TIGIT antibodies, their preparation methods and applications

Abstract

This invention belongs to the field of biotechnology, and particularly relates to TIGIT antibodies, their preparation methods, and applications. The TIGIT antibody consists of a heavy chain and a light chain; the heavy chain comprises: a first heavy chain with an amino acid sequence similar to SEQ ID NO: 1, a second heavy chain with an amino acid sequence similar to SEQ ID NO: 2, or a third heavy chain with an amino acid sequence similar to SEQ ID NO: 3; the light chain comprises: a first light chain with an amino acid sequence similar to SEQ ID NO: 4, a second light chain with an amino acid sequence similar to SEQ ID NO: 5, or a third light chain with an amino acid sequence similar to SEQ ID NO: 6. This invention provides a TIGIT antibody with high specificity, strong competitive activity, and excellent thermostability, which significantly promotes IFN-γ secretion by cells and exhibits excellent tumor-suppressive effects.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to TIGIT antibodies, their preparation methods and applications. Background Technology

[0002] TIGIT is a co-inhibitory receptor widely expressed on T cells and NK cells. When TIGIT binds to its ligand PVR / PVRL2 on tumor cells, it inhibits the immune killing activity of CD8+ T cells or NK cells through signaling pathways, thereby promoting tumor cell escape.

[0003] Currently, TIGIT is considered one of the most promising and potential targets, with multiple pieces of evidence supporting its crucial role in limiting tumor adaptive and innate immunity. Therefore, this invention designs and studies TIGIT antibodies. However, most current TIGIT antibodies have certain limitations, such as weak competitive activity or poor thermal stability. Summary of the Invention

[0004] To address the shortcomings of existing TIGIT antibodies, this invention provides a novel TIGIT antibody, its preparation method, and its applications. The objective of this invention is to provide a TIGIT antibody with strong competitive activity, high specificity, and improved thermostability, along with its preparation method and applications.

[0005] To achieve the above objectives, the present invention adopts the following technical solution.

[0006] TIGIT antibody,

[0007] The TIGIT antibody is composed of heavy and light chains;

[0008] The heavy chain includes at least one of the following:

[0009] The first heavy chain (huVH1) has an amino acid sequence that is 85-100% identical to SEQ ID NO: 1;

[0010] The second chain (huVH2) has an amino acid sequence that is 85-100% identical to SEQ ID NO: 2;

[0011] The third chain (huVH3) has an amino acid sequence that is 85-100% identical to SEQ ID NO: 3;

[0012] The light chain comprises at least one of the following:

[0013] The first light chain (huVL1) has an amino acid sequence that is 85-100% identical to SEQ ID NO: 4;

[0014] The second light chain (huVL2) has an amino acid sequence that is 85-100% identical to SEQ ID NO: 5;

[0015] The third light chain (huVL3) has an amino acid sequence that is 85-100% identical to SEQ ID NO: 6.

[0016] As a preferred option

[0017] The amino acid sequence of the huVH1 is shown in SEQ ID NO: 1;

[0018] The amino acid sequence of the huVH2 is shown in SEQ ID NO: 2;

[0019] The amino acid sequence of the huVH3 is shown in SEQ ID NO: 3;

[0020] The amino acid sequence of huVL1 is shown in SEQ ID NO: 4;

[0021] The amino acid sequence of the huVL2 is shown in SEQ ID NO: 5;

[0022] The amino acid sequence of the huVL3 is shown in SEQ ID NO: 6.

[0023] As a preferred option

[0024] The antibody is a humanized antibody.

[0025] As a preferred option

[0026] The TIGIT antibody binds to human TIGIT recombinant protein (TIGIT-ECD-mFc).

[0027] Preparation method of TIGIT antibody

[0028] The method includes:

[0029] 1) Hybridoma antibody sequencing: Rat hybridoma cells were cultured, purified RNA was obtained, reverse transcribed into cDNA as a template, and then the target fragment was introduced for in vitro amplification using specific PCR. The positive PCR product was ligated into a T vector for plasmid recombination and transformation. Positive clones were selected for culture and sequencing through blue-white screening and PCR verification to obtain the original sequence of the rat antibody.

[0030] 2) Human sequence construction: Based on the original mouse antibody sequence obtained in step 1), the original mouse antibody sequence was mutated into human heavy chain sequence and human light chain sequence by using 3D modeling and database comparison.

[0031] 3) Antibody construction: Combine the human heavy chain sequence and human light chain sequence obtained in step 2) to form a humanized antibody;

[0032] 4) Antibody expression: The humanized antibody obtained in step 3) was expressed in the Expi293 mammalian expression system;

[0033] 5) Antibody purification: After expression in step 4), the cell supernatant is collected and purified to a purity of ≥90%.

[0034] As a preferred option

[0035] Step 1) The method for culturing the murine hybridoma cells includes:

[0036] 1-1) Using human TIGIT recombinant protein to immunize experimental mice with target protein, B cells that can secrete specific target protein antibodies were generated in the mice.

[0037] 1-2) After euthanizing the experimental mice, spleen cells were harvested for mouse cell fusion to obtain fused cells;

[0038] 1-3) The obtained fusion cells were detected by ELISA, and fusion cells that could competitively bind human TIGIT recombinant protein were screened for subcloning;

[0039] 1-4) After subcloning for at least two cycles, 11A12 clones that showed strong binding to TIGIT-his and strong competition with PVR-hFc were identified by ELISA and used to establish cell lines to obtain murine hybridoma cells.

[0040] As a preferred option

[0041] Step 1) The original sequence of the murine antibody includes:

[0042] Heavy chain nucleic acid sequence, heavy chain amino acid sequence, light chain nucleic acid sequence, and light chain amino acid sequence.

[0043] As a preferred option

[0044] The heavy chain nucleic acid sequence is shown in SEQ ID NO: 7;

[0045] The light chain nucleic acid sequence is shown in SEQ ID NO: 8.

[0046] As a preferred option

[0047] The heavy chain amino acid sequence is shown in SEQ ID NO: 9;

[0048] The light chain amino acid sequence is shown in SEQ ID NO: 10.

[0049] Applications of TIGIT antibodies

[0050] The TIGIT antibody is used to prepare pharmaceuticals and / or pharmaceutical compositions;

[0051] The drug and / or drug composition are used to treat diseases associated with human TIGIT;

[0052] The disease in question is a tumor, cancer, immune disease, or infectious disease.

[0053] The beneficial effects of this invention are as follows:

[0054] This invention provides a highly specific, competitively active, and thermostable TIGIT antibody, along with a detailed preparation method. Furthermore, experiments have shown that... Figure 10 The TIGIT antibody of the present invention, as shown, has a significant promoting effect on the secretion of IFN-γ by cells, and as... Figure 11 As shown, it exhibits excellent tumor suppression effects in the pharmacodynamic evaluation experiment of subcutaneous transplanted tumor model. Attached Figure Description

[0055] Figure 1 This is a schematic diagram showing the purification results of the murine hybridoma antibody described in Example 2;

[0056] Figure 2 The results are for the determination of the binding activity of 11A12 murine hybridoma antibody to antigen by ELISA.

[0057] Figure 3 The results are for the ELISA assay to determine the competitive activity of 11A12 murine hybridoma antibody against PVR-hFc ligand;

[0058] Figure 4 This is a schematic diagram showing the purification results of the 11A12 humanized antibody;

[0059] Figure 5 The results of ELISA assay for determining the binding activity of 11A12 humanized antibody with TIGIT-his;

[0060] Figure 6 The results are for the FACS assay to determine the binding activity of the 11A12 humanized antibody with 293F-TIGIT.

[0061] Figure 7 The results are for the ELISA assay to determine the competitive activity of the 11A12 humanized antibody against PVR-mFc.

[0062] Figure 8 The results of the FACS assay for determining the competitive activity of 11A12 humanized antibody against PVR-mFc;

[0063] Figure 9This is a schematic diagram of the fluorescence signal for detecting the thermostability of the 11A12 humanized antibody;

[0064] Figure 10 The results of the assay for the promotion of IFN-γ secretion in cell supernatant by the 11A12 humanized antibody;

[0065] Figure 11 This is a schematic diagram showing the efficacy test results of the 11A12 antibody in a Hepa1-6 cell mouse xenograft model. Detailed Implementation

[0066] The present invention will be further described clearly and in detail below with reference to specific embodiments and the accompanying drawings. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below are generally only some, not all, of the embodiments of the present invention. Therefore, all other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0067] Unless otherwise specified, all raw materials used in the embodiments of the present invention are commercially available or obtainable by those skilled in the art; unless otherwise specified, all methods used in the embodiments of the present invention are methods mastered by those skilled in the art.

[0068] Example 1

[0069] Mouse immunization test:

[0070] Primary immunization: On April 16, 2021, three Bal B / C mice (Shanghai Xipu-Bikai Experimental Animal Co., Ltd., females 8 weeks old) were immunized with TIGIT-ECD-mFc antigen to produce B cells that secrete specific target protein antibodies (labeled as 11A12 murine antibody).

[0071] Boost immunity:

[0072] On June 17, 2021, mice 54 and 55 were given a booster immunization via subcutaneous injection for subsequent cell fusion.

[0073] Example 2

[0074] Hybridoma fusion assay:

[0075] Cell fusion: On July 2, 2021, immunized mice No. 54 and No. 55 obtained in Example 1 were euthanized and spleen cells were collected. Cell fusion of immunized mice was performed according to the standard operating procedure for cell fusion based on the principle of SP2 / 0:spleen cells = 1:5. The fusion number was marked as H006 fusion cell plate.

[0076] Cell screening: Fusion cells were detected by ELISA, and 31 wells with strong binding to TIGIT-ECD-his and strong competition for PVR-hFC ligand were selected for subcloning.

[0077] Subcloning: Positive cells were cloned by detecting subcloned cells, and the cells were further purified. After two cycles of subcloning, the 11A12 clones that strongly bind to TIGIT-his (OD450=1.780) and strongly compete with PVR-hFc (OD450=0.289) were used to establish and detect hybridoma cells.

[0078] Purification: Hybridoma cells were expanded, and the cell supernatant was collected. Antibody purification was performed according to standard operating procedures for protein purification. The purification results are as follows: Figure 1 As shown, from Figure 1 As can be seen, the purity of the 11A12 murine antibody in the obtained murine hybridoma cells is >90%.

[0079] Example 3

[0080] Hybridoma antibody activity detection:

[0081] The binding activity of 11A12 murine antibody to TIGIT-his was detected using ELISA:

[0082] Using TIGIT-his as the target antigen, an ELISA method was employed. The test plate was coated with 0.5 μg / mL TIGIT-his. The concentration gradient of the purified 11A12 murine antibody sample from Example 2 was set from 0.00004 to 1.2 μg / mL. The binding activity OD value was measured to detect the affinity between the 11A12 murine antibody and the target antigen. The experimental results are as follows: Figure 2 As shown;

[0083] from Figure 2 As can be seen, the OD value increases significantly with increasing sample concentration, and clear upper and lower plateaus are generated very quickly. The window is large, and the 11A12 murine antibody of this invention has strong affinity for binding to the antigen. The half maximum effect concentration (EC50) was calculated and obtained to be EC50=0.003711 μg / mL. It can be seen that the 11A12 murine antibody has great pharmacological potential.

[0084] The competitive activity of 11A12 murine antibody against PVR-hFc was detected using ELISA:

[0085] The test plate was coated with 2.0 µg / ml TIGIT-his. The concentration gradient of the purified 11A12 murine antibody sample from Example 2 was set to 0.00004–1.2 μg / mL. The OD value was measured to detect the competitive activity between the 11A12 murine antibody and 3.0 µg / ml PVR-hFc ligand. The experimental results are as follows: Figure 3 As shown;

[0086] from Figure 3 The curve shows a clear trend in the OD value of the 11A12 murine antibody, with distinct upper and lower plateaus. The half-inhibitory concentration (IC50) was analyzed and calculated to obtain an IC50 of 0.02558 μg / mL, indicating that the 11A12 murine antibody has strong competitive activity against PVR-hFc.

[0087] Example 4

[0088] Hybridoma antibody sequencing:

[0089] Hybridoma cells were cultured to obtain a small amount of purified high-purity RNA, which was reverse transcribed into cDNA. This cDNA was then used as a template for in vitro amplification of the target fragment using specific PCR primers. The positive PCR product was ligated into a T-vector for plasmid recombination and transformation. Positive clones were selected through blue-white screening and PCR verification, cultured, and sequenced. The sequencing results were analyzed, and the correct sequences were determined, completing the antibody sequencing.

[0090] The sequencing results are shown in the table below:

[0091] Biological information sequencing results Serial Number Mouse antibody heavy chain nucleic acid sequencing results (Nucleic acid sequence) GAGGTGAAGCTGGTGGAGAGCGGCGGCCTGGTGCAACCAGGAGGAAGCATGAAGCTGAGCTGCGTGGCCAGCGGCTTTACCTTCTCCAACTACTGGATGAACTGGGTGAGACAGAGCCCCGAGAAGGGCCTGGAGTGGGTGAGCGAGATCAGGCTGAAGAGCAACAGATACGCCACA CACTTTGTGGAGTCCGTGAAGGGCAGATTCACCATTCCAGGGACGACAGCAAGAGCACAGTGTACCTGCAGATGAATAACCTGAGGACAGAGGATACCGGCATCTACTACTGCACAAGACTGTACTACGGCAACTACTTCGACTACTGGGGCCAGGGCACCAGCCTGACCGTGAGCAGC SEQ ID NO: 7 Protein sequence of mouse antibody heavy chain. EVKLVESGGGLVQPGGSMKLSCVASGFTFSNYWMNWVRQSPEKGLEWVSEIRLKSNRYATHFVESVKGRFTISRDDSKSTVYLQMNNLRTEDTGIYYCTRLYYGNYFDYWGQGTSLTVSS SEQ ID NO: 9 Nucleic acid sequence of mouse antibody light chain. GATATCCTGATGACACAGAGCCCCGCCAGCCTGTCCGCCTCCGTTGGAGAGACCGTGACCATCACCTGTAGAGCCACAGAGAACATCTACGGCTACCTGGCCTGGTACCAGCAGAAGCAGGGCAAGAGCCCTCAGCTGCTGGTGTACAATGCCAAGACCC TGGCCGAGGGCGTGAGCTCCAGGCTGAGCGGAAGCGGCTCCGGCACACAGTTTTCCCTGAAGATCAAGAGCCTGCAGCCTGAGGATTTCGGCGACTACTACTGCCAGTACCACTACGATAATATCTTCACATTTGGCACAGGCACAAAGGTGGAGCTGAAG SEQ ID NO: 8 Mouse-derived antibody light chain amino acid sequencing results (Protein sequence) DILMTQSPASLSASVGETVTITCRATENIYGYLAWYQQKQGKSPQLLVYNAKTLAEGVSSRLSGSGSGTQFSLKIKSLQPEDFGDYYCQYHYDNIFTFGTGTKVELK SEQ ID NO: 10

[0092] Example 5

[0093] Using 3D modeling and database comparison, the original mouse sequence was mutated into a human sequence. The original 11A12 mouse antibody sequence was designed into multiple humanized amino acids (huVH1, huVH2, huVH3, huVL1, huVL2, huVL3), and the designed sequences were combined to form the 11A12 humanized antibody, which was then expressed in the Expi 293 mammalian expression system.

[0094] The sequencing results of the above humanized amino acid sequences are shown in the table below.

[0095] Biological information sequencing results Sequence number 11A12_huVH1 EVQLVESGGGLVKPGGSMKLSCVASGFTFSNYWMNWVRQAPGKGLEWVSEIRLKSNRYATHFVESVKGRFTISRDDSKSTVYLQMNSLKTEDTAVYYCTRLYYGNYFDYWGQGTTVTVSS SEQ ID NO: 1 11A12_huVH2 EVQLVESGGGLVKPGGSLRLSCVASGFTFSNYWMNWVRQAPGKGLEWVSEIRLKSNRYATHFVESVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTRLYYGNYFDYWGQGTTVTVSS SEQ ID NO: 2 11A12_huVHIII EVQLVESGGGLVKPGGSLRLSCAASGFTFSNYWMNWVRQAPGKGLEWVSEIRLKSNRYATHYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTRLYYGNYFDYWGQGTTVTVSS SEQ ID NO: 3 11A12_huVL1 DIQMTQSPSTLSASVGDRVTITCRATENIYGYLAWYQQKPGKAPKLLVYNAKTLAEGVSSRLSGSGSGTEFTLTISSLQPDDFATYYCQYHYDNIFTFGQGTKLEIK [[ID=十六]]SEQ ID NO: 4 11A12_huVL2 DIQMTQSPSTLSASVGDRVTITCRATENIYGYLAWYQQKPGKAPKLLVYNAKTLAEGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQYHYDNIFTFGQGTKLEIK SEQ ID NO: 5 11A12_huVL3 DIQMTQSPSTLSASVGDRVTITCRATENIYGYLAWYQQKPGKAPKLLIYNAKTLESGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQYHYDNIFTFGQGTKLEIK SEQ ID NO: 6

[0096] The above-mentioned 11A12 humanized antibody was purified: The 11A12 series humanized antibody expressed in Expi 293 cells was collected, and the cell supernatant was purified according to standard protein purification procedures. The experimental results were characterized, and the characteristics are shown below. Figure 4As shown in the figure. The experimental results indicate that the purity of the purified 11A12 humanized antibody is >90%.

[0097] Example 6

[0098] 11A12 Humanized Antibody Activity Assay:

[0099] The binding activity of 11A12 humanized antibody to TIGIT-his was detected using ELISA:

[0100] Using TIGIT-his as the target antigen, an ELISA method was employed. The test plate was coated with 0.5 μg / mL TIGIT-his. The concentration gradient of the purified 11A12 humanized antibody sample from Example 5 was set from 0.00004 to 1.2 μg / mL. The binding activity OD value was measured to detect the affinity between the 11A12 humanized antibody and the target antigen. The experimental results are as follows: Figure 5 As shown;

[0101] from Figure 5 It can be seen that as the sample concentration increases, the OD value increases significantly and produces clear upper and lower plateaus very quickly. The window is large, and the 11A12 murine antibody of this invention has strong affinity for antigen binding and has great potential for efficacy.

[0102] The binding activity of the 11A12 humanized antibody to 293F-TIGIT cells was detected using FACS:

[0103] The purified 11A12 humanized antibody sample from Example 5 was set at a concentration gradient of 0.01–15 μg / mL and incubated with 293F-TIGIT cells. The binding signal intensity was measured using FACS to detect the binding activity of the humanized antibody to the target protein. The detection results are as follows: Figure 6 As shown. From Figure 6 As can be seen, the 11A12-H3 / L1 human antibody performed best, while the 11A12-H2 / L1 and 11A12-H1 / L1 human antibodies were second only to the 11A12-H3 / L1 human antibody, indicating that the TIGIT antibody of this invention with the light chain huVL1 had the best effect.

[0104] Further, the competitive activity of the 11A12 series humanized antibodies against PVR-mFc was detected using ELISA:

[0105] The test plate was coated with 2.0 µg / ml TIGIT-his. The concentration gradient of the 11A12 humanized antibody sample purified in Example 5 was set to 0.00004-1.2 μg / mL. The OD value was measured to detect the competitive activity of the 11A12 series humanized antibodies against 9.0 µg / ml PVR-mFc ligand.

[0106] Determining structures such as Figure 7 As shown. From Figure 7 It can be clearly seen that the OD value of the 11A12 humanized antibody shows a clear trend, with distinct upper and lower plateaus on the curve, indicating strong competitive activity;

[0107] The competitive activity of 11A12 humanized antibody against PVR-mFc was detected by FACS:

[0108] Using the FACS method, 293F-TIGIT cells were uniformly seeded into 96-well cell plates. A constant concentration of PVR-mFc and a concentration gradient of 11A12 humanized antibody sample purified in Example 5 were set at 0.00390625–30 μg / mL to bind together with the cells. Then, 3 µg / mL of Alex488-labeled goat anti-mouse secondary antibody was added to each well. After binding, the competitive activity OD value was measured to detect the competitiveness between 11A12 humanized antibody and PVR-mFc.

[0109] Test results are as follows Figure 8 As shown, Figure 8 The 11A12-H / L sample is the parent sample, from... Figure 8 Each of the samples is highly competitive.

[0110] Example 7

[0111] Thermostability assay of humanized antibodies:

[0112] The thermal stability of proteins was monitored by detecting fluorescence signal intensity using the Protein Thermal Shift™ dye kit and real-time quantitative PCR. The fluorescence signal intensity for detecting the thermal stability of the 11A12 humanized antibody was as follows: Figure 9 As shown in the table, the results also show that the Tm values ​​of the 11A12 series humanized antibodies are all higher than those of the parent antibody. Among them, 11A12-H3 / L2 has the highest thermal stability, followed by 11A12-H3 / L3 and 11A12-H3 / L1. The thermal stability of these three antibodies is significantly improved, while the thermal stability of the other four antibodies is slightly improved compared to the parent antibody.

[0113] The measurement results are shown in the table below:

[0114] Antibody Tm value 1 Tm value 2 Average 11A12-H / L 68.56 68.66 68.61 11A12-H1 / L1 70.29 70.33 70.31 11A12-H2 / L1 70.79 70.78 70.785 11A12-H3 / L1 76.22 76.21 76.215 11A12-H2 / L2 71.88 71.89 71.885 11A12-H3 / L3 76.58 76.56 76.57 11A12-H2 / L3 71.01 71.05 71.03 11A12-H3 / L2 77.07 76.98 77.025

[0115] Example 8

[0116] Humanized antibody MLR experiment:

[0117] All sample concentration gradients were set up and incubated with fully differentiated cells. OD values ​​were measured using an ELISA kit to detect IFN-γ secretion in the cell supernatant. Test results are as follows: Figure 10 As shown in the figure, both groups of antibodies 11A12 have a significant promoting effect on the secretion of IFN-γ by cells.

[0118] Example 9

[0119] The TIGIT antibody of this invention was used in a Hepa1-6 mouse subcutaneous hepatocellular carcinoma xenograft model for pharmacodynamic evaluation.

[0120] A Hepa1-6 subcutaneous xenograft model was established, and the tumor volume was allowed to grow to ~120 mm. 3 Mice were randomly divided into 4 groups of 8 mice each. The average tumor volume after grouping was 120 mm. 3 The tumor growth curves of the control group and each experimental group are as follows: Figure 11 As shown: On day 21 after grouping, which is the end of the experiment, the average tumor volume of the Group-1 (Vehicle) control group was 2392.7 mm. 3 The mean tumor volume in Group-2 (11A12) was 1475.8 mm. 3 The tumor volume inhibition rate (TGI) was 40.3%; the mean tumor volume of Group-3 (PD-1 antibody) was 1133.4 mm. 3 The tumor volume inhibition rate (TGI) was 60.7%; the mean tumor volume of Group-4 (11A12+PD-1 antibody) was 555.9 mm. 3 The tumor volume inhibition rate (TGI) was 80.8%.

[0121] As can be seen from the above pharmacodynamic evaluation experiments, the antibody of the present invention has significant pharmacodynamic effects in inhibiting and / or treating TIGIT-related diseases, and can produce good positive effects.

Claims

1. A TIGIT antibody, characterized in that, The TIGIT antibody is composed of heavy and light chains; The heavy chain is any of the following: The first chain, whose amino acid sequence is shown in SEQ ID NO: 1; The second chain has the amino acid sequence shown in SEQ ID NO: 2; The third chain has the amino acid sequence shown in SEQ ID NO: 3; The light chain is any of the following: The first light chain has the amino acid sequence shown in SEQ ID NO: 4; The second light chain has the amino acid sequence shown in SEQ ID NO: 5; The third light chain has the amino acid sequence shown in SEQ ID NO:

6.

2. The application of the TIGIT antibody according to claim 1, characterized in that, The TIGIT antibody is used to prepare pharmaceuticals and / or pharmaceutical compositions; The drug and / or drug composition are used to treat diseases associated with human TIGIT; The disease in question is liver cancer.

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