CN112979809A

CN112979809A - Targeting molecule for reversing anti-PD-1 antibody treatment resistance

Info

Publication number: CN112979809A
Application number: CN202110208260.8A
Authority: CN
Inventors: 赵健; 郎小玲; 耿树生
Original assignee: Beijing Zhaoderivative Technology Co ltd
Current assignee: Beijing Zhaoderivative Technology Co ltd
Priority date: 2021-02-24
Filing date: 2021-02-24
Publication date: 2021-06-18
Anticipated expiration: 2041-02-24
Also published as: CN112979809B

Abstract

The invention discloses a targeting molecule for reversing the treatment resistance of an anti-PD-1 antibody, which is a monoclonal antibody for blocking the function of TIM-3. The light chain variable region comprises a heavy chain and a light chain, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO.7, and the amino acid sequence of the light chain variable region is shown as SEQ ID NO. 8. The monoclonal antibody can be specifically combined with TIM-3 antigen, can specifically block a TIM-3 mediated signal channel, and can synergistically enhance the function of resisting the immune disorder of the PD-1 antibody, thereby becoming a new medicament for treating tumor immunotherapy, chronic virus infectious diseases and autoimmune diseases.

Description

Targeting molecule for reversing anti-PD-1 antibody treatment resistance

Technical Field

The invention belongs to the fields of tumor treatment and molecular immunology, relates to a targeting molecule for reversing the treatment resistance of an anti-PD-1 antibody, and particularly relates to a monoclonal antibody specifically binding to TIM-3.

Background

Tim-3 gene and Tim-3 structural protein: the mouse Tim-3 gene is positioned on chromosome 11, and the total length of the transcript is 1015 basic groups; the human Tim-3 gene is located on chromosome 5, and the total length of the transcript is 1116 bases. The Tim family has at least four members, which are Tim-1, Tim-2, Tim-3 and Tim-4. Wherein Tim-1 is the receptor of hepatitis A virus, Tim-2 can be expressed in Th2 cell specifically, Tim-3 protein is the surface mark of activated Th1 cell, Tim-4 is the ligand molecule of Tim-1 and is expressed in mature DC.

Current data indicate that the mouse Tim-3 molecule consists of 281 amino acids, which are associated with Th 1-related responses and diseases; the human Tim-3 molecule is a type I membrane protein consisting of 301 amino acids, the extracellular portion of which includes a cysteine-rich Ig-like region containing 4 conserved cysteines and a mucin region rich in threonine, serine and proline. The intracellular region contains about 42-77 amino acids, which is the most highly conserved region in mouse and human homologues.

Link between Tim-3 gene and immune system related diseases and functions: the human Tim family is located at 5q33.2, a region that is susceptible to asthma. Certain atopy, such as asthma and allergic rhinitis, are most closely related to the polymorphic site of Tim-3 gene-574T/G. In patients with allergic rhinitis, the genotype and the allele frequency of the Tim-3 gene at the-1516G/T, -574T/G and 4259G/T sites are remarkably different from those of normal people. The research on the relationship between two single nucleotide polymorphic sites of the Tim-3 promoter region and allergic asthma proves that the Tim-3 promoter region polymorphic sites and allergic diseases are closely related.

Autoimmune disease is a disease associated with an inflammatory response. If the inflammatory response is down-regulated, the development of autoimmune disease can be reduced or even prevented. Some receptor molecules (e.g., CTLA-4), cytokines (e.g., TGF-. beta.), and regulatory cells (e.g., CD4+ CD25+ T cells) together maintain the immune balance of the body. The Tim family of proteins are a major regulatory molecule for inflammatory responses, providing inhibitory signals, down-regulating inflammatory responses, and reducing the incidence of autoimmune diseases and allergic reactions. Fri-sancho-Kiss and the like can induce the expression of a co-stimulatory molecule CD80 to be reduced in macrophages and mast cells and can also reduce the level of CD4+ T cell CTLA-4 by reducing the expression of Tim-3 signals of virus-infected mice, finally results in the reduction of regulatory T cells and the increase of heart inflammatory responses, and shows that the Tim-3 signals play an important role in regulating adaptive immune responses.

Immune factors are one of the important factors affecting the survival and expansion of tumor cells in vivo. Transplant recipients who chronically take immunosuppressive agents are often at risk of developing certain malignancies because they are unable to activate a large number of functional T cells. It is generally considered that T cell immunity is the main content of tumor immunity, and if cytotoxic T Cells (CTLs) with sufficient specificity against tumor cells can be generated in vivo, the tumor cells are difficult to survive. Tim-3 is a specific surface marker for Th1 type cells and is not expressed on naive T cells, B cells, DCs and hematopoietic cells. Tim-3 is presumed to regulate tumor immunity by regulating Th1 type cell response.

Tim-3/Tim-3L immunoreaction with Th: the ligand expression of Tim-3 (Tim-3L) is much broader than Tim-3. The studies by Zhu et al found that Tim-3L was highly expressed on TK-1, a mouse lymphoma cell of CD8+, and was identified as Galectin-9 (Galectin-9). Galectin-9 is a soluble glycoprotein A adhered to a cell membrane, can induce thymocytes and peripheral CD4+ and CD8+ T cells to die, and plays an important role in regulating the dynamic balance of immune cells and inflammatory response. In addition, the research finds that the Tim-3/Galectin-9 pathway is blocked, the survival effect of prolonging the allogeneic skin transplantation mediated by CD4+ CD25+ regulatory T cells is eliminated, and the fact that the Tim-3L is not only expressed on CD4+ CD25+ regulatory T cells is proved, and the function of the CD4+ CD25+ regulatory T cells is influenced by the Tim-3/Galectin-9 pathway is proved. Galectin-9 is also present on the surface of a few CD11+ dendritic cells and CDllb + monocytes in the spleen, and Tim-3L may also be expressed on macrophages and APC. Tim-3 binds to Tim-3L (Galectin-9) thereby down-regulating the Thl-type immune response and may modulate CD4+ CD25+ regulatory T cells. To understand the mechanism of Tim-3 binding to its ligand to inhibit the effector function of Th1 cells, Kuchroo et al added human Galectin-9 to differentiated mature Thl and Th2 cells and found abnormal calcium influx and rapid Thl cell death, while Th2 and Tim-3-/-Thl cells did not have this effect, thus confirming that the action of Galectin-9 is dependent on binding to Tim-3. Abnormalities in the Tim-3/Tim-3L signaling pathway are present in many diseases in which the Thl response is aberrant. Autoimmune Encephalomyelitis (EAE) is a typical Thl type immune response, and application of Tim-3 antibodies in a mouse EAE model by Monney et al can accelerate the onset of the disease, increase the severity of the disease and increase the mortality. The WangF et al research shows that recombinant Galectin-9 can selectively delete Tim3+ Th1 cells, reduce IFN-gamma secretion, obviously inhibit allograft rejection and increase the survival rate of allograft skin when being administered to an allograft skin transplantation mouse.

Tim-3/Tim-3L and tumor immunity: it is believed that in tumor immunity, T lymphocytes are the core, and CD8+ T cells can directly act as effector cells to specifically kill cancer cells, and are called killer T lymphocytes (CTLs). Yet another type of tumoricidal cell is an NK cell, which spontaneously kills target cells without antigen pre-sensitization and has a cytotoxic effect. CD4+ T cells are of less interest, but they also play an important role in regulating tumor immunity in the body. CD4+ T cell Thl cell secretes IL-2, IFN-gamma and TNF-alpha, etc., which can promote cellular immunity directly or indirectly by activating the cytotoxicity and phagocytosis of CTL cell, NK cell and macrophage, so that they are very important for the anti-tumor immune response of the body. In advanced tumor patients, mainly Thl cells are inhibited, and thus tumor immunotherapy tries to shift Thl/Th2 to Thl. Tim-3 is a specific surface marker for Thl-type cells and is not expressed on naive T cells, B cells, DCs and hematopoietic cells. It is further speculated that Tim-3 may modulate tumor immunity by modulating the Thl cell response.

The research finds that: a large amount of Galectin-9 is expressed in a exosome secreted by nasopharyngeal carcinoma cells, and the Galectin-9 can be prevented from being cut by protease when the Galectin-9 is expressed by inserting into the exosome. The exosome secreted by the nasopharyngeal carcinoma cells can induce a great deal of apoptosis of specific nasopharyngeal carcinoma CD4+ T cells, and the effect can be inhibited by an anti-Tim-3 antibody or an anti-Galectin-9 antibody, so that the blockage of the pathway Tim-3/Galectin-9 is proved, the inhibition reaction of the nasopharyngeal carcinoma exosome on Thl immune response can be relieved, the anti-tumor effect of the T cells is maintained, and the effect of clinical immunotherapy on the nasopharyngeal carcinoma is improved. The study of Zoltan et al found: tim-3 is highly expressed on both human mast cells and melanoma cells, and interestingly Galectin-9 is also expressed on mast cells, but not on tumor cells; they also found that the increase of the expression level of Tim-3 in TGF-beta 1-stimulated mast cells can inhibit the anti-tumor capability of the body, and the tumor promotion effect is probably dependent on the combination of Tim-3 expressed on the mast cells and Galetin-9 expressed on self mast cells, so that the Tim-3 is considered to promote the development of melanoma, a tumor cell. Geng and the like construct a eukaryotic expression plasmid psTim-3 of sTim-3, and the growth of melanoma cells in vivo of animals transfected with the expression plasmid is accelerated, which is probably related to that the sTim-3 can inhibit the production of cytokines IL-2, IFN-gamma and TNF-beta in vivo; the activity of anti-tumor CTL cells is reduced, the number of infiltrating lymphocytes in tumor tissues is reduced, and the result shows that the sTim-3 can obviously reduce the anti-tumor immunity of T cells; the gene expression of Thl cell factors is also found to be down-regulated in the real-time quantitative PCR monitoring of the gene expression of the tumor microenvironment, while the expression of genes Foxp-3, IL-10 and TGF-beta related to regulatory T cells is basically unchanged. Thus, it was suggested that sTim-3 might be involved in negative regulation of T cell mediated immune responses.

At present, no Tim-3 antibody is on the market at home and abroad, so that detection and diagnosis and treatment articles based on the antibody with independent intellectual property rights are established and developed, and the method has important practical significance for the intervention of various related diseases.

Disclosure of Invention

It is an object of the present invention to provide a targeting molecule that binds to TIM-3 and acts by blocking the binding of TIM-3 to its ligand.

In order to achieve the purpose, the invention adopts the following technical scheme:

the present invention provides an isolated targeting molecule comprising:

(1) heavy chain CDR1 shown in SEQ ID NO. 1, heavy chain CDR2 shown in SEQ ID NO. 2, and heavy chain CDR3 shown in SEQ ID NO. 3; and/or

(2) Light chain CDR1 shown in SEQ ID NO.4, light chain CDR2 shown in SEQ ID NO. 5, and light chain CDR3 shown in SEQ ID NO. 6.

As one aspect of the present invention, the targeting molecule of the present invention comprises:

(1) a heavy chain variable region having an amino acid sequence set forth in SEQ ID NO. 7; and/or

(2) And a light chain variable region having an amino acid sequence set forth in SEQ ID NO 8.

(1) a heavy chain having an amino acid sequence shown in SEQ ID NO 17;

(2) and the light chain has an amino acid sequence shown as SEQ ID NO. 18.

The CDRs in the targeting molecules of the present invention are not limited to the specific sequences of VH and VL mentioned above, and may include variants of these sequences that retain the ability to specifically bind to TIM-3. Such variants may be derived from the specific sequences mentioned above by the skilled artisan using techniques well known in the art. For example, amino acid substitutions, deletions or additions may be made in the FRs and/or CDRs. While changes in FRs are generally designed to improve the stability and immunogenicity of antibodies, changes in CDRs are generally designed to increase the affinity of an antibody for its target. FRs variants also include naturally occurring immunoglobulin allotypes. Such changes that increase affinity can be determined empirically by conventional techniques, including altering CDRs and testing the affinity of an antibody for its target. For example, conservative amino acid substitutions may be made within any of the CDRs disclosed. Various modifications may be made in accordance with the methods described in antibody engineering, 2 nd edition, Oxford University Press, ed by Borrebaeck, 1995. These include, but are not limited to, nucleotide sequences that have been altered by the substitution of different codons within the sequence that encode functionally equivalent amino acid residues, thereby producing "silent" changes. For example, non-polar amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Amino acid substitutions within a sequence may be selected from other members of the class to which the amino acid belongs. In addition, any natural residues in the polypeptide may also be substituted with alanine.

"targeting molecules" as used herein refer to antibody molecules and immunologically active fragments, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen. The antibody molecules of the invention may be of any type (e.g. IgG, IgE, IgM, IgD, IgA), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1, hIgA2) or subclass. Immunologically active portions of antibody molecules include, but are not limited to, Fab 'and F (ab') 2, Fd, single chain fv (scfv), single chain antibodies, disulfide linked fv (sdfv), and single domain antibodies comprising a VL or VH domain. Antigen-binding antibody fragments, including single chain antibodies, may include variable regions alone or in combination with all or a portion of: hinge region, CH1, CH2 and CH3 domain. The invention also includes antigen binding fragments comprising any combination of the variable region and the hinge, CH1, CH2, and CH3 domains.

Targeting molecules of the invention also include targeting molecules recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugation) to the polypeptide.

Examples of recombinant fusion of polypeptides include fusion of a targeting molecule of the invention with a tag sequence including, but not limited to, an HA tag, a 6XHis tag, a c-Myc tag, a flag tag.

Examples of chemical conjugation include conjugating the targeting molecule of the present invention to a detectable substance. Detection may be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomography scans, and non-radioactive paramagnetic metal ions. The detectable substance may be coupled or conjugated directly to the antibody (or fragment thereof) or indirectly via an intermediate, such as a linker as is known in the art, using techniques known in the art. See, e.g., U.S. Pat. No.4,741,900 for metal ions that can be conjugated to antibodies for use as diagnostic agents of the present invention. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include umbelliferylKetones, fluorescein isothiocyanate, rhodamine, dichlortriazinylamine fluorescein, dansyl chloride or phycoerythrin; examples of luminescent materials include luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; examples of suitable radioactive materials include¹²⁵I、¹³¹I、¹¹¹In or⁹⁹Tc。

The targeting molecules of the present invention also include functional variants of the targeting molecules described above. A variant molecule is considered to be a functional variant of a targeting molecule of the invention if the variant competes with the parent targeting molecule for specific binding to TIM-3 or a protein fragment thereof. In other words, the functional variant is still able to bind to TIM-3 or a protein fragment thereof. The functional variants may have conservative sequence modifications, including amino acid substitutions, additions, and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and can comprise natural as well as unnatural amino acids. Furthermore, functional variants may comprise a truncation of the amino acid sequence at the amino terminus or the carboxy terminus or both. The functional variants of the invention may have the same or different, higher or lower binding affinity than the parent targeting molecule, but still bind to TIM-3 or a fragment thereof. Hereinafter, when the term "targeting molecule" is used, it also covers functional variants of said targeting molecule.

As a further aspect of the invention, the invention also provides a polynucleotide of a targeting molecule as described above.

The polynucleotide sequence encoding the heavy chain CDR1 is shown in SEQ ID NO. 9, the polynucleotide sequence encoding the heavy chain CDR2 is shown in SEQ ID NO. 10, the polynucleotide sequence encoding the heavy chain CDR3 is shown in SEQ ID NO. 11, the polynucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO. 12, and the polynucleotide sequence encoding the heavy chain is shown in SEQ ID NO. 19; the polynucleotide sequence encoding light chain CDR1 is set forth in SEQ ID NO. 13, the polynucleotide sequence encoding light chain CDR2 is set forth in SEQ ID NO. 14, the polynucleotide sequence encoding light chain CDR3 is set forth in SEQ ID NO. 15, the polynucleotide sequence encoding light chain variable region is set forth in SEQ ID NO. 16, and the polynucleotide sequence encoding light chain is set forth in SEQ ID NO. 20.

The invention also includes polynucleotides that hybridize under stringent or less stringent conditions to a polynucleotide encoding a targeting molecule of the invention.

Those skilled in the art will appreciate that functional variants of these polynucleotides are also part of the present invention. A functional variant is a nucleic acid sequence that can be directly translated using standard genetic code to provide the same amino acid sequence as translated from a parent nucleic acid molecule.

The polynucleotide sequence can be obtained and the nucleotide sequence of the polynucleotide determined using any method known in the art. For example, if the nucleotide sequence of an antibody is known, a polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides.

Alternatively, polynucleotides encoding the antibody may be produced from nucleic acids from a suitable source. If no clone containing a nucleic acid encoding a particular antibody is available, but the sequence of the antibody molecule is known, then the nucleic acid encoding the immunoglobulin can be obtained by chemical synthesis, or by PCR amplification from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing the antibody, e.g., hybridoma cells selected for expression of the antibody of the invention, or a nucleic acid isolated therefrom, preferably poly A + RNA) using synthetic primers hybridizable to the 3 'and 5' ends of the sequence, or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify a cDNA clone encoding the antibody, e.g., from a cDNA library. The amplified nucleic acid produced by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

Once the nucleotide sequence of the antibody and the corresponding amino acid sequence are determined, the nucleotide sequence of the antibody can be processed using methods well known in the art for processing nucleotide sequences, such as recombinant DNA techniques, site-directed mutagenesis, PCR, and the like, to generate antibodies having different amino acid sequences, such as amino acid substitutions, deletions, and/or insertions.

The amino acid sequences of the heavy and/or light chain variable domains can be examined to identify the sequence of the CDRs by well-known methods, for example, by comparison with known amino acid sequences of other heavy and light chain variable regions to determine regions of high variability of the sequences. Using conventional recombinant DNA techniques, one or more CDRs can be inserted into a framework region, for example into a human framework region to humanize a non-human antibody, as described above. The framework regions may be naturally occurring or common framework regions, and preferably human framework regions (see, e.g., the list of human framework regions in 293 Tzhiaeta l., J.mol.biol.278: 457-. Preferably, the polynucleotides produced by the combination of framework regions and CDRs encode antibodies that specifically bind to the polypeptides of the invention. Preferably, as discussed above, one or more amino acid substitutions may be made within the framework regions, and preferably the amino acid substitutions improve binding of the antibody to its antigen. In addition, one or more variable region cysteine residues involved in an intrachain disulfide bond may be substituted or deleted by these methods to produce an antibody molecule lacking one or more intrachain disulfide bonds. Other variations on the polynucleotides are encompassed by the present invention and are within the skill of those in the art.

The present invention also provides a recombinant vector comprising the polynucleotide as defined above; preferably, the recombinant vector is constructed by inserting the polynucleotide described above into a ZY-CDMO vector.

Recombinant vectors containing nucleotide sequences encoding the targeting molecules of the invention can be prepared using well-known techniques. The vector includes a nucleotide sequence operably linked to a suitable transcription or translation regulating nucleotide sequence, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, enhancers, mRNA ribosome binding sites, and/or other suitable sequences that control transcription and translation initiation and termination. Nucleotide sequences are "operably linked" when the regulatory sequence is functionally related to the nucleotide sequence of a suitable polypeptide. Thus, a promoter nucleotide sequence is operably linked to, for example, an antibody heavy chain sequence if it controls the transcription of the appropriate nucleotide sequence.

The present invention also provides a host cell comprising a polynucleotide as described above or a recombinant vector as described above.

Host cells useful in the present invention include, but are not limited to, microorganisms such as bacteria (e.g., escherichia coli, bacillus subtilis) transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing antibody coding sequences; yeast such as Saccharomyces (Saccharomyces), Pichia (Pichia)) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus (CaMV); Tobacco Mosaic Virus (TMV); or transformed with recombinant plasmid expression vectors containing antibody coding sequences (e.g., Ti plasmid), or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) carrying recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., the metallothionein promoter) or promoters derived from mammalian viruses (e.g., the adenovirus late promoter, the vaccinia virus 7.5K promoter).

In a particular embodiment of the invention, the host cell is a mammalian cell, more preferably a CHO cell.

Transformation and transfection of host cells with recombinant DNA may be carried out by conventional techniques well known to those skilled in the art. Some transformation, transfection methods that may be employed include, but are not limited to: conventional chemical methods such as calcium phosphate co-precipitation, PEI transfection, and conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like.

The resulting transformants can be cultured by conventional methods to express the targeting molecules of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The targeting molecules of the present invention are preferably produced using mammalian cells, which typically require culture in serum-containing media. After the serum-free adaptation process of the cells is required, the cells can be normally grown in a serum-free medium.

The present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a targeting molecule as described above.

Preferably, the pharmaceutical composition comprises a therapeutically effective amount of the targeting molecule as described above; more preferably, the pharmaceutical composition comprises a therapeutically effective amount of the targeting molecule as described above, a therapeutically effective amount of at least one of the following antibodies: an antibody or antigen-binding fragment thereof against a negative immune regulator molecule on a T cell membrane, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof; most preferably, the negative immune regulator molecules on the T cell membrane include CD25, Foxp3, CTLA-4, GITR.

The invention also provides a pharmaceutical package or kit comprising one or more containers containing one or more components of the pharmaceutical composition of the invention. Optionally accompanying these containers may be instructions in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which instructions reflect certification by the agency regulating the manufacture, use or sale of pharmaceuticals for human administration.

The invention also provides a test product comprising a targeting molecule as hereinbefore described.

The detection product includes, but is not limited to, a detection reagent, a kit, a chip or a test paper. Any assay product capable of detecting TIM-3 that includes the aforementioned targeting molecules is included within the scope of the present invention.

The present invention also provides a method for detecting TIM-3 levels for non-diagnostic purposes, comprising the steps of:

(1) obtaining a sample containing TIM-3;

(2) contacting the sample obtained in step (1) with a targeting molecule as described above;

(3) detecting the binding reaction of the sample and the targeting molecule.

The invention also provides a method of making the targeting molecule comprising culturing a host cell comprising the vector of the invention under conditions suitable to cause expression of the protein from the DNA encoding the antibody molecule of the invention, and isolating the targeting molecule.

The invention also provides the use of a targeting molecule as hereinbefore described, which comprises any one of:

(1) use in the preparation of a test product as hereinbefore described;

(2) use in the preparation of a pharmaceutical composition as hereinbefore described;

(3) the application of the compound in preparing a medicament for blocking the binding of TIM-3 and a ligand of TIM-3;

(4) use in the manufacture of a medicament for modulating TIM-3 activity or level;

(5) the application in preparing the medicine for relieving the immunosuppression of PD-1 to the organism;

(6) the application in preparing the medicine for activating T cells;

(7) the application in preparing the medicine for promoting the expression of IFN-gamma in T lymphocyte;

(8) the application in preparing the medicine for reversing acquired resistance of anti-PD-1 immunotherapy;

(9) the application in preparing the medicine for enhancing the anti-tumor immune response;

(10) the application in preparing the medicine for reversing the peripheral tolerance of the organism;

(11) the application in preparing antineoplastic drugs;

(12) the application in preparing medicine for treating immune disorder.

The invention also provides the use of a pharmaceutical composition as hereinbefore described, which comprises any one of:

(1) the application in preparing the medicine for activating T cells;

(2) the application in preparing the medicine for promoting the expression of IFN-gamma in T lymphocyte;

(3) the application in preparing the medicine for relieving the immunosuppression of PD-1 to the organism;

(4) the application in preparing the medicine for reversing acquired resistance of anti-PD-1 immunotherapy;

(5) the application in preparing the medicine for enhancing the anti-tumor immune response;

(6) the application in preparing the medicine for reversing the peripheral tolerance of the organism;

(7) the application in preparing antineoplastic drugs;

(8) the application in preparing medicine for treating immune disorder.

The targeting molecule can also be combined with other medicines with the same or complementary functions, the combined application effect can be the sum of the functions of the targeting molecule and other medicines, and can also be far greater than the sum of the functions of the targeting molecule and other medicines, and the situation shows that the targeting molecule and other medicines generate synergistic action.

Other drugs with the same or complementary functions include known and unknown targeted antineoplastic agents, and may also include any chemotherapeutic agent or compound.

The targeted antitumor drugs include, but are not limited to, 17-AAG, 2-deoxyglucose, Abiraterone (Abiraterone), ABT-263, AC-220, AT-406, AZD4547, AZD5363, AZD7762, BI-2536, Birinapant, BMS-754807, Bortezomib (Bortezomib), BX-795, Cabozantinib (Cabozantinib), CAL-101, Carfilzomib (Carfilzomib), crizotinib (crizotinib), danesertib, Dasatinib (Dasatinib), Dovitinib, Elesclomol, Embelint, Entinostat (Entinostat) (MS-275), Enzastaurin, Everolimus (Everolis), Foretinib, fulvestris (Funetlestt), Nevirant, Netinc-49391, MK-7235, NV-7241, MK-599, MK-367241, ML7211, ML4135235, ML4153, MLNPK-364153, MLNPK-367211, MLIVibP, MLIVibe, SAb, Kb-3552, MLIVibb, Kb, NV-3675, MLIVibb, MLV-3675, MLIVibb-3, MLV-2 52, MLIVibb, MLV-3, MLIVibb, MLV-3, MLV-429286A 3, and MLIVibb, PD-173074, PH-797804, PRT062607, R-406, Refametinib, Regorafenib (Regorafenib), SCH900776, sgi-1776, Sorafenib (Sorafenib), Sunitinib (Sunitinib), TAE684, Temsirolimus (Temsimus), TG-101348, Tidegluusib, Tipifarnib, Tivantinib, Tormentifene, Tozatiliib, Trametinib (Trametinib), Tretinoin (Tretinoin), Triptolide, Valdecoxib (Valdecoxib), Victorib (Victorgium), Volertib, Vorinostat (Vorinostat), YM-155, CHIR-99021, NVP-99209398, BGP 0314.

The chemotherapeutic agent includes, but is not limited to, altretamine, aminoglutethimide, anastrozole, azacitidine, bendamustine, busulfan, cabazitaxel, capecitabine, carboplatin, cisplatin, cladribine, clofarabine, cyclophosphamide, cytarabine, dacarbazine, daunorubicin, decitabine, docetaxel, doxorubicin, epirubicin, etoposide, exemestane, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lenalidomide, letrozole, leucovorin, lomustine, 6-mercaptopurine, mesna, methotrexate, mitotane, mitoxantrone, oxaliplatin, paclitaxel, nelarabine, pemetrexed, pralatrexate, procaine, ritin, streptozotocin, temozolomide, teniposide, thioguanine, azathioprine, doxycycline, topotecan, vinblastine, vinorelbine, zoledronic acid.

Drawings

FIG. 1 shows a physical map of a ZY-CDMO vector;

FIG. 2 is a graph showing the results of detection of antibody affinity activity by ELISA;

FIG. 3 shows a statistical chart for the detection of the effect of antibodies on IFN-. gamma.secretion by T cells using ELISA.

Detailed Description

Interpretation of terms

The term "immune disorder" as used herein refers to immunosuppression of the body. Non-limiting examples of immune disorders that can be used in the present invention include, but are not limited to, rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, crohn's disease, systemic lupus erythematosus, type I diabetes, transplant rejection, graft versus host disease, hyperproliferative immune disorders, tumors, and infectious diseases. The type of tumor that the targeting molecule of the present invention can be used to treat is not particularly limited, and any solid, non-solid, malignant or benign tumor is included within the scope of the present invention. Examples of tumors include, but are not limited to: skin cancer, leukemia, adrenocortical cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, tracheal and bronchial tumors, lymphoma, tumors of the nervous system, cervical cancer, intestinal cancer, anal cancer, endometrial cancer, esophageal cancer, nasopharyngeal cancer, ovarian cancer, sarcoma, eye cancer, malignant fibrous histiocytic carcinoma, gallbladder cancer, stomach cancer, colorectal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, blastoma, head and neck cancer, liver cancer, hypopharynx cancer, melanoma, pancreatic cancer, kidney cancer, larynx cancer, lip cancer, oral cancer, oropharyngeal cancer, lung cancer, mesothelioma, myeloma, parathyroid cancer, penile cancer, eosinophilic tumor, pituitary tumor, prostate cancer, retinoblastoma, salivary gland cancer, skin cancer, testicular cancer, thymoma, thyroid cancer, urinary tract cancer, vaginal cancer, vulval cancer.

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population in which the individual antibodies comprised are identical except for a few naturally occurring mutations that may be present. The modifier "monoclonal" indicates only the identity of the antibody and is obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring any particular method for producing the antibody.

The term "variable" as used herein means that certain portions of the variable regions of an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. Variability is concentrated in three segments of the light and heavy chain variable regions, called Complementarity Determining Regions (CDRs) or hypervariable regions. The variable regions of native heavy and light chains each comprise four FR regions (the more conserved portions of the variable regions) in a substantially β -sheet configuration, joined by three CDRs forming a connecting loop, which may form part of a β -sheet structure. The CDRs in each chain are held together tightly by the FR regions and form the antigen binding site of the antibody with the CDRs of the other chain. The constant regions are not directly involved in the binding of antibodies to antigens, but they exhibit different effector functions, such as participation in antibody-dependent cytotoxicity of antibodies.

The terms "treatment" and "method of treatment" refer to both therapeutic treatment and prophylactic/preventative measures. Those in need of treatment may include those already suffering from a particular medical disorder, as well as those who may eventually suffer from the disorder (i.e., those in need of prophylactic measures).

As used herein, "sample" encompasses a variety of sample types, including blood and other bodily fluid samples of biological origin, solid tissue samples such as biopsy tissue samples or tissue cultures, or cells derived therefrom or progeny thereof. The term also includes samples that have been treated by any means after they have been obtained, for example by treating with reagents, solubilizing, or enriching certain components such as proteins or polynucleotides. The term encompasses various clinical samples obtained from any species, also including cultured cells, cell supernatants and cell lysates.

The term "isolated" refers to a molecule that is substantially free from its natural environment. For example, an isolated protein is substantially free of cellular material or other proteins from the cell or tissue source from which it is derived. The term "isolated" also refers to a formulation wherein the isolated protein is sufficiently pure to be capable of being administered as a pharmaceutical composition, or at least 70-80% (w/w) pure, more preferably at least 80-90% (w/w) pure, even more preferably 90-95% pure, and most preferably at least 95%, 96%, 97%, 98%, 99% or 100% (w/w) pure.

The term "specifically binds" refers to the formation of a complex of two molecules that is relatively stable under physiological conditions. Specific binding is characterized by high affinity and low to moderate binding capacity, as opposed to non-specific binding, which typically has low affinity and moderate to high binding capacity. In general, binding is considered specific when the affinity constant KA is greater than 106M-1 or more preferably greater than 108M-1. If necessary, non-specific binding can be reduced by changing the binding conditions without substantially affecting specific binding. The skilled artisan can optimize suitable binding conditions, such as concentration of antibody, ionic strength of the solution, temperature, time allowed for binding, concentration of blocking agent (e.g., serum albumin, milk casein), and the like, using routine techniques.

Embodiments of the present invention will be described in detail with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the invention only and should not be taken as limiting the scope of the invention. The examples do not show the specific techniques or conditions, and the techniques or conditions are described in the literature in the art (for example, refer to molecular cloning, a laboratory Manual, third edition, scientific Press, written by J. SammBruker et al, Huang Petang et al) or according to the product instructions. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

EXAMPLE 1 construction of anti-TIM-3 antibody expression vector

And screening phage antibody libraries at the early stage to obtain a plurality of antibodies with stronger binding force with TIM antigen, wherein one antibody is researched and numbered as No. 37.

(1) An antibody heavy chain sequence (wherein, the heavy chain nucleic acid sequence of the 37# antibody is shown as SEQ ID NO: 19) and a light chain sequence (wherein, the light chain nucleic acid sequence of the 37# antibody is shown as SEQ ID NO: 20) are synthesized by using a gene synthesis method, and the fragments are cloned into a ZY-CDMO vector by using a molecular cloning method, wherein the map of the ZY-CDMO vector is shown as figure 1.

The specific operation is as follows: introducing an EcoRI enzyme cutting site at the 5 'end of the light chain and an XbaI enzyme cutting site at the 3' end by conventional PCR, and inserting the EcoRI enzyme cutting site and the XbaI enzyme cutting site between the EcoRI and the XbaI enzyme cutting site of the ZY-CDMO vector; introducing HindIII enzyme cutting site at the 5 'end of the heavy chain, introducing PmeI enzyme cutting site at the 3' end, inserting the enzyme cutting sites between HindIII and PmeI of the ZY-CDMO vector, and constructing the eukaryotic expression vector.

EXAMPLE 2 expression and purification of anti-TIM-3 antibodies

Inoculating 30ml of cell culture shake flask with density of 0.5X 10⁶cells/ml CHO cells, when cell density is up to 2X 10⁶cells/ml, cell viability above 95%, cell count at 1 × 10⁷cells/ml density was inoculated in the electrotransfer buffer,add 40. mu.g plasmid to the cuvette, add 0.7ml cell suspension, and finally add the electrotransfer buffer to 0.8ml, mix gently without air bubbles. Shocking once at 300V and 900 μ F, placing the shocking cup in ice box, ice-cooling for 5min, diluting to 6ml culture medium, and adding CO at 37 deg.C₂Recovering for 48 hr in incubator, adding 75 μ M MSX into seed culture medium, inoculating 96-well plate, cloning, and screening, adding monoclonal cells into 96-well plate by limiting dilution method, adding 5% CO at 37 deg.C₂Culturing in an incubator, performing ELISA verification when the cells are amplified to a certain number, and selecting positive clones and sequentially amplifying the positive clones to a 24-pore plate, a 6-pore plate and a T25 square bottle. And culturing and screening for 6-8 weeks to obtain a monoclonal cell strain capable of efficiently expressing the anti-TIM-3 antibody.

The monoclonal cell strain is subjected to screening culture medium multi-step amplification culture, and the inoculation density is 0.5 multiplied by 10⁶cells/ml, after 2 weeks, cells were harvested by centrifugation in 125ml shake flasks at 37 ℃ with 5% CO₂Fed Batch culture was carried out for 12 days on a shaker at 125rpm, and during the period, 10% supplemented medium was added to the culture medium at the initial volume on days 3, 5, 7 and 9, while maintaining the glucose concentration at 3g/L to 5g/L, and after the completion of the culture, the supernatant was harvested and purified. TIM-3 antibody was isolated and purified using AKTA (GE). The eluate, which was passed through a Protein A affinity column (MabSelect Sure) at pH ranging from 3.4 to 3.6 (monitored at 280 nm), was first collected, adjusted to pH 8.0, applied to an anion exchange chromatography column (Q-Sepharose FF), monitored at 280nm, and the sample was collected. Adjusting the pH value of the collected solution to 5.5, loading the solution to cation exchange chromatography (Poros) to collect a sample, and performing ultrafiltration and concentration to obtain an antibody against TIM-3.

Example 3 detection of affinity Activity of anti-TIM-3 antibodies for TIM-3 antigen

The affinity is detected by ELISA, and the specific steps are as follows:

coating: ELISA plates were taken and the target protein human TIM-3 (Nano Biological, 10390-H08H) was diluted with pH9.6 coating to 0.5. mu.g/ml, 100. mu.l/well coated overnight at 4 ℃ in a wet box.

The well was discarded and the plate inverted to pat the residual liquid dry on paper towel and washed 3 times with 300. mu.l/well of washing solution (0.1% PBST).

And (3) sealing: add blocking solution (2% M-PBS) 300. mu.l/well and let stand at 37 ℃ for 1.0 h.

Adding a primary antibody: the anti-TIM-3 antibodies (37#, 67#, 75#) were diluted to 1mg/ml with DPBS, and then 10-fold dilutions were performed in sequence to give 7 concentration gradients, and the diluted samples were added to the corresponding wells at 100. mu.l/well and left to stand at 37 ℃ for 1.0 h.

DPBS was also added as a negative control. The dilution method is as follows:

sheep anti-human-HRP secondary antibody: diluted with DPBS at a ratio of 1:3000, 100. mu.l/well, and left to stand at 37 ℃ for 1.0 h.

Color development: TMB color development, 100. mu.l/well, room temperature development for 10 min.

And (4) terminating: 2N H₂SO₄100 μ l/well, OD450nm by microplate reader.

As a result:

as shown in FIG. 2, the anti-TIM-3 antibody of the present invention had an EC50 of 0.02785. mu.g/ml.

Example 4 affinity kinetics of anti-TIM antibodies with TIM-3 antigen

Surface plasmon resonance biosensors were used to measure binding kinetics and affinity of antibodies to TIM-3 antigens. Unless otherwise indicated, all reagents and materials were purchased from GE corporation and measurements were made at 25 ℃. Affinity analysis was performed by SPR (Biacore T200) instrument, coupling anti-human IgG Fc antibody to CM5 chip by amino coupling, flowing the antibody to be tested at a flow rate of 30 μ L/min, and capturing the antibody to be tested with the anti-human IgG Fc antibody coupled to the chip; after analyte (TIM-3) gradient dilution (100nM, 50nM, 25nM, 12.5nM, 6.25nM, 3.13nM and 0nM), the flow rate was 30 μ L/min, the binding time of the antibody to be tested to the analyte was 120s, and the dissociation time was 1200 s; HBS-EP was used as the running buffer throughout the experiment and the chip was regenerated with a 60 second pulse of 10mM glycine HCl, pH 2.1. The data were fit to a 1:1 binding model to determine the equilibrium dissociation constant, KD.

As a result: equilibrium dissociation constant KD of 0.98X 10^-9M。

Example 5 Effect of anti-TIM-3 antibodies on INF-gamma secretion by immune cells

Human Peripheral Blood Mononuclear Cells (PBMC) were separated from 300mL of fresh blood cells using a human lymphocyte separation medium, and mononuclear cells were separated from PBMC using a monocyte magnetic bead sorting kit (Meitian whirlpool, Cat. 130-. Subjecting the isolated monocytes to a temperature of 2.5X 10^4/The wells were plated in 96-well plates in 1640 medium containing 10% serum, 250U/mL human GM-CSF and 500U/mL human IL-4, the medium was changed every three days for seven days, and on the seventh day the monocytes were differentiated into dendritic cells (DC cells).

CD4+ T cells were simultaneously isolated on day seven from PBMCs of another fresh blood fraction by CD4+ T cell magnetic bead sorting (anti-biotin magnetic beads (America whirlwind company: 130-. 2 x 10 to⁵CD4+ T cells/well with dendritic cells according to 5: 1, and inoculated into a 96-well cell culture plate, while adding 10. mu.g/mL anti-TIM-3 antibody and 0. mu.g/mL and 2. mu.g/mL nivolumab antibody, respectively, and after culturing for five days, the supernatant was collected and the expression of IFN-. gamma.in the supernatant was detected using an IFN-. gamma.detection kit (purchased from BioLegend, Cat. 430104).

PBMC isolation procedure was as follows:

(1) 300mL of fresh blood was drawn (heparin anticoagulated) and diluted with an equal volume of saline.

(2) 5mL of the human peripheral blood lymphocyte separation medium was added to a 15mL centrifuge tube, and then the diluted blood was slowly added to the upper layer of the lymphocyte separation medium, followed by centrifugation at 2000rpm for 20 min.

(3) After centrifugation, the buffy coat layer was aspirated by a pipette into a tube containing 10mL of physiological saline, and centrifuged at 1500rpm for 10min, which was repeated twice.

(4) Counting under microscope and carrying out next experiment.

The magnetic bead sorting of the monocytes was as follows:

(1) every 10 th⁷Each PBMC cell was resuspended in 30. mu.L sorting Buffer, 10. mu.L FcR Blocking Reagent and 10. mu.L Biotin-Antibody Cocktail were added and mixed well, and incubated on ice for 10min in the absence of light.

(2) Every 10 th⁷30 μ L of sorting Buffer and 20 μ L of Anti-Biotin MicroBeads were added to each PBMC cell, mixed well, and incubated on ice for 15min in the dark.

(3) 2mL/10 of the solution was added⁷Sorting Buffer of individual cells, centrifuge at 300g for 10 min.

(4) Adding 500 mu L/10⁸The individual cell sorting Buffer resuspended the cells, prepared beads, placed on the magnet, and the column washed with 300mL of sorting Buffer. The cell suspension was added to the column.

(5) The cell fluid left was collected and the column was washed twice with 3mL of sorting Buffer.

The magnetic bead sorting procedure for CD4+ T cells was as follows:

(1) every 10 th⁷Each PBMC Cell was resuspended in 40. mu.L sorting Buffer, 10. mu.L CD4+ T Cell Biotin-Antibody Cocktail were added, and incubated on ice for 5 min.

(2) Every 10 th⁷mu.L of sorting Buffer and 20. mu.L of CD4+ T Cell MicroBead Cocktail were added to each PBMC Cell, mixed well and incubated on ice for 10min in the absence of light.

(3) The beads were prepared, placed on a magnetic pole, and the column was washed with 300mL sorting Buffer. The cell suspension was added to the column.

(4) The cell fluid that flowed down was collected and the column was washed three times with 3mL of sorting Buffer.

The ELISA detection procedure for IFN-. gamma.was as follows:

(1) mu.L of diluted Capture antibodies was added to each well, and the ELISA plates were sealed and incubated overnight at 4 ℃.

(2) The plate was washed 4 times with plate washer, 200. mu.L of 1 × Assay dilution A was added to each well, the ELISA plates were sealed and incubated on a shaker for 1h at room temperature.

(3) The plate was washed 4 times with plate washer, 100. mu.L of diluted standards and test samples were added to each well, the ELISA plates were sealed and incubated on a shaker for 2h at room temperature.

(4) The plate was washed 4 times with plate washing machine, 100. mu.L of diluted Detection Antibody was added to each well, the ELISA plate was sealed, and incubated on a shaker at room temperature for 1 h.

(5) The plate was washed 4 times with plate washer, 100. mu.L of diluted Avidin-HRP was added to each well, the ELISA plate was sealed and incubated on a shaker for 30min at room temperature.

(6) And washing the plate by a plate washing machine for 5 times, adding 100 mu L of mixed TMB color development liquid into each hole, and incubating for 20min in a dark place.

(7) Add 100. mu.L of Stop Solution to each well and read the absorbance of the ELISA plate at 450nm on the microplate.

Results

As shown in FIG. 3, the anti-TIM-3 antibody significantly enhanced the IFN-. gamma.secretion ability of CD4+ T cells when human IgG was used as a negative control, and the synergistic effect of the anti-TIM-3 antibody and nivolumab was superior to the sum of the effects of the anti-TIM-3 antibody and nivolumab used alone. The results show that the antibody of the invention can greatly cooperate with the PD-1 antibody to activate T cells to obviously secrete INF-gamma.

Although only specific embodiments of the present invention have been described above, it will be understood by those skilled in the art that these are by way of illustration only, and that the scope of the invention is defined by the appended claims. Various changes or modifications to these embodiments may be made by those skilled in the art without departing from the principle and spirit of the invention, and these changes or modifications are within the scope of the invention.

Sequence listing

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ggccaggcgc cgcgcctgct gatttataat gcgagccaac gctccaccgg cattccggcg 180

cgctttagcg gcagcggcag cggcaccgat tttaccctga ccattagcag cctggaaccg 240

gaagattttg cggtgtatta ttgccagcag cgcttctcct ggccgccgac ctttggccag 300

ggcaccaaac tggaaattaa acgtacggtg gctgcaccat ctgtcttcat cttcccgcca 360

tctgatgagc agttgaaatc tggaactgcc tctgttgtgt gcctgctgaa taacttctat 420

cccagagagg ccaaagtaca gtggaaggtg gataacgccc tccaatcggg taactcccag 480

gagagtgtca cagagcagga cagcaaggac agcacctaca gcctcagcag caccctgacg 540

ctgagcaaag cagactacga gaaacacaaa gtctacgcct gcgaagtcac ccatcagggc 600

ctgagctcgc ccgtcacaaa gagcttcaac aggggagagt gt 642

Claims

1. An isolated targeting molecule, wherein said targeting molecule comprises:

(2) Light chain CDR1 shown in SEQ ID NO.4, light chain CDR2 shown in SEQ ID NO. 5, and light chain CDR3 shown in SEQ ID NO. 6;

preferably, the targeting molecule comprises:

(2) A light chain variable region having an amino acid sequence set forth in SEQ ID NO 8;

preferably, the targeting molecule comprises:

(1) a heavy chain, wherein the variable region of the heavy chain has an amino acid sequence shown in SEQ ID NO. 17;

and/or

(2) And the light chain has an amino acid sequence shown as SEQ ID NO. 18.

2. A polynucleotide encoding the targeting molecule of claim 1; preferably, the polynucleotide sequence encoding heavy chain CDR1 is set forth in SEQ ID NO. 9, the polynucleotide sequence encoding heavy chain CDR2 is set forth in SEQ ID NO. 10, the polynucleotide sequence encoding heavy chain CDR3 is set forth in SEQ ID NO. 11, the polynucleotide sequence encoding heavy chain variable region is set forth in SEQ ID NO. 12, and the polynucleotide sequence encoding heavy chain is set forth in SEQ ID NO. 19; the polynucleotide sequence encoding light chain CDR1 is set forth in SEQ ID NO. 13, the polynucleotide sequence encoding light chain CDR2 is set forth in SEQ ID NO. 14, the polynucleotide sequence encoding light chain CDR3 is set forth in SEQ ID NO. 15, the polynucleotide sequence encoding light chain variable region is set forth in SEQ ID NO. 16, and the polynucleotide sequence encoding light chain is set forth in SEQ ID NO. 20.

3. A recombinant vector comprising the polynucleotide of claim 2; preferably, the recombinant vector is constructed by inserting the polynucleotide of claim 2 into a ZY-CDMO vector.

4. The method of constructing a recombinant vector according to claim 3, wherein the recombinant vector is constructed by inserting the polynucleotide according to claim 2 into a ZY-CDMO vector.

5. A host cell comprising the polynucleotide of claim 2 or the recombinant vector of claim 3.

6. A pharmaceutical composition or TIM-3 detection product comprising a therapeutically effective amount of the targeting molecule of claim 1; preferably, the pharmaceutical composition comprises a therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof of claim 1; more preferably, the pharmaceutical composition comprises a therapeutically effective amount of the monoclonal antibody or antigen-binding fragment thereof of claim 1, a therapeutically effective amount of at least one of the following antibodies: an antibody or antigen-binding fragment thereof against a negative immune regulator molecule on a T cell membrane, an anti-PD-1 antibody or antigen-binding fragment thereof, an anti-PD-L1 antibody or antigen-binding fragment thereof; most preferably, the negative immune regulator molecules on the T cell membrane include CD25, Foxp3, CTLA-4, GITR.

7. A method for detecting TIM-3 levels for non-diagnostic purposes, said method comprising the steps of:

(1) obtaining a sample containing TIM-3;

(2) contacting the sample obtained in step (1) with the targeting molecule of claim 1;

(3) detecting binding of a sample to the targeting molecule of claim 1.

8. The use of the targeting molecule of claim 1, comprising any one of:

(1) use in the preparation of a test product according to claim 6;

(2) use in the preparation of a pharmaceutical composition according to claim 6;

(6) the application in preparing the medicine for activating T cells;

(11) the application in preparing antineoplastic drugs;

(12) the application in preparing medicine for treating immune disorder.

9. The pharmaceutical composition for use of claim 6, comprising any one of:

(1) the application in preparing the medicine for activating T cells;

(7) the application in preparing antineoplastic drugs;

(8) the application in preparing medicine for treating immune disorder.

10. Use according to claim 8 or 9, wherein the immune disorder comprises rheumatoid arthritis, multiple sclerosis, inflammatory bowel disease, crohn's disease, systemic lupus erythematosus, type I diabetes, transplant rejection, graft versus host disease, hyperproliferative immune disorders, tumors, or infectious diseases.