CN110563845A

CN110563845A - anti-IGSF 9 antibody, pharmaceutical composition and application thereof

Info

Publication number: CN110563845A
Application number: CN201910867146.9A
Authority: CN
Inventors: 李尊岭; 邵淑红; 王耀鹏; 孙大庆; 刘祎璠
Original assignee: Binzhou Medical College
Current assignee: Binzhou Medical College
Priority date: 2019-09-12
Filing date: 2019-09-12
Publication date: 2019-12-13

Abstract

The application provides an anti-IGSF 9 antibody, a pharmaceutical composition and application thereof, belonging to the technical field of antibody engineering and biological engineering. Screening research shows that the IGSF9 protein has high expression characteristic on the surfaces of various tumor cells; IGSF9 protein generates an immunosuppressive microenvironment by virtue of an intracellular ITIM motif (LQYLSL sequence) and promotes tumor growth; the blocking antibody recognizes an epitope on the surface of IGSF9 protein, can block an IGSF9 signal channel, removes the inhibition effect on immune cells, has good effects of inhibiting and treating tumors, and is used for preparing bifunctional antibody conjugates, drug conjugates and drug compositions; or the IGSF9 protein is used as a tumor marker to prepare a diagnostic reagent with good effect.

Description

anti-IGSF 9 antibody, pharmaceutical composition and application thereof

Technical Field

The invention relates to the technical field of antibody engineering and biological engineering, in particular to an anti-IGSF 9 antibody, a pharmaceutical composition and application thereof.

Background

To date, tumor immunotherapy is the most likely method for curing tumors. The antibody blocks the immunotherapy mediated by a Programmed cell death receptor (PD-1), can obviously inhibit the growth of tumors such as melanoma, non-small cell lung cancer and the like, and improves the five-year survival rate of patients; however, only about 20% of patients respond well to PD-1 blocking antibodies, indicating that most tumors have immune evasion that is independent of the PD-1/PD-L1 signaling pathway; therefore, the research on a new mechanism of tumor immune evasion is the basis for developing tumor immunotherapy drugs.

Disclosure of Invention

The first aspect of the application discloses an anti-IGSF 9 antibody, which can recognize IGSF9 protein of tissues or cells, and the amino acid sequence of the IGSF9 protein is shown as SEQ ID NO. 1.

IGSF9 is a new cloned gene, which is mainly expressed in central nervous tissue during mammalian embryonic development and is involved in the adhesion of nerve cells, the transmission of inhibitory neurotransmitters and the movement of nerve cells. The IGSF9 protein comprises a signal peptide, an extracellular region, a transmembrane region, and an intracellular region; the canonical ITIM motif of the sequence of intracellular domains 908-914(LQYLSL) suggests that IGSF9 may function as an immunosuppressive receptor. And the extracellular region comprises 5 Ig structural domains and 2 Fn3 structural domains, and the extracellular region can be used as an antigen epitope recognized by an antibody. The expression of IGSF9 in tumor tissues is detected by immunohistochemistry, the expression of IGSF9 in lung cancer, liver cancer, breast cancer, endometrial cancer and ovarian cancer is higher, and the analysis of variance results show that: the expression of IGSF9 is related to the differentiation of tumors, the higher the expression of IGSF9, the worse the differentiation of tumors; the multi-factor survival analysis result shows that: IGSF9 protein can be used as a new tumor marker to evaluate prognosis of tumor patients. In addition, the in vivo and in vitro experiment results prove that: IGSF9 protein depends on its intracellular ITIM motif, and through paracrine route, inhibits T cell proliferation, and has the function of immunosuppression receptor; the mediated T cell proliferation inhibition is a novel tumor immune evasion mechanism.

Therefore, the anti-IGSF 9 blocking antibody is combined with IGSF9 protein of tumor cells to promote T cell proliferation and achieve the aim of inhibiting and treating tumors.

In some embodiments of the aforementioned first aspect, the anti-IGSF 9 antibody recognizes an extracellular epitope domain of said IGSF9 protein, preferably, the amino acid sequence of the extracellular domain is as set forth in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No. 7.

An Immunoreceptor tyrosine-based inhibition motif (ITIM) consists of 6 conserved amino acid sequences (I/L/V-x-x-Y-x-L/V), wherein phosphorylation of tyrosine residues can recruit downstream phosphatases SHP-1, SHP-2 or SHIP-1, so as to regulate the activity of immune cells; since phosphatases are primarily directed at inhibiting immune cell activity, membrane receptors containing ITIM sequences are collectively referred to as immunosuppressive receptors. To date, the intracellular domain of 109 membrane proteins from the human proteome has been identified as containing ITIM sequences, and these 109 membrane proteins are collectively referred to as immunosuppressive receptors. The expression of the 109 proteins in tumor tissues is inquired by a prophase application of a Proteinatlas database, and the Immunoglobulin superfamily member 9(Immunoglobulin super family member9, IGSF9) is found to be highly expressed in all tumor tissues, and is possibly a new and unreported tumor marker molecule.

the ITIM motif contained in IGSF9 proteins of the present application has the sequence: LQYLSL, and the antibody recognizes the extracellular region part of IGSF9 protein, and the extracellular region of IGSF9 protein comprises a sequence with an amino acid sequence shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO. 7.

The IGSF9 protein has 5 immunoglobulin-like domains (Ig) in the extracellular region, which are Ig1 to Ig5, and are the main ligand binding regions; there are two further FN3 domains (Fibronectin3, FN3), the function of which is unknown. The IGSF9 protein extracellular region Ig1 structural domain is from 24 th to 132 th of an IGSF9 protein amino acid sequence, and the sequence is shown as SEQ ID NO. 2; the Ig2 structural domain of the extracellular region of the IGSF9 protein is from 154 th to 220 th of an amino acid sequence of the IGSF9 protein, and the sequence is shown as SEQ ID NO. 3; the IGSF9 protein extracellular region Ig3 structural domain is from 226 th to 305 th of an amino acid sequence of IGSF9 protein, and the sequence is shown as SEQ ID NO. 4; the IGSF9 protein extracellular region Ig4 structural domain is from 325 th to 388 th of an amino acid sequence of IGSF9 protein, and the sequence is shown as SEQ ID NO. 5; the IGSF9 protein extracellular region Ig5 structural domain is from 420 th to 484 th of an IGSF9 protein amino acid sequence, and the sequence is shown as SEQ ID NO. 6; the FN3 structural domain of the extracellular domain of the IGSF9 protein is from 495 th to 580 th of an amino acid sequence of the IGSF9 protein, and the sequence is shown as SEQ ID NO. 7.

the amino acid sequence of SEQ ID NO.2 is:

pevvsvv gragesvvlg cdllppagrp plhviewlrfgfllpifiqf glyspridpd yvgrvrlqkg aslqieglrv edqgwyecrv ffldqhipeddfangswvhl tv；

The amino acid sequence of SEQ ID NO.3 is:

pqfqe tppavlevqe lepvtlrcva rgsplphvtw klrgkdlgqgqgqvqvqngt lrirrvergs sgvytcqass tegsathatq llv；

The amino acid sequence of SEQ ID No.4 is:

ppviv vppknstvnasqdvslacha eaypanltys wfqdninvfh isrlqprvri lvdgslrlla tqpddagcytcvpsn；

the amino acid sequence of SEQ ID No.5 is:

vircpvranp pllfvswtkdgkalqldkfp gwsqgtegsl iialgnedal geysctpyns lgta；

The amino acid sequence of SEQ ID No.6 is:

llipc saqgdpppvv swtkvgrglq gqaqvdsnss lilrpltkea hghwecsasn avarvatstn；

The amino acid sequence of SEQ ID NO.7 is:

vtnvsvvalp kganvswepg fdggylqrfsvwytplakrp drmhhdwvsl avpvgaahll vpglqphtqy qfsvlaqnkl gsgpfs；

In some embodiments of the foregoing first aspect, the tissue comprises tumor tissue comprising lung cancer tissue, liver cancer tissue, breast cancer tissue, endometrial cancer tissue, and ovarian cancer tissue.

A second aspect of the present application discloses bifunctional antibody conjugates comprising the aforementioned blocking antibody of anti-IGSF 9 antibody and a second biologically functional fragment; the anti-IGSF 9 antibody and the second biologically functional fragment are linked by a linker protein or a disulfide bond.

The bifunctional antibody conjugates can be bispecific antibody molecules including anti-IGSF 9 antibodies.

in some embodiments of the foregoing second aspect, the second biologically functional fragment specifically binds to a receptor or ligand selected from the group consisting of: PD-1, PD-L1, VGFR, PD-L2, TIM3, TIGIT, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, nkp80 and LAG-3.

of course, the second biologically functional fragment may also be a single domain antibody, a single chain antibody.

In some embodiments of the foregoing second aspect, the linker protein is an Fc structural protein or a (GGGGS) n protein; wherein n is an integer from 1 to 20.

Linking the anti-IGSF 9 antibody and the second biologically functional fragment through the Fc domain protein, such that the Fc domain protein is linked to the anti-IGSF 9 antibody, allows the Fc domain protein linked to the anti-IGSF 9 antibody to function simultaneously.

Alternatively, the (GGGGS) n protein may be used as a linker protein, and the (GGGGS) n protein may be used to allow the Fc domain protein to bind to the anti-IGSF 9 antibody and function simultaneously.

a third aspect of the present application discloses a drug conjugate comprising a cytotoxic molecule, a linker and the aforementioned anti-IGSF 9 antibody.

In some embodiments of the foregoing third aspect, the cytotoxic molecule comprises calicheamicin, duocarmycin, the microtubule inhibitor paclitaxel, dolastatin, and maytansine.

Calicheamicins (CLM) are enediyne antitumor antibiotics isolated from rare actinomyces micromonospora fermentation broth. The calicheamicin also has nonspecific damage effect on cell RNA, and the conjugate Mylotarg of calicheamicin and anti-CD 33 monoclonal antibody is the first monoclonal antibody-oriented drug approved by FDA for tumor treatment and is already clinically applied.

paclitaxel is one of the most effective chemotherapeutic drugs in clinical application, binds to tubulin, inhibits cell mitosis, and thereby inhibits tumor growth; however, because of lack of targeting, paclitaxel has larger toxic and side effects and has more obvious inhibition effect on normal cells with active metabolism. By linking the anti-IGSF 9 antibody with paclitaxel to form a conjugate, paclitaxel is targeted to tumor tissues and more specifically kills tumor cells.

The microtubule inhibitor dolastatin is one of marine biotoxins, is found in digestive glands of sea hares of the urogastic mollusk long tail, the back and the anus, and is named as dolastatin. Is a linear depsipeptide natural cytotoxic protein and has very little content in organisms. Has high anticancer effect. It is a nonapeptide and is present in very small amounts in organisms. The Pettit research group has identified 18 short chain peptide compounds containing special amino acids, which are named dolastatins 1-18. Wherein the structures of Dolastatin3, Dolastatin10(D10), Dolastatin15 were determined. D10 is the most studied, and D10 is generally used as representative of dolastatin.

Maytansine is an alkaloid, which is present in the genus maytansinoid of the family Celastraceae and its relatives. In 1972, the content of S.M. Kupoch was first isolated from Maytenus ovatus (or called Maytenus dentata) collected in Africa, and was two million. Maytansine has remarkable therapeutic effects on tumors such as L-1210, P-388 leukemia, S-180, W-256, Lewis lung cancer and in vitro nasopharyngeal carcinoma.

In a fourth aspect of the present application, a pharmaceutical composition is disclosed, comprising the aforementioned anti-IGSF 9 antibody, a buffer, an osmolality adjusting agent, and a surfactant; the buffer solution is selected from a citric acid buffer solution, a histidine buffer solution, an acetate buffer solution, a succinate buffer solution or a Tris buffer solution; the osmotic pressure regulator is sodium chloride; the surfactant is selected from polysorbate 80 or polysorbate 20.

The fifth aspect of the application discloses the application of IGSF9 protein as a tumor diagnosis marker in the preparation of tumor diagnosis reagents or kits.

Definition of terms

In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art. Also, the procedures of cell culture, biochemistry, nucleic acid chemistry, immunological laboratories and the like used herein are all conventional procedures widely used in the corresponding fields. Meanwhile, in order to better understand the present invention, the definitions and explanations of related terms are provided below.

As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one Light Chain (LC) and one Heavy Chain (HC). Antibody light chains can be classified as K (kappa) and lambda (lambda) light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE, respectively. Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, and the heavy chain also contains a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of 3 domains (CHl, CH2, and CH 3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit a variety of effector functions, such as may mediate binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). The VH and VL regions can also be subdivided into regions of high denaturation, called Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, called Framework Regions (FRs). Each VH and VL are composed of, in the following order: FRI, CDRl, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from the amino terminus to the terminal of the immediately adjacent base. The variable domains (VH and VL) of each heavy/light chain pair form the 5-position of the antigen-binding portion, respectively. The assignment of amino acids in each region or domain may follow Kabat, Sequences of Proteins of immunological Interest (national institutes of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J.mol.biol.196: 901-917; chothia et al (1989) Nature342: 878-883.

As used herein, the terms "monoclonal antibody", "mAb" have the same meaning and are used interchangeably to refer to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on the antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies that typically recognize different epitopes on an antigen. Furthermore, the modifier "monoclonal" is used merely to indicate that the antibody is characterized as being obtained from a population of highly homologous antibodies, and is not to be construed as requiring production of the antibody by any particular method.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed. The strength or affinity of a specific binding interaction may be the equilibrium dissociation constant (K) of the interaction_D) And (4) showing. In the present invention, the term "K_D"refers to the dissociation equilibrium constant for a particular antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the more tight the antibody-antigen binding and the higher the affinity between the antibody and the antigen. In certain embodiments, an antibody that specifically binds to (or is specific for) an antigen means that the antibody has less than about 10^-9M, e.g. less than about 10^-9M、10^-10M、10^-11M or 10^-12M or less affinity (K)_D) Binding the antigen. In certain embodiments, when K_DIs less than or equal to 10^-9M (preferably ≦ 10)^-10M), an antibody or antigen-binding fragment thereof of the invention is said to specifically bind IGSF 9.

The terms "cancer", "tumor" and "tumors" are used interchangeably and refer to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in vivo. Unregulated cell division may lead to the formation of malignant tumors or cells that invade adjacent tissues and may migrate to remote sites in the body through the lymphatic system or the bloodstream. Cancer includes benign and malignant cancers as well as dormant tumors or micrometastases. Cancer also includes hematologic malignancies.

Exemplary such tumors include, but are not limited to, solid tumors, hematological tumors (e.g., leukemia, lymphoma, 30 myeloma, e.g., multiple myeloma), and metastatic, refractory, or recurrent lesions of cancer; for example, including, but not limited to, esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, renal cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, bile duct cancer, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, leukemia. The term "hematological malignancy" includes lymphoma, leukemia, myeloma or lymphoid malignancies, as well as spleen cancer and lymph node tumors. Exemplary lymphomas include B-cell lymphomas and T-cell lymphomas. B cell lymphomas, including, for example, hodgkin lymphoma. T cell lymphomas, including, for example, cutaneous T cell lymphomas. Hematological malignancies also include leukemias, such as secondary leukemia or acute lymphocytic leukemia. Hematological malignancies also include myelomas (e.g., multiple myeloma) and other hematological and/or B-cell or T-cell related cancers.

Compared with the prior art, the beneficial effect of this application is: the application discovers that IGSF9 protein, particularly LQYLSL of an intracellular region of IGSF9 protein as an ITIM motif has the characteristic of high expression in various cancer cells through screening research; the antibody is used for identifying IGSF9 protein, particularly identifying the epitope of LQYLSL of IGSF9 protein, has good effects of inhibiting tumor and treating tumor, and is used for preparing bifunctional antibody conjugate, drug conjugate and drug composition; or the IGSF9 protein is used as a tumor marker to prepare a diagnostic reagent with good effect.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 shows the expression of IGSF9 protein in lung, breast, liver, endometrial, ovarian and corresponding normal tissues according to Experimental example 1 of the present invention;

FIG. 2 is a diagram showing the results of the co-culture of the present invention in experimental example 2 in which cells are in contact with each other;

FIG. 3 shows the results of cell-to-cell isolation co-culture in Experimental example 2;

FIG. 4 shows that IGSF9 of Experimental example 3 of the present invention promotes tumor growth in vivo. Mice vaccinated with LL2-XZ201 and LL2-IGSF9 showed no significant change in body weight (A), but mice vaccinated with LL2-IGSF9 showed significantly enhanced luciferase activity (B), significant increase in tumor volume and weight (CD);

FIG. 5 is a graph showing the effect of IGSF9 of example 3 on the level of immune cells in tumor-bearing mice.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory Press, 1989, and F.M. Ausubel et al, eds. molecular biology laboratory Manual, 3 rd edition, John Wiley & Sons, Inc., 1995; the use of restriction enzymes follows the conditions recommended by the product manufacturer. The examples are given by way of illustration and are not intended to limit the scope of the invention as claimed.

Example 1

In the present example, the expression of IGSF9 protein in lung cancer, liver cancer, breast cancer, endometrial cancer and ovarian cancer was detected by immunohistochemistry.

As a result, IGSF9 was found to be highly expressed in these tumor tissues, but not expressed or expressed less in the corresponding normal tissues, as shown in FIG. 1.

The immunohistochemical staining results show that: in 48 lung adenocarcinoma tissues, IGSF9 expression was detected, with weak positive, moderate intensity and strong positive expression rates: 35.4%, 45.8% and 18.75%; chi-square test showed that expression of IGSF9 correlates with tumor differentiation; multifactorial anova has shown that IGSF9 can be used as an independent factor in assessing the prognosis of patients with lung adenocarcinoma.

In 40 squamous cell lung carcinoma samples, the expression rates of IGSF9 with weak positive, moderate intensity and strong positive were: 65%, 30% and 5%; statistical analysis shows that the expression of IGSF9 is related to lung squamous carcinoma differentiation and can be used as an independent factor for prognosis of patients with lung squamous carcinoma.

IGSF9 was expressed in all (47) small cell lung cancer tissues with weak positive, moderate intensity and strong positive expression rates: 36.17%, 31.91% and 31.91%, and is related to the differentiation and clinical stage of the small cell lung cancer patients, and can also be used as an independent factor to evaluate the prognosis of the small cell lung cancer patients.

IGSF9 was expressed in all non-triple negative breast cancer (72 cases), triple negative breast cancer (57 cases), hepatocellular carcinoma (94 cases), cholangiocarcinoma (15 cases), endometrial cancer (97 cases), and ovarian cancer (55 cases) specimens, and the degree of expression correlated with tumor differentiation.

The combination of the above results shows that: IGSF9 protein is not expressed or is expressed in normal tissues and is expressed in tumor tissues, so that the IGSF9 protein is a novel tumor marker; the expression degree of IGSF9 protein is related to tumor differentiation, the higher the expression is, the worse the tumor differentiation is, and IGSF9 protein can be used as an independent factor to evaluate the prognosis of lung cancer, liver cancer, breast cancer, endometrial cancer and ovarian cancer.

Example 2

The present example provides a gene knockout method to verify the relationship between high expression of IGSF9 protein in tumor tissues and immunosuppressive function.

Knock off the expression of the IGSF9 gene in H69 cells using CRISP-Cas9 (IGSF9 knock out, IGSF 9-KO); on the basis of IGSF9-KO cells, IGSF9 gene (IGSF9 reseue, IGSF9-Res) is overexpressed, and meanwhile, truncated somatic cells (IGSF9-ECD) which only contain IGSF9 Extracellular domain (ECD) but do not contain intracellular domain are constructed. The above cells (IGSF9-WT, KO, Res, ECD) were co-cultured with normal human CD3+ T cells in a U-shaped 96-well plate. CD3+ T cells were stained with 5- (6) -Carboxyfluorescein diacetate (5-and 6-Carboxyfluorescein diacetate, CFSE), the dilution factor of CFSE representing cell proliferation.

The results are shown in fig. 2 and 3, and flow cytometry analysis showed that: the proliferation of CD3+ T cells in the IGSF9-KO group was significantly higher than that of the IGSF9 wild-type group (IGSF 9-WT); restoring expression of IGSF9 in IGSF9-KO cells, enabling restoration of IGSF 9-mediated T cell proliferation inhibition; however, IGSF9-ECD cells failed to significantly restore T cell proliferation inhibition, suggesting that IGSF9 may inhibit T cell proliferation by a paracrine pathway rather than by cell-cell contact. To verify this conclusion, we performed isolated T cell co-cultures using 96-well plates in Transwell, placing CFSE-labeled CD3+ T cells in the 96-well plates and H69 cells in the upper layers of the Transwell chambers, consistent with cell-cell contact results, further indicating that IGSF9 inhibits T cell proliferation via the paracrine pathway.

Example 3

This example demonstrates that IGSF9 protein inhibits T cell proliferation and promotes escape of tumor immune cells in vivo.

the LL2 cells expressing the empty vector were designated (LL2-XZ201) by cloning a murine IGSF9 gene (mIGSF9) which is highly homologous to a human IGSF9 gene (hIGSF9) and overexpressed in LL2 cells (designated LL2-mIGSF 9). Will be 5X 10⁶The individual cells were inoculated subcutaneously into C57BL/6 mice, tumor size was measured every 3 days, when the tumor volume exceeded 2cm3, the mice were sacrificed under anesthesia, tumors were dissected, and single cell suspensions of peripheral blood, bone marrow, spleen and liver were prepared and the levels of immune cells in each tissue were analyzed by flow cytometry. The results show that: there was no significant change in body weight of LL2-mIGSF9 mice compared to control LL2-XZ201 (fig. 4A), but there was a significant increase in tumor volume, weight (fig. 4B, fig. 4C, and fig. 4D), indicating that mIGSF9 was able to promote tumor cell growth. Two possible reasons for this were to analyze that mIGSF9 directly promoted the proliferation of LL2 cells, and that mIGSF9 promoted the proliferation of LL2 cells by inhibiting the activity of T cells. And drawing a cell growth curve by the Ing.

The results are shown in FIG. 4, where the body weights of mice vaccinated with LL2-XZ201 and LL2-IGSF9 were not significantly changed (FIG. 4A), but the luciferase activity of mice vaccinated with LL2-IGSF9 was significantly enhanced (FIG. 4B), and the tumor volume and weight were significantly increased (FIG. 4C and FIG. 4D). mIGSF9 had no significant function to promote tumor cell proliferation (fig. 4E); it is shown that mIGSF9 can promote tumor proliferation by inhibiting the activity of T cells.

As shown in fig. 5, by analyzing the levels of immune cells in various tissues, it was found that mice inoculated with LL2-mIGSF9 cells had significantly reduced levels of CD3+/CD4+, CD3+/CD8+ T cells in tumor tissues and spleen, compared to mice inoculated with LL2-XZ201 cells, while there was no significant difference in the content of CD3+ T cells in peripheral blood, bone marrow and liver (fig. 5A and 5B); in contrast, the levels of Treg (CD4+/CD25+) cells were also significantly reduced in tumor tissues and spleen (fig. 5C), indicating that mIGSF9 directly inhibited the proliferation of CD3+ T cells, rather than CD3+/CD8+ effector T cells by increasing the level of Treg cells. Macrophage (Mac1+/F4/80+), Granulytic MDSC (Ly6G +/Ly6C low) and Monocytic MDSC (Ly6G-/Ly6C high) did not differ significantly between the two groups (FIGS. 5D and 5E).

The combination of the above results shows that: the IGSF9 protein can inhibit cellular immunity through a paracrine pathway, and is a novel tumor immune escape mechanism.

the IGSF9 protein is highly expressed in tumor tissues, so the IGSF9 protein can be used as a tumor marker; therefore, the IGSF9 protein can be used as a tumor diagnosis marker in the preparation of tumor diagnosis reagents or kits.

The present example provides an antibody, an anti-IGSF 9 antibody recognizes IGSF9 protein of tissues or cells (including but not limited to lung cancer tissue, liver cancer tissue, breast cancer tissue, endometrial cancer tissue and ovarian cancer tissue), and the full-length amino acid sequence of IGSF9 protein is shown in SEQ ID No. 1.

Further, the anti-IGSF 9 antibody recognizes an extracellular region of IGSF9 protein, preferably, the anti-IGSF 9 antibody recognizes a sequence shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 or SEQ ID NO.7 of the extracellular region of IGSF9 protein.

The amino acid sequence of SEQ ID NO.2 is:

The amino acid sequence of SEQ ID NO.3 is:

the amino acid sequence of SEQ ID No.4 is:

The amino acid sequence of SEQ ID No.5 is:

vircpvranp pllfvswtkdgkalqldkfp gwsqgtegsl iialgnedal geysctpyns lgta；

The amino acid sequence of SEQ ID No.6 is:

llipc saqgdpppvv swtkvgrglq gqaqvdsnss lilrpltkea hghwecsasn avarvatstn；

the amino acid sequence of SEQ ID NO.7 is:

vtnvsvvalp kganvswepg fdggylqrfsvwytplakrp drmhhdwvsl avpvgaahll vpglqphtqy qfsvlaqnkl gsgpfs。

This example also provides a bifunctional antibody conjugate comprising an anti-IGSF 9 antibody as described above and a second biologically functional fragment; the anti-IGSF 9 antibody and the second biologically functional fragment are linked by a linker protein or a disulfide bond; the second biologically functional fragment specifically binds to a receptor or ligand selected from the group consisting of: PD-1, PD-L1, VGFR, PD-L2, TIM3, TIGIT, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, nkp80 and LAG-3. Wherein the linker protein is Fc structural protein or (GGGGS) n protein; wherein n is an integer from 1 to 20. The connection through Fc structural protein or (GGGGS) n protein does not cause steric hindrance, and the functions of the anti-IGSF 9 antibody and the second biological functional fragment can be ensured.

The present embodiments also provide a drug conjugate comprising a cytotoxic molecule, a linker, and the aforementioned anti-IGSF 9 antibody; cytotoxic molecules include calicheamicin, duocarmycin, the microtubule inhibitor paclitaxel, dolastatin, and maytansine.

This embodiment also provides a drug conjugate comprising the aforementioned anti-IGSF 9 antibody, a buffer, an osmolality adjusting agent, and a surfactant; the buffer solution is selected from a citric acid buffer solution, a histidine buffer solution, an acetate buffer solution, a succinate buffer solution or a Tris buffer solution; the osmotic pressure regulator is sodium chloride; the surfactant is selected from polysorbate 80 or polysorbate 20; the pH value of the buffer solution is 4.5 to 6.5; the concentration is 5mM to 100 mM.

The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Sequence listing

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Arg Pro Pro Leu His Val Ile Glu Trp Leu Arg Phe Gly Phe Leu Leu

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Gly Leu Arg Val Glu Asp Gln Gly Trp Tyr Glu Cys Arg Val Phe Phe

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Leu Asp Gln His Ile Pro Glu Asp Asp Phe Ala Asn Gly Ser Trp Val

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180 185 190

Ile Arg Arg Val Glu Arg Gly Ser Ser Gly Val Tyr Thr Cys Gln Ala

195 200 205

Ser Ser Thr Glu Gly Ser Ala Thr His Ala Thr Gln Leu Leu Val Leu

210 215 220

Gly Pro Pro Val Ile Val Val Pro Pro Lys Asn Ser Thr Val Asn Ala

225 230 235 240

Ser Gln Asp Val Ser Leu Ala Cys His Ala Glu Ala Tyr Pro Ala Asn

245 250 255

Leu Thr Tyr Ser Trp Phe Gln Asp Asn Ile Asn Val Phe His Ile Ser

260 265 270

Arg Leu Gln Pro Arg Val Arg Ile Leu Val Asp Gly Ser Leu Arg Leu

275 280 285

Leu Ala Thr Gln Pro Asp Asp Ala Gly Cys Tyr Thr Cys Val Pro Ser

290 295 300

Asn Gly Leu Leu His Pro Pro Ser Ala Ser Ala Tyr Leu Thr Val Leu

305 310 315 320

Tyr Pro Ala Gln Val Thr Ala Met Pro Pro Glu Thr Pro Leu Pro Ile

325 330 335

Gly Met Pro Gly Val Ile Arg Cys Pro Val Arg Ala Asn Pro Pro Leu

340 345 350

Leu Phe Val Ser Trp Thr Lys Asp Gly Lys Ala Leu Gln Leu Asp Lys

355 360 365

Phe Pro Gly Trp Ser Gln Gly Thr Glu Gly Ser Leu Ile Ile Ala Leu

370 375 380

Gly Asn Glu Asp Ala Leu Gly Glu Tyr Ser Cys Thr Pro Tyr Asn Ser

385 390 395 400

Leu Gly Thr Ala Gly Pro Ser Pro Val Thr Arg Val Leu Leu Lys Ala

405 410 415

Pro Pro Ala Phe Ile Glu Arg Pro Lys Glu Glu Tyr Phe Gln Glu Val

420 425 430

Gly Arg Glu Leu Leu Ile Pro Cys Ser Ala Gln Gly Asp Pro Pro Pro

435 440 445

Val Val Ser Trp Thr Lys Val Gly Arg Gly Leu Gln Gly Gln Ala Gln

450 455 460

Val Asp Ser Asn Ser Ser Leu Ile Leu Arg Pro Leu Thr Lys Glu Ala

465 470 475 480

His Gly His Trp Glu Cys Ser Ala Ser Asn Ala Val Ala Arg Val Ala

485 490 495

Thr Ser Thr Asn Val Tyr Val Leu Gly Thr Ser Pro His Val Val Thr

500 505 510

Asn Val Ser Val Val Ala Leu Pro Lys Gly Ala Asn Val Ser Trp Glu

515 520 525

Pro Gly Phe Asp Gly Gly Tyr Leu Gln Arg Phe Ser Val Trp Tyr Thr

530 535 540

Pro Leu Ala Lys Arg Pro Asp Arg Met His His Asp Trp Val Ser Leu

545 550 555 560

Ala Val Pro Val Gly Ala Ala His Leu Leu Val Pro Gly Leu Gln Pro

565 570 575

His Thr Gln Tyr Gln Phe Ser Val Leu Ala Gln Asn Lys Leu Gly Ser

580 585 590

Gly Pro Phe Ser Glu Ile Val Leu Ser Ala Pro Glu Gly Leu Pro Thr

595 600 605

Thr Pro Ala Ala Pro Gly Leu Pro Pro Thr Glu Ile Pro Pro Pro Leu

610 615 620

Ser Pro Pro Arg Gly Leu Val Ala Val Arg Thr Pro Arg Gly Val Leu

625 630 635 640

Leu His Trp Asp Pro Pro Glu Leu Val Pro Lys Arg Leu Asp Gly Tyr

645 650 655

Val Leu Glu Gly Arg Gln Gly Ser Gln Gly Trp Glu Val Leu Asp Pro

660 665 670

Ala Val Ala Gly Thr Glu Thr Glu Leu Leu Val Pro Gly Leu Ile Lys

675 680 685

Asp Val Leu Tyr Glu Phe Arg Leu Val Ala Phe Ala Gly Ser Phe Val

690 695 700

Ser Asp Pro Ser Asn Thr Ala Asn Val Ser Thr Ser Gly Leu Glu Val

705 710 715 720

Tyr Pro Ser Arg Thr Gln Leu Pro Gly Leu Leu Pro Gln Pro Val Leu

725 730 735

Ala Gly Val Val Gly Gly Val Cys Phe Leu Gly Val Ala Val Leu Val

740 745 750

Ser Ile Leu Ala Gly Cys Leu Leu Asn Arg Arg Arg Ala Ala Arg Arg

755 760 765

Arg Arg Lys Arg Leu Arg Gln Asp Pro Pro Leu Ile Phe Ser Pro Thr

770 775 780

Gly Lys Ser Ala Ala Pro Ser Ala Leu Gly Ser Gly Ser Pro Asp Ser

785 790 795 800

Val Ala Lys Leu Lys Leu Gln Gly Ser Pro Val Pro Ser Leu Arg Gln

805 810 815

Ser Leu Leu Trp Gly Asp Pro Ala Gly Thr Pro Ser Pro His Pro Asp

820 825 830

Pro Pro Ser Ser Arg Gly Pro Leu Pro Leu Glu Pro Ile Cys Arg Gly

835 840 845

Pro Asp Gly Arg Phe Val Met Gly Pro Thr Val Ala Ala Pro Gln Glu

850 855 860

Arg Ser Gly Arg Glu Gln Ala Glu Pro Arg Thr Pro Ala Gln Arg Leu

865 870 875 880

Ala Arg Ser Phe Asp Cys Ser Ser Ser Ser Pro Ser Gly Ala Pro Gln

885 890 895

Pro Leu Cys Ile Glu Asp Ile Ser Pro Val Ala Pro Pro Pro Ala Ala

900 905 910

Pro Pro Ser Pro Leu Pro Gly Pro Gly Pro Leu Leu Gln Tyr Leu Ser

915 920 925

Leu Pro Phe Phe Arg Glu Met Asn Val Asp Gly Asp Trp Pro Pro Leu

930 935 940

Glu Glu Pro Ser Pro Ala Ala Pro Pro Asp Tyr Met Asp Thr Arg Arg

945 950 955 960

Cys Pro Thr Ser Ser Phe Leu Arg Ser Pro Glu Thr Pro Pro Val Ser

965 970 975

Pro Arg Glu Ser Leu Pro Gly Ala Val Val Gly Ala Gly Ala Thr Ala

980 985 990

Glu Pro Pro Tyr Thr Ala Leu Ala Asp Trp Thr Leu Arg Glu Arg Leu

995 1000 1005

Leu Pro Gly Leu Leu Pro Ala Ala Pro Arg Gly Ser Leu Thr Ser Gln

1010 1015 1020

Ser Ser Gly Arg Gly Ser Ala Ser Phe Leu Arg Pro Pro Ser Thr Ala

1025 1030 1035 1040

Pro Ser Ala Gly Gly Ser Tyr Leu Ser Pro Ala Pro Gly Asp Thr Ser

1045 1050 1055

Ser Trp Ala Ser Gly Pro Glu Arg Trp Pro Arg Arg Glu His Val Val

1060 1065 1070

Thr Val Ser Lys Arg Arg Asn Thr Ser Val Asp Glu Asn Tyr Glu Trp

1075 1080 1085

Asp Ser Glu Phe Pro Gly Asp Met Glu Leu Leu Glu Thr Leu His Leu

1090 1095 1100

Gly Leu Ala Ser Ser Arg Leu Arg Pro Glu Ala Glu Pro Glu Leu Gly

1105 1110 1115 1120

Val Lys Thr Pro Glu Glu Gly Cys Leu Leu Asn Thr Ala His Val Thr

1125 1130 1135

Gly Pro Glu Ala Arg Cys Ala Ala Leu Arg Glu Glu Phe Leu Ala Phe

1140 1145 1150

Arg Arg Arg Arg Asp Ala Thr Arg Ala Arg Leu Pro Ala Tyr Arg Gln

1155 1160 1165

Pro Val Pro His Pro Glu Gln Ala Thr Leu Leu

1170 1175

<210> 2

<211> 109

<212> PRT

<213> Homo sapiens

<400> 2

Pro Glu Val Val Ser Val Val Gly Arg Ala Gly Glu Ser Val Val Leu

1 5 10 15

Gly Cys Asp Leu Leu Pro Pro Ala Gly Arg Pro Pro Leu His Val Ile

20 25 30

Glu Trp Leu Arg Phe Gly Phe Leu Leu Pro Ile Phe Ile Gln Phe Gly

35 40 45

Leu Tyr Ser Pro Arg Ile Asp Pro Asp Tyr Val Gly Arg Val Arg Leu

50 55 60

Gln Lys Gly Ala Ser Leu Gln Ile Glu Gly Leu Arg Val Glu Asp Gln

65 70 75 80

Gly Trp Tyr Glu Cys Arg Val Phe Phe Leu Asp Gln His Ile Pro Glu

85 90 95

Asp Asp Phe Ala Asn Gly Ser Trp Val His Leu Thr Val

100 105

<210> 3

<211> 88

<212> PRT

<213> Homo sapiens

<400> 3

Pro Gln Phe Gln Glu Thr Pro Pro Ala Val Leu Glu Val Gln Glu Leu

1 5 10 15

Glu Pro Val Thr Leu Arg Cys Val Ala Arg Gly Ser Pro Leu Pro His

20 25 30

Val Thr Trp Lys Leu Arg Gly Lys Asp Leu Gly Gln Gly Gln Gly Gln

35 40 45

Val Gln Val Gln Asn Gly Thr Leu Arg Ile Arg Arg Val Glu Arg Gly

50 55 60

Ser Ser Gly Val Tyr Thr Cys Gln Ala Ser Ser Thr Glu Gly Ser Ala

65 70 75 80

Thr His Ala Thr Gln Leu Leu Val

85

<210> 4

<211> 80

<212> PRT

<213> Homo sapiens

<400> 4

Pro Pro Val Ile Val Val Pro Pro Lys Asn Ser Thr Val Asn Ala Ser

1 5 10 15

Gln Asp Val Ser Leu Ala Cys His Ala Glu Ala Tyr Pro Ala Asn Leu

20 25 30

Thr Tyr Ser Trp Phe Gln Asp Asn Ile Asn Val Phe His Ile Ser Arg

35 40 45

Leu Gln Pro Arg Val Arg Ile Leu Val Asp Gly Ser Leu Arg Leu Leu

50 55 60

Ala Thr Gln Pro Asp Asp Ala Gly Cys Tyr Thr Cys Val Pro Ser Asn

65 70 75 80

<210> 5

<211> 64

<212> PRT

<213> Homo sapiens

<400> 5

Val Ile Arg Cys Pro Val Arg Ala Asn Pro Pro Leu Leu Phe Val Ser

1 5 10 15

Trp Thr Lys Asp Gly Lys Ala Leu Gln Leu Asp Lys Phe Pro Gly Trp

20 25 30

Ser Gln Gly Thr Glu Gly Ser Leu Ile Ile Ala Leu Gly Asn Glu Asp

35 40 45

Ala Leu Gly Glu Tyr Ser Cys Thr Pro Tyr Asn Ser Leu Gly Thr Ala

50 55 60

<210> 6

<211> 65

<212> PRT

<213> Homo sapiens

<400> 6

Leu Leu Ile Pro Cys Ser Ala Gln Gly Asp Pro Pro Pro Val Val Ser

1 5 10 15

Trp Thr Lys Val Gly Arg Gly Leu Gln Gly Gln Ala Gln Val Asp Ser

20 25 30

Asn Ser Ser Leu Ile Leu Arg Pro Leu Thr Lys Glu Ala His Gly His

35 40 45

Trp Glu Cys Ser Ala Ser Asn Ala Val Ala Arg Val Ala Thr Ser Thr

50 55 60

Asn

65

<210> 7

<211> 86

<212> PRT

<213> Homo sapiens

<400> 7

Val Thr Asn Val Ser Val Val Ala Leu Pro Lys Gly Ala Asn Val Ser

1 5 10 15

Trp Glu Pro Gly Phe Asp Gly Gly Tyr Leu Gln Arg Phe Ser Val Trp

20 25 30

Tyr Thr Pro Leu Ala Lys Arg Pro Asp Arg Met His His Asp Trp Val

35 40 45

Ser Leu Ala Val Pro Val Gly Ala Ala His Leu Leu Val Pro Gly Leu

50 55 60

Gln Pro His Thr Gln Tyr Gln Phe Ser Val Leu Ala Gln Asn Lys Leu

65 70 75 80

Gly Ser Gly Pro Phe Ser

85

Claims

1. The anti-IGSF 9 antibody, wherein the anti-IGSF 9 antibody recognizes IGSF9 protein of tissue or cell, and the amino acid sequence of the IGSF9 protein is shown in SEQ ID NO. 1.

2. The anti-IGSF 9 antibody of claim 1, wherein the anti-IGSF 9 antibody recognizes the extracellular domain of IGSF9 protein, preferably the amino acid sequence of the extracellular domain is as shown in SEQ ID No.2, SEQ ID No.3, SEQ ID No.4, SEQ ID No.5, SEQ ID No.6 or SEQ ID No. 7.

3. The anti-IGSF 9 antibody of claim 1, wherein the tissue comprises tumor tissue comprising lung cancer tissue, liver cancer tissue, breast cancer tissue, endometrial cancer tissue, and ovarian cancer tissue.

4. A bifunctional antibody conjugate comprising an anti-IGSF 9 antibody of any one of claims 1-3 and a second biologically functional fragment; the anti-IGSF 9 antibody and the second biologically functional fragment are linked by a linker protein or a disulfide bond.

5. The bifunctional antibody conjugate of claim 4, wherein the second biologically functional fragment specifically binds to a receptor or ligand selected from the group consisting of: PD-1, PD-L1, VGFR, PD-L2, TIM3, TIGIT, VISTA, CTLA-4, OX40, BTLA, 4-1BB, CD96, CD27, CD28, nkp80 and LAG-3.

6. The bifunctional antibody conjugate of claim 4, wherein the linker protein is an Fc structural protein or a (GGGGS) n protein; wherein n is an integer from 1 to 20.

7. a drug conjugate comprising a cytotoxic molecule, a linker and the anti-IGSF 9 antibody of any one of claims 1-3.

8. The drug conjugate according to claim 7, characterized in that the cytotoxic molecule comprises calicheamicin, duocarmycin, the microtubule inhibitors dolastatin and maytansine.

9. A pharmaceutical composition comprising the anti-IGSF 9 antibody of any one of claims 1-3, a buffer, an osmolality adjusting agent, and a surfactant; the buffer solution is selected from a citric acid buffer solution, a histidine buffer solution, an acetate buffer solution, a succinate buffer solution or a Tris buffer solution; the osmotic pressure regulator is sodium chloride; the surfactant is selected from polysorbate 80 or polysorbate 20.

The IGSF9 protein is used as a tumor diagnosis marker in the preparation of tumor diagnosis reagents or kits.