is at positions 27, 28, 29, 30, 31, 32 of SEQ ID NO: 2. Bit or position 33, the end point is at position 42, 43 , 44, 45, 46, 47 or 48 of SEQ ID NO: 2.
13. The use according to claim 11, wherein the sequence of the polypeptide is SEQ ID NO:4.
14. The use according to claim 11, wherein the cancer is selected from the group consisting of: ovarian cancer; lung cancer; gastric cancer; breast cancer; liver cancer; pancreatic cancer; skin cancer; malignant melanoma; head and neck cancer; sarcoma; cholangiocarcinoma; bladder cancer; Cancer; colon cancer; small intestine cancer; testicular cancer; placental choriocarcinoma; cervical cancer; testicular cancer; uterine cancer; prostate cancer; multiple myeloma; malignant lymphoma;
15. The use according to claim 11, wherein the cancer cells of the cancer express TM4SF1.
16. An isolated TM4SF1 extracellular loop 1 (ECL1) polypeptide having the sequence of a contiguous fragment of SEQ ID No: 2 and having a starting point at positions 27, 28, 29, 30, 31, 32 of SEQ ID NO: Or 33, the end point is at position 42, 43 , 44, 45, 46, 47 or 48 of SEQ ID NO: 2.
17. The polypeptide according to claim 16, which has tumor suppressing activity.
18. The polypeptide of claim 16 having the sequence of SEQ ID NO:4.
19. An antibody that specifically binds to extracellular loop 1 (ECL1) of TM4SF1.
20. The antibody according to claim 19, which is a chimeric antibody, a humanized antibody or a human antibody.
21. The antibody of claim 19 which is a monoclonal antibody.
22. The antibody of claim 19 which is a bispecific antibody.
23. The antibody according to claim 19, wherein said antibody specifically binds to an extracellular loop 1 (ECL1) polypeptide of TM4SF1 having the sequence of SEQ ID NO: 2 and a starting point at SEQ ID NO: 2 is 27, 28, 29, 30, 31, 32 or 33, and the end point is 42, 43, 44, 45, 46, 47 of SEQ ID NO: 2. Or 48.
24. The antibody of claim 19, wherein the antibody specifically binds to an extracellular loop 1 (ECL1) polypeptide of TM4SF1 having the sequence SEQ ID NO:4.
25. The antibody of claim 19, wherein the antibody comprises a heavy chain and a light chain, wherein

    (i) the heavy chain comprises three CDR regions having the sequences set forth in SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9, respectively;

    (ii) The light chain comprises three CDR regions having the sequences shown in SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 12, respectively.
26. The antibody according to claim 19, which is secreted by a hybridoma cell, and the sample of the hybridoma cell has been deposited with the China Center for Type Culture Collection on July 13, 2017 under the accession number CCTCC NO: C2017110.
27. An antibody-drug conjugate comprising the antibody according to any one of claims 19-26 and a therapeutic agent, preferably the therapeutic agent is selected from the group consisting of a cytotoxic drug, an immunopotentiator, and a radioisotope, more preferably The therapeutic agent is selected from the group consisting of a dolastatin peptide and derivatives thereof, and most preferably the therapeutic agent is selected from the group consisting of MMAE and MMAF.
28. A pharmaceutical composition comprising the polypeptide of any one of claims 16-18, the antibody of any one of claims 19-26 and/or the conjugate of claim 27, and a pharmaceutically acceptable carrier.
29. A method of screening for cancer therapeutics, comprising:

    (i) contacting the substance to be tested with extracellular loop 1 (ECL1) of TM4SF1;

    (ii) determining whether the substance to be tested specifically binds to extracellular loop 1 (ECL1) of TM4SF1;

    Wherein the specific binding of the substance to be tested to the extracellular loop 1 (ECL1) of TM4SF1 indicates that the substance to be tested can be used as a cancer therapeutic drug.

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