Antibodies targeting TIM3 and uses thereof

Abstract

The invention provides an antibody targeting TIM3 and application of the antibody. The amino acid sequences of the six CDRs of the TIM3 targeting antibody are as follows: SEQ ID NO: 1 to SEQ ID NO: 6. The TIM3 antibody disclosed by the invention can be used for regulating / improving T cell immunocompetence independently or in combination with other active ingredients.

Description

Technical Field

[0001] The present invention relates to biopharmaceuticals (antibody drugs) and genetic engineering, and in particular to antibodies targeting immune checkpoint TIM3 and applications thereof. Background Art

[0002] T cell immunoglobulin and mucin domain-containing protein 3 (TIM3), also known as hepatitis A virus cellular receptor 2 (HAVCR2), is a type of T cell surface inhibitory molecule. It is a type I transmembrane protein belonging to the TIM immunoregulatory protein family. It is mainly expressed on a variety of immune cells such as T cells, monocytes, macrophages and dendritic cells, and is also expressed on a variety of tumor cells. TIM3 was originally identified as a receptor expressed on helper T cells (CD4+Th1) and cytotoxic T cells (CD8+ T) that can produce gamma interferon. It is expressed in a variety of mouse and human immune cells. The TIM3 gene is located at 11B1.1 in the mouse genome and 5q33.2 in the human genome. The human TIM3 protein consists of 302 amino acids, and the mouse homolog contains 281 amino acid residues (63% homology with humans). TIM3 belongs to the immunoglobulin superfamily (IgSF) and consists of 7 exons that encode its full-length protein sequence, which is characterized by a common structural organization consisting of an amino-terminal immunoglobulin variable domain (V domain) with five non-covalent cysteines, a mucin stalk, a transmembrane domain and a cytoplasmic tail. It has an N-terminal Ig variable region (IgV)-like domain, a membrane-proximal mucin-like domain containing an O-linked glycosylation site (glycosylated mucin domain), a single transmembrane region and a C-terminal cytoplasmic tail. There is also an N-linked glycosylation site between the mucin and the transmembrane.

[0003] The main ligands of TIM3 include galectin-9, high mobility group protein 1 (HMGBl), carcinoembryonic antigen-related adhesion molecule 1 (CEACAM1), and phosphatidylserine (PS), but the mechanism by which TIM3 interacts with its ligands to cause immunosuppression is still not very clear.

[0004] TIM3 can be expressed in a variety of immune cells, including type 1 T helper cells (Th1) and type 17 T helper cells (Th17) in CD4+T cells, CD8+T cells, tumor infiltrating lymphocytes (TILs), regulatory T cells (Tregs), innate immune cells (such as NK cells) and FoxP3+Treg cells. Studies have shown that the key to hindering anti-tumor immunity is T cell dysfunction or T cell exhaustion. This cell state is characterized by impaired production of pro-inflammatory cytokines, and cytotoxicity develops with continued exposure to antigens. TIM3 inhibits anti-tumor immunity mainly by mediating T cell exhaustion. Th1 and Th17 cells participate in anti-tumor cell-mediated immunity through pro-inflammatory cytokines, while CD8+T cells destroy potential threats by binding to target antigens, so TIM3 promotes the occurrence of immune tolerance. After T cell activation, TIM3 is upregulated in CD4+T cells and CD8+T cells that release IFN-γ. In human PBMCs, TIM3 is expressed on all mature CD56dimCD16+NK cells. Immature CD56brightCD16 NK cells can induce TIM3 expression under the stimulation of IL-12, IL-15, and IL-18, indicating that TIM3 is a marker of NK cell maturity. T cell exhaustion inhibits anti-tumor immunity, which can also be caused by impaired Stat5 and p38 signaling pathways in TIM3+CD8+ T cells. Blocking the TIM3 pathway can increase the production of interferon-γ (IFN-γ) in T cells and enhance anti-tumor immune response. In in vitro and in vivo models, the expression of CD8+TIM3+T cells is correlated with the expression of PD-1. In addition, the expression of TIM3 on natural immune cells can inhibit natural anti-tumor immunity and inhibit the proliferation and production of cytokines such as interleukin-2 (IL-2). PD-1 and TIM3 positive CD8+T cells produce less IFN-γ than TIM3 negative CD8+T cells, and anti-TIM3 antibodies can also increase IFN-γ in peripheral blood NK cells. Mast cells expressing TIM3 can be activated by IgE high affinity receptor (FcepsilonRI) containing immunoreceptor tyrosine-based activation motif (ITAM), and the signal transduction pathway used is similar to that of T cells.TIM3 acts proximally to the receptor to enhance Lyn-dependent kinase signaling, which regulates immediate degranulation and late cytokine production downstream of FcepsilonRI ligation (Wolf, Y.; AC Anderson and VK Kuchroo, TIM3 comes of age as an inhibitory receptor. Nature reviews Immunology, 2020. 20(3): 173-185).

[0005] A large amount of evidence supports that blocking the TIM3 signaling pathway can enhance the effect of PD-1 antibody in treating cancer, which has promoted the development of anti-TIM3 blocking antibodies as anti-tumor immune drugs in the hope of being used in the treatment of tumors. Currently, several clinical trials using anti-TIM3 antibodies in cancer patients are underway.

[0006] Therefore, the development of TIM3 antibodies has important clinical significance. Summary of the invention

[0007] One object of the present invention is to provide a molecule that specifically binds to TIM3.

[0008] Another object of the present invention is to provide an antibody targeting TIM3.

[0009] Another object of the present invention is to provide related applications of TIM3 antibodies.

[0010] In order to develop a binding molecule that specifically binds to TIM3, the present invention uses TIM3 fusion protein to immunize BALB / c mice, screens hybridoma clones that secrete antibodies that can specifically bind to TIM3 through hybridoma fusion technology and cell ELISA (using 293T-TIM3 cells as positive cells and 293T cells as negative cells), isolates an antibody that can specifically bind to TIM3, and identifies the antibody light chain and heavy chain variable region sequences by sequencing.

[0011] According to one aspect of the present invention, the present invention provides a molecule that can immunospecifically bind to TIM3, and is therefore also referred to as a TIM3 binding molecule in the present invention. Specifically, the molecule includes a TIM3 antibody or a biologically active fragment (antigen binding fragment) derived from the antibody that can immunospecifically bind to TIM3, wherein the amino acid sequence of the six CDRs of the TIM3 antibody includes:

[0012] Heavy chain CDR1: GYSFTGYNMN (SEQ ID NO: 1);

[0013] Heavy chain CDR2: IIDPYYGTTNYNQKFRG (SEQ ID NO: 2);

[0014] Heavy chain CDR3: GGNSFDY (SEQ ID NO: 3);

[0015] Light chain CDR1: TASSILSSNYLH (SEQ ID NO: 4);

[0016] Light chain CDR2: STSYLAS (SEQ ID NO: 5);

[0017] Light chain CDR3: HQFHPSPRT (SEQ ID NO: 6).

[0018] In the present invention, "immunospecific binding" means that if such binding exhibits the specificity and affinity of an antibody binding to its cognate antigen, then a molecule is said to be able to "immunospecifically bind" to another molecule. If such binding involves the antigen recognition site of the immunoglobulin molecule, then the antibody is said to be able to "immunospecifically bind" to a target region or structure ("epitope") of the antigen (and in particular human TIM3). An antibody that immunospecifically binds to a specific antigen may bind to other antigens with lower affinity if the other antigen has been identified by, for example, immunoassay, The antibody may bind to other molecules in a non-immunospecific manner, such as to Fc receptors (FcRs), by virtue of binding domains in other regions / domains of the molecule that are not involved in the antigen recognition site, such as the Fc region.

[0019] In the present invention, a molecule "capable of immunospecifically binding to TIM3" means that the molecule has the ability to weaken or block the binding of TIM3 to the ligand by binding to TIM3.

[0020] According to a specific embodiment of the present invention, the biologically active fragment capable of immunospecifically binding to TIM3 derived from the antibody of the present invention comprises the amino acid sequence of the six CDRs of the TIM3 antibody.

[0021] According to a specific embodiment of the present invention, the antibody of the present invention may be an animal-derived antibody, or may be a chimeric antibody, a humanized antibody or a human antibody.

[0022] In the present invention, "antibody" is intended to mean an immunoglobulin molecule having a "variable region" antigen recognition site. The term "variable region" is intended to distinguish this domain of the immunoglobulin from domains widely shared by antibodies (e.g., an antibody Fc domain). In the present invention, the term "antigen-binding fragment" of an antibody refers to one or more portions of an antibody that include the complementarity determining region ("CDR") of the antibody and optionally framework residues that include the "variable region" antigen recognition site of the antibody and exhibit the ability to immunospecifically bind to an antigen. Such fragments include Fab', F(ab') and 2 , Fv, single chain (ScFv), and mutants thereof, naturally occurring variants, and fusion proteins comprising the "variable region" antigen recognition site of the antibody and a heterologous protein (e.g., a toxin, an antigen recognition site for a different antigen, an enzyme, a receptor or a receptor ligand, etc.). In the present invention, the term "fragment" refers to a peptide or polypeptide that includes an amino acid sequence of at least 3 or 4 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues.

[0023] Human chimeric antibodies, humanized antibodies or human antibodies are particularly preferred for use in humans, however, antibodies of animal origin may be advantageously employed for many purposes (e.g., in vitro or in situ detection assays, acute in vivo use, etc.). Fully human antibodies are particularly desirable for therapeutic treatment of human subjects. Human antibodies can be manufactured by a variety of methods known in the art, including the above-mentioned phage display methods using antibody libraries derived from human immunoglobulin sequences.

[0024] According to a specific embodiment of the present invention, the present invention provides an antibody that binds to TIM3, named Tim3-1#, whose variable region amino acid sequence is as follows (CDR sequences are shown in bold and underlined):

[0025] Heavy Chain:

[0026]

[0027] Light chain:

[0028]

[0029] According to a specific embodiment of the present invention, the TIM3 binding molecule of the present invention comprises:

[0030] (1) a heavy chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence with substantially equivalent functions formed by replacing, deleting or adding one or more amino acids based on the amino acid sequence; and / or

[0031] (2) a light chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence with substantially equivalent functions formed by replacing, deleting or adding one or more amino acids on the basis of the amino acid sequence.

[0032] As used herein, the term "substantially" in the context of binding or exhibiting an effect is intended to indicate that the observed effect is physiologically or therapeutically relevant. Thus, for example, a molecule is capable of substantially blocking the activity of CD47 if the extent of the blockade is physiologically or therapeutically relevant (e.g., if such extent is greater than 60% of complete blockade, greater than 70% of complete blockade, greater than 75% of complete blockade, greater than 80% of complete blockade, greater than 85% of complete blockade, greater than 90% of complete blockade, greater than 95% of complete blockade, or greater than 97% of complete blockade). Similarly, a molecule is said to have substantially the same immunospecificity and / or characterization as another molecule if such immunospecificity and characterization are greater than 60% identical, greater than 70% identical, greater than 75% identical, greater than 80% identical, greater than 85% identical, greater than 90% identical, greater than 95% identical, or greater than 97% identical.

[0033] In some specific embodiments of the present invention, the present invention demonstrates the specific affinity of the TIM3 antibody of the present invention through ELISA experiments, SPR experiments and flow cytometry experiments.

[0034] The TIM3 antibody of the present invention has the ability to specifically bind to TIM3 at both the protein and cell levels, and has high affinity. The activity of the antibody TIM3 of the present invention is mainly reflected in the ability to activate T cells and enhance the expression and secretion of the cytokine IFN-γ.

[0035] According to another aspect of the present invention, the present invention also provides a nucleic acid molecule encoding the TIM3 binding molecule of the present invention (eg, the TIM3 antibody or a biologically active fragment derived from the antibody that can specifically bind to TIM3).

[0036] According to another aspect of the present invention, the present invention also provides a vector containing the above-mentioned nucleic acid molecule of the present invention.

[0037] According to another aspect of the present invention, the present invention also provides a cell containing the above nucleic acid molecule of the present invention or containing the above vector.

[0038] According to another aspect of the present invention, the present invention also provides a pharmaceutical composition comprising the TIM3 binding molecule described in the present invention (e.g., a TIM3 antibody or a biologically active fragment derived from the antibody that can specifically bind to TIM3), and a pharmaceutically acceptable carrier or excipient.

[0039] According to another aspect of the present invention, the present invention also provides the use of the TIM3 binding molecule or the pharmaceutical composition in the preparation of a therapeutic agent for treating a disease in a subject, wherein the disease includes a disease related to TIM3 expression in cells and tissues.

[0040] According to another aspect of the present invention, the present invention also provides the use of the TIM3 binding molecule in the preparation of a diagnostic agent for diagnosing a disease in a subject, wherein the disease includes a disease related to TIM3 expression in cells and tissues.

[0041] According to a specific embodiment of the present invention, the TIM3 binding molecule of the present invention can be used alone as an active ingredient or in combination with other active ingredients. For example, the TIM3 binding molecule can be used in combination with a PD1 inhibitor (e.g., an anti-PD1 antibody) to intervene in and regulate T cell activity. The targeted TIM3 antibody of the present invention has the ability to synergize with a PD1 inhibitor.

[0042] In summary, the present invention provides a specific antibody targeting TIM3, determines the sequence of the antibody variable region, and provides its application in regulating / enhancing T cell immune activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 Western Blot analysis shows the stable expression of recombinant human TIM3 in 293T cells.

[0044] Figure 2 The results of cell ELISA test of hybridoma cell supernatant, positive cells (293T-TIM3) and control cells (293T) are shown.

[0045] Figure 3 The TIM3 antibody was purified and then electrophoresed and identified by Coomassie Brilliant Blue staining.

[0046] Figure 4 The results of cell ELISA assay of purified antibodies with positive cells (293T-TIM3) and negative control cells (293T) are shown.

[0047] Figure 5 Flow cytometry shows the binding of antibody #1 (targeting TIM3) to positive cells (293T-TIM3) and control cells (293T).

[0048] Figure 6 The SPR detection shows the affinity of TIM3 1# antibody to TIM3.

[0049] Figure 7 The functional activity experiment showing TIM3 antibody activated CD4+T cells (MLR system, ELISA detection).

[0050] Figure 8 Experiment showing the functional activity of TIM3 1# antibody and its combination with other drugs in activating CD4+T cells (MLR system, ELISA detection).

[0051] Fig. 9 It showed that the combination of TIM3 1# antibody and PD-1 antibody can synergistically inhibit the growth of tumors in tumor-bearing mice. DETAILED DESCRIPTION

[0052] In order to have a clearer understanding of the technical features, purposes and beneficial effects of the present invention, the technical scheme of the present invention is described in detail below in conjunction with specific embodiments and accompanying drawings. Those skilled in the art will appreciate that the embodiments describe the present invention by way of example and are not intended to limit the scope of protection claimed in the present invention. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other. All public cases and other references mentioned herein are incorporated herein by reference in their entirety. In the embodiments, each raw reagent material is commercially available, and the experimental method without specifying specific conditions is a conventional method and conventional conditions well known in the art, or according to the conditions recommended by the manufacturer. The reagents or instruments used without specifying the manufacturer are all conventional products that can be obtained commercially.

[0053] Main reagents and instruments: human lymphocyte separation solution Histopaque-1077 (Cat#10771-6×100ML, Sigma Aldrich); CD14+ Microbeads (Cat#130-050-201), CD4+ T cell isolation kit (Cat#130-096-533), Mo-DC Generation Toolbox I (Cat#130-093-568), MidiMACS separator (Cat#130-042-302), LS chromatography column (Cat#130-042-401) were purchased from Miltenyi Biotech; human IFN-γ DuoSet ELISA (DY285B) was purchased from R&D Systems; TMB substrate reaction solution (D0022, Suzhou Yaco Technology Co., Ltd.); PD-1 inhibitor Opdivo (NDC 0003-3772-11, Bristol-Myers Squibb Co., Ltd. Squibb); Freund's complete adjuvant and incomplete adjuvant were purchased from Sigma; PEG was purchased from Fluka; TIM3 protein (Cat#10390-H38H, SinoBiological); Anti-GAPDH (10494-1-AP, Proteintech); flow cytometer (Accuri C6, BD); MicroPlate Reader (BIO-RAD).

[0054] Cell lines and culture: Human renal epithelial cell lines 293T and 293T-TIM3 cell lines (KYinno co., Ltd. Beijing, China); RPMI-1640 medium (#11875500BT), DMDM ​​(#SH30243.01) and fetal bovine serum (FBS; #SV30087.02, Gibco, USA); penicillin / streptomycin solution (SV30082.01) were purchased from Beijing Sunshine Biotechnology Co., Ltd.; Mo-DC differentiation medium (130-094-812) and Mo-DC maturation medium (130-094-813) were purchased from Miltenyi Biotec; the culture medium was supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin and incubated at 37°C, 5% CO 2 Next cultivation.

[0055] Example 1: Screening of monoclonal hybridoma antibodies targeting TIM3

[0056] Western blot analysis identifies TIM3 expression in 293T-TIM3 cell line

[0057] 293T-TIM3 cells are tool cells used in the present invention to screen and identify the specificity of TIM3 antibodies. To verify the expression of TIM3 in 293T cells, it is identified by Western blotting experiments. 293T / 293T-TIM3 cells were lysed with lysis buffer (1× protease inhibitor mixture from Roche, 2mM EDTA, 2mM dithiothreitol, 150mM NaCl, 50mM Tris-HCl, pH 7.0 and 1% SDS). The protein concentration used in the experiment was quantitatively detected by BCA protein detection kit (Thermo Fisher Scientific, USA). After gel electrophoresis and Western blotting of the cell lysate, the protein bands were qualitatively and quantitatively identified by Image Pro Plus 6.0 software. After the negative cells (293T) and the positive cells (293T-TIM3) were lysed, the total protein was extracted and two loading systems (total protein amount 30μg and 15μg) were detected respectively. The TIM3 protein band was detected at 55kDa. The TIM3 protein content in the two negative cells (293T) and the positive cells (293T-TIM3) was qualitatively and quantitatively detected, that is, the negative cells (293T) did not contain TIM3, and the positive cells (293T-TIM3) contained TIM3. The results met the experimental requirements. Figure 1 Western Blot analysis shows the stable expression of recombinant human TIM3 in 293T cells.

[0058] Immunization of BALC / c mice

[0059] Take 6-8 week old BALB / c mice, collect blood from the tail vein of the immunized mice to keep the background serum. Take 50μg TIM3 protein, add Freund's complete adjuvant and inject it subcutaneously at multiple points on the back to complete the initial immunization. 3 weeks later, take 50μg TIM3 protein, add Freund's incomplete adjuvant and inject it subcutaneously at multiple points on the back to complete the second immunization. 3 weeks later, take 50μg TIM3 protein, add Freund's incomplete adjuvant and inject it subcutaneously at multiple points on the back to complete the third immunization. 2 weeks later, take 50μg TIM3 protein and inject it intraperitoneally for a booster immunization. Take the mouse spleen 3 days after the booster immunization and perform a fusion experiment with SP 2 / 0 myeloma cells.

[0060] Fusion Experiment

[0061] Mouse spleen was taken to prepare spleen cell suspension. Myeloma cells and spleen cells were mixed at a ratio of 1:10 and fused using 50% PEG-DMEM. The fused cells were plated on a 96-well plate and cultured in a 37°C, 5% CO2 incubator. After 7-10 days, clones were grown and tested.

[0062] Cell ELISA assay

[0063] 293T / 293T-TIM3 cells were plated in 96-well plates, with 25,000 cells per well. After 24 hours, 25% glutaraldehyde was added to the culture medium at a ratio of 1:200 and fixed at room temperature for 20 minutes. After washing, 5% skim milk powder was used to block the cells at room temperature for 2 hours. After washing, the cells were set aside for later use.

[0064] Add 50 μL / well of the hybridoma cell supernatant or antibody to be tested to the prepared 96-well plate and incubate at 4°C overnight. After washing, add HRP-labeled goat anti-mouse secondary antibody and incubate at room temperature for 1 hour. After washing, add TMB for color development. Use 12.5% ​​H 2 SO 4 After the reaction was terminated, the OD490 value was read by MicroPlate Reader.

[0065] Screening and establishment of positive monoclonal hybridoma cell lines

[0066] The hybridoma cell supernatant was added to the prepared 96-well plate at 50 μL / well, and the hybridoma clones that showed positive reaction with 293T-TIM3 cells and negative reaction with 293T cells were screened by cell ELISA experiment and monocloned by limiting dilution method. After three monoclonal clonings, positive clones were selected to establish hybridoma cell lines.

[0067] Figure 2 The results of cell ELISA detection of 10 hybridoma cell supernatants (named 1#, 2#, 3#, 4#, 5#, 6#, 7#, 8#, 9#, and 10#, respectively) screened and obtained in this example and positive cells (293T-TIM3) and control cells (293T) are shown.

[0068] Hybridoma cell sequencing

[0069] Experimental methods:

[0070] Take 1×10 7 RNA was extracted from each cell, and reverse transcription was performed using Oligo dT primers to prepare cDNA. The prepared cDNA was used as a template and PCR amplification was performed using the above primers. After the PCR product was recovered by gel, it was connected to the T vector. After the transformation experiment, the plate with uniform single spots was sent for sequencing.

[0071] In this example, it was found that among the 10 antibodies that bind to TIM3 obtained by screening, 1# has a significant and unexpected activation activity of CD4+T cell function, and is named Tim3-1# (or TIM3 1# or 1# antibody) in the present invention, and its variable region amino acid sequence is as follows (CDR sequences are shown in bold and underlined):

[0072] Heavy Chain:

[0073]

[0074] Light chain:

[0075]

[0076] Example 2: Purification and identification of TIM3 monoclonal antibodies

[0077] Flow cytometry and surface plasmon resonance technology (SPROpenSPR, Nicoya, V3.01) were used to detect and identify the binding characteristics of the TIM3-targeting antibody (Tim3-1#) with TIM3 and the competitive inhibitory activity of inhibiting the binding of the ligand Galectin-9 with TIM3.

[0078] Ascites preparation: BALB / c mice were first intraperitoneally injected with 0.5 mL of liquid paraffin, and then 1×10 6 The ascites can be collected 7-10 days after the hybridoma cells are inoculated.

[0079] Monoclonal antibody purification: The collected ascites was purified using Protein G, eluted with pH 2.8 glycine-HCl, dialyzed against PBS, embedded in sucrose and concentrated, and subjected to 10% SDS-PAGE electrophoresis and identified by Coomassie Brilliant Blue staining.

[0080] Figure 3 The electrophoresis and Coomassie Brilliant Blue staining of purified TIM3 antibodies are shown. Lane 1-10 are TIM3 1#-10# purified antibodies, Lane 11 is the control, Lane 12 is the marker, and Lane 13-15 are BSA 0.5μg / mL, 1μg / mL and 2μg / mL.

[0081] Monoclonal antibody cell ELISA experiment: TIM3 monoclonal antibody was diluted to 1 μg / mL using 5% skim milk powder, and the rest was the same as the above cell ELISA experiment.

[0082] Figure 4 The results of cell ELISA detection of purified TIM3 antibody with positive cells (293T-TIM3) and negative control cells (293T) are shown.

[0083] Flow cytometry: 293T-TIM3 and 293T cells (negative control cells) were washed with PBS buffer and counted. The cells were then resuspended in PBS and transferred to autoclaved EP tubes, each containing 100 μl of cell suspension (2×10 6Cells were collected and purified, filtered and sterilized TIM3 antibody was added to the cell suspension at a final concentration of 2 μg / mL. After incubation at 37°C for 1 hour, the cells were washed twice with PBS. The cells were resuspended to 100 μl, FITC-labeled goat anti-mouse fluorescent secondary antibody was added, and the cells were incubated at room temperature in the dark for 1 hour. After washing with PBS twice, the cells were resuspended in 200 μL PBS for flow cytometry analysis.

[0084] Figure 5 Flow cytometry was used to detect the binding of TIM3 1# antibody to positive cells (293T-TIM3) and control cells (293T).

[0085] SPR experiment: The affinity identification between the TIM3-targeting antibody and TIM3 was performed by immobilizing the TIM3 protein (50 μg / mL) on the surface of the nanogold sensor chip after EDC / NHS activation. The TIM3-targeting antibody was diluted and dissolved in a buffer solution (PBS, pH 7.4) to a specific concentration. According to the instrument instructions, the test sample was injected at a flow rate of 20 μL / min for about 4 minutes. The KD, ka and kd values ​​were calculated and analyzed by Trace Drawer Evaluation (version 2.0) software.

[0086] Figure 6 The SPR test results of the affinity of TIM3 1# antibody to TIM3 are shown. The test concentrations are 5.6μg / mL, 17μg / mL and 50μg / mL, and the KD value is 1.03×10 -7 M,ka is 9.89×10 3 Ms -1 , Kd is 1.02×10 -3 S -1 .

[0087] Example 3: Identification of in vitro functional activity of antibodies targeting TIM3 and their combination with other drugs

[0088] The identification of the functional activity of the antibody targeting TIM3 is completed by evaluating the expression level of T cell activation and secretion of IFN-γ cytokines. First, dendritic cells (DCs) and CD4+T cells are obtained by magnetic bead sorting, and then the expression of IFN-γ is detected by mixed lymphocyte reaction (MLR) experiment and ELISA experiment simulating the physiological environment of the human body to identify the biological activity of the antibody targeting TIM3.

[0089] Isolation and purification of dendritic cells (DCs) and CD4+ T cells

[0090] 1. Blood separation: Prepare 20 mL of whole blood from healthy subjects and cool it to room temperature (25°C), mix it with physiological saline or 1×PBS for a total of about 36 mL, and mix it by inversion; prepare 4 15 mL centrifuge tubes, and add 5 mL / tube of cell separation solution. Divide the whole blood mixture into centrifuge tubes containing lymphocyte separation solution, and add about 10 mL to each tube.

[0091] 2. Centrifugation: centrifuge at 1500rpm (centrifugal force 400g), room temperature for 40min to separate the cell layers. (supernatant - mononuclear cell layer - separation fluid - red blood cells)

[0092] 3. Collect the mononuclear cell layer: Use a fine pipette to aspirate the PBMC buffy coat layer (lymphocytes and monocytes, etc.) and add an equal volume of PBS;

[0093] 4. Wash cells with PBS and count them: Collect the buffy coat cells and dilute to 40-45 mL with PBS, centrifuge at 1500 rpm for 10 min. Aspirate the supernatant and continue washing and lysis as needed (aspirate the supernatant after centrifugation, be careful not to aspirate the cells, and leave a little liquid at the bottom of the tube. Add 2 mL of red blood cell lysis buffer and lyse on ice for 5-10 min. After lysis, dilute the centrifuge tube with PBS to 40-45 mL, centrifuge at 1000 rpm for 10 min. Aspirate the upper layer of liquid, pour off the supernatant, resuspend the cell pellet in 5 mL of buffer, and count the PBMCs. Then centrifuge at 1200 rpm for 10 min; if there are not many red blood cells, lysis is not required).

[0094] 5. Incubate on ice: discard the supernatant and add pre-cooled buffer (80 μL / 10 7 cells) (stored on ice for incubation and at room temperature for column), CD14 MicroBeads (20 μL / 10 7 cells) and incubated on ice for 10 min.

[0095] 6. Prepare magnetic bead sorting: When centrifuging, set up the stage, install the filter, prepare two magnetic bead filters and push tubes, four centrifuge tubes, and elution buffer. After centrifugation, discard the supernatant and resuspend the cell pellet in 500μL buffer (500μL / 10 8 cells) by pipetting evenly to remove as many bubbles as possible.

[0096] 7. Column: Wash the column first, add 3mL Buffer to the column for cleaning. Then, add the cell mixture incubated on ice and blow it evenly without bubbles into the filter column. After adding, wash it with Buffer 3 times, 3mL / time. Do not let the liquid in the column drain during this period.

[0097] 8. Collect cells: remove the LS separation column together with the separator from the base, add 3 mL of elution solution, collect the eluted portion, and repeat twice; the bound portion in the column, i.e. the eluted portion pushed down, is the DC cells; take 10 μl of the unbound portion and count it for use in separating CD4+ T cells.

[0098] 9. Centrifuge DC cells and cells to be separated at 1500 rpm for 10 min.

[0099] 10. Add special culture medium to DC and culture in incubator: discard the supernatant and resuspend the cell pellet in DC cell differentiation medium (10mL / 10 7 The cells were cultured in 25 mL culture flasks at 37°C with RMPI 1640+10% FCS, IL-4 20 ng / mL, GM-CSF 50 ng / mL. The cells were added with liquid every 3 days, the liquid was changed every 7 days, and the cells were harvested after 10 days.

[0100] 11.CD4+ T cell isolation:

[0101] 1) Counting and centrifugation: Take the CD4+ T cell suspension and count it. Centrifuge at 1500 rpm, 4°C, 10 min;

[0102] 2) Incubation: Discard the supernatant and resuspend the cell pellet in pre-cooled buffer (40 μL / 10 7 cells), and then add CD4+ T Cell Biotin-Antibody Cocktail (10μL / 10 7 cells), mix well, and incubate at 4°C or on ice for 5 min; then add pre-cooled buffer (30 μL / 10 7 cells), CD4+ T Cell MicroBead Cocktail (20μL / 10 7 cells), mix well, and incubate at 4°C or on ice for 15 min;

[0103] 3) Washing and centrifugation: Add 10 mL of Buffer to wash once, centrifuge at 1500 rpm, room temperature, for 10 min; discard the supernatant, and resuspend the cell pellet in 500 μL Buffer (500 μL / 10 8 );

[0104] 4) Filtration: Take out the LS separation column, install and stick it in the separator adsorbed on the base, add 3 mL of eluent to rinse once; add 500 μL of cell suspension to the LS column, add 3 mL of eluent, repeat 3 times; collect the unbound portion;

[0105] 5) Collect CD4+ T cells: Collect the unbound fraction from the column as CD4+ T cells. Centrifuge, discard the supernatant, and resuspend the cell pellet in basal culture medium (RMPI 1640 + 10% FCS) for immediate use.

[0106] Mixed lymphocyte reaction (MLR)

[0107] Dendritic cell (DC) culture process: After isolation, in Mo-DC differentiation medium (10 7 Dendritic cells (DCs) were cultured in 10 mL of medium (cells / 10 mL medium), and 10 mL of the same medium (cells cultured in differentiation medium) was added to the cells 3 days later. The cells were incubated at 37°C with 5% CO 2 On day 7, DCs were resuspended in Mo-DC maturation medium and cultured for another 3 days by adding Mo-DC differentiation medium. Mature DCs and freshly isolated T cells (DCs and T cells from different donors) were mixed for mixed lymphocyte reaction. Approximately 1×10 4 DC and 1×10 5 T cells were mixed and cultured in 200 μL RPMI-1640 medium plus 10% FBS.

[0108] To evaluate the activity and synergy of TIM3-targeted antibodies (TIM3-targeted antibodies and PD-1 inhibitor Opdivo), the corresponding concentrations of antibodies and Opdivo were added to the corresponding wells to interact with the cells. The expression level of secreted IFN-γ in the cell supernatant was measured on the fifth day.

[0109] IFN-γ expression level detection (ELISA)

[0110] Briefly, the capture antibody dilution was coated overnight (24 h) at room temperature in a volume of 100 μL at a concentration of 2 μg / mL on a 96-well plate. On the next day, the capture antibody solution was aspirated and washed with 300 μL of PBST (0.05% 20, pH 7.2-7.4) and washed the wells 3 times. After that, the test wells were blocked with 300 μL of 1% BSA-PBS for 1 hour. After sealing, the test wells were washed in the same manner as before, and 100 μL of test sample or standard (in 1% BSA-PBS) was pipetted into each well for 2 hours. After that, the detection antibody (125 ng / mL, 100 μL) was added to each well according to the procedure of the Human IFN-γ Duo Set ELISA Kit and incubated for 2 hours. At the end of each step, each well was washed in the same manner as above. Then 100 μL of HRP-labeled streptavidin was added and incubated for 20 minutes. After TMB color development was completed, the test wells were blocked with 12.5% ​​H2 SO 4 (50 μL / well) and read on MicroPlate Reader at 450 / 570 nm. All experimental test wells were repeated.

[0111] Detailed experimental operation process

[0112] Reagent preparation

[0113] 1. Wash Buffer: PBST; i.e., 0.05% Tween-20 prepared with 1×PBS (PH7.2-7.4) (500 mL PBS + 250 μl Tween20).

[0114] 2. Block Buffer: 1% BSA; dissolve BSA in PBS (pH 7.2-7.4) to a final concentration of 1%, and filter through a 0.2 μm filter membrane (0.5 g BSA dissolved in 50 mL PBS).

[0115] 3. Reagent Diluent: Same as the above blocking solution.

[0116] 4. The Streptavidin-HRP kit is provided as a stock solution and stored at 4°C (diluted 1:40 with diluent, i.e., blocking solution).

[0117] 5. Preparation of mouse anti-human IFN-γ coating antibody (Capture) storage solution: Centrifuge the freeze-dried antibody to be used in the kit (120 μg of anti-human IFN-γ antibody dry powder in each tube, add 0.5 mL 1×PBS to dissolve, and prepare the storage solution so that the final concentration of anti-IFN-γ coating antibody is 2 μg / mL.

[0118] 6. Preparation of biotinylated goat anti-human IFN-γ detection antibody storage solution: Centrifuge the freeze-dried biotinylated goat anti-human IFN-γ antibody to be used in the kit (each tube contains 7.5 μg of biotinylated anti-human IFN-γ antibody), dissolve it in 1 mL of reagent diluent, i.e., blocking solution 1% BSA, to prepare 7500 ng / mL storage solution, and use the final concentration of 125 ng / mL.

[0119] 7. Standard: IFN-γ 95ng / bottle, dissolve in 0.5mL reagent diluent 1% BSA, IFN-γ is 190ng / mL. Dilution can be made according to the actual situation of the experiment.

[0120] 8. Substrate Solution: TMB, used directly.

[0121] 9. Reaction termination solution: 12.5% ​​HO 2 SO 4 .

[0122] Experimental Procedure

[0123] Day 1

[0124] 1. Antibody coating: The final concentration of anti-INF-γ coating antibody is 2 μg / mL, 50 μL / well, coating overnight at room temperature.

[0125] the next day

[0126] 2. Blocking: The next day, block with blocking solution at room temperature for 1 hour, and then wash the plate three times with PBST.

[0127] 3. Add culture supernatant and standard: Add the cell culture supernatant and standard to be tested, 50 μL / well, incubate at room temperature for 2 hours, and wash the plate 3 times with PBST.

[0128] 4. Add biotinylated anti-human INF-γ detection antibody: dilute the biotinylated sheep anti-human INF-γ storage solution (7500ng / mL) detection antibody 1:60 with diluent, add 50μL to each well, incubate at room temperature for 2h, and wash the plate 3 times with PBST. (The final concentration of anti-human INF-γ is 125ng / mL).

[0129] 5. Add HRP-labeled streptavidin: dilute HRP-labeled streptavidin at 1:40 with diluent, add 50 μL / well, incubate at room temperature for 20 min, and wash the plate 3 times with PBST.

[0130] 6. Color development: Add 100 μL / well of TMB color development substrate and incubate at room temperature away from light for 5-20 min (depending on the actual situation).

[0131] 7. Stop color development: add 12.5% ​​HO 2 SO 4 50 μL / well.

[0132] 8. Reading on the machine: The microplate reader reads the 450nm / 570nm test.

[0133] Figure 7The ELISA test shows the biological activity identification of TIM3 antibody in the MLR system (activating T cells to express INF-γ level). As shown in the figure, NC is the negative control for DCs mixed with T cells, Opdivo is the positive control for anti-PD1 antibody, and the experimental group is DCs mixed with T cells and then TIM3 antibody is added. The test results show that compared with the control group, the INF-γ expression level of TIM31# antibody in the experimental group was significantly increased, indicating that TIM3 1# antibody has the best activity and can promote the expression and secretion of INF-γ in T cells.

[0134] Figure 8 It shows that the combination of TIM3 1# antibody and other drugs can synergistically activate the functional activity of CD4+T cells (MLR system, ELISA test). The test results show that after the combination of TIM3 1# antibody (50μg / mL) and anti-PD1 antibody Opdivo (2μg / mL), the expression level of INF-γ was significantly increased compared with the TIM3 antibody group alone or the Opdivo antibody group alone, indicating that the two have a good synergistic effect.

[0135] Example 4: Antibodies targeting TIM3 synergistically inhibit tumor growth in vivo in combination with PD-1 antibodies

[0136] The in vivo functional activity of the antibody targeting TIM3 was identified by establishing a tumor-bearing animal model using PD1 / TIM3 double humanized mice. Mouse colorectal cancer cells MC38 were inoculated subcutaneously in the right anterior rib of female B6-hPD1 / hTIM3 mice. 3 The mice were divided into four groups: Vehicle group, hTIM3 antibody group (i.e., 1# antibody 10 mg / kg), hPD1 antibody group (1 mg / kg), and hTIM3 antibody (10 mg / kg) + hPD1 antibody group (1 mg / kg). The mice were intraperitoneally injected twice a week for 3 weeks. The tumor volume and body weight were measured every week. At the end of the experiment, the mice were euthanized, the tumors were dissected and weighed. The tumor growth curve was drawn and the tumor inhibition rate was calculated. Fig. 9 The results showed that the combination of TIM3 1# antibody and PD-1 antibody can synergistically inhibit tumor growth in tumor-bearing mice. The tumor inhibition rate of TIM3 1# antibody was 5%, the tumor inhibition rate of PD-1 antibody was 6%, and the tumor inhibition rate of the combined administration of TIM3 1# antibody and PD-1 antibody was as high as 67%.

[0137] The description presented in the above exemplary embodiments is only used to illustrate the technical solution of the present invention, and is not intended to be exhaustive, nor is it intended to limit the present invention to the precise form described. Obviously, it is possible for a person of ordinary skill in the art to make many changes and variations based on the above teachings. The exemplary embodiments are selected and described to explain the specific principles of the present invention and its practical application, so that other technicians in the field can easily understand, implement and use the various exemplary embodiments of the present invention and its various selected forms and modified forms. The scope of protection of the present invention is intended to be limited by the scope of protection requested and its equivalent form.

Claims

1. A molecule comprising a TIM3 antibody or a biologically active fragment derived from the antibody that can immunospecifically bind to TIM3, wherein the amino acid sequence of the six CDRs of the TIM3 antibody include: Heavy chain CDR1: GYSFTGYNMN (SEQ ID NO: 1); Heavy chain CDR2: IIDPYYGTTNYNQKFRG (SEQ ID NO: 2); Heavy chain CDR3: GGNSFDY (SEQ ID NO: 3); Light chain CDR1: TASSILSSNYLH (SEQ ID NO: 4); Light chain CDR2: STSYLAS (SEQ ID NO: 5); Light chain CDR3: HQFHPSPRT (SEQ ID NO: 6).

2. The molecule according to claim 1, wherein the biologically active fragment capable of specifically binding to TIM3 derived from the antibody comprises the amino acid sequence of the six CDRs of the TIM3 antibody.

3. The molecule according to claim 1, wherein the antibody is an animal-derived antibody, a chimeric antibody, a humanized antibody or a human antibody.

4. The molecule according to claim 1, include: (1) a heavy chain variable region, wherein the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 7, or an amino acid sequence with equivalent functions formed by replacing, deleting or adding one or more amino acids on the basis of the amino acid sequence; and / or (2) a light chain variable region, wherein the light chain variable region has the amino acid sequence shown in SEQ ID NO: 8, or an amino acid sequence with equivalent function formed by replacing, deleting or adding one or more amino acids on the basis of the amino acid sequence.

5. A nucleic acid molecule encoding the amino acid sequence of the molecule according to any one of claims 1 to 4.

6. A vector or cell containing the nucleic acid molecule according to claim 5.

7. A pharmaceutical composition comprising the molecule of any one of claims 1 to 4, and a pharmaceutically acceptable carrier or excipient.

8. Use of the molecule according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 7 in the preparation of a therapeutic agent for treating a disease in a subject, or a diagnostic agent for diagnosing a disease in a subject, in, The diseases include diseases associated with TIM3 expression in cells and / or tissues.

9. Use of the molecule according to any one of claims 1 to 4 or the pharmaceutical composition according to claim 7 in the preparation of an agent for intervening and / or regulating T cell activity.

10. The use according to claim 8 or 9, in, The molecule according to any one of claims 1 to 4 is used alone as an active ingredient or in combination with other active ingredients; Preferably, the other active ingredient is a PD1 inhibitor.

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