Anti-TNF-α antibody, its products and applications

Abstract

The present invention belongs to the field of antibodies, and particularly relates to anti-TNF-α antibodies and their products and applications. The present invention utilizes a canine natural phage antibody library and screens out TNF-α antibodies specific to the specific targets of canine inflammatory bowel disease. ELISA analysis and BLI affinity detection show that the anti-TNF-α antibody can bind to TNF-α. The L929 cytotoxicity experiment demonstrates that the anti-TNF-α antibody has the ability to neutralize the cytotoxicity of this cytokine. By constructing an in vivo TNBS-induced enteritis model, the present invention finds that this antibody can have a good therapeutic effect in treating diseases related to TNF-α.

Description

Technical Field

[0001] The present invention belongs to the field of antibodies, and specifically relates to anti-TNF-α antibodies and their products and applications. Background Art

[0002] Tumor necrosis factor α (TNF-α) is a pro-inflammatory cytokine produced by macrophages or monocytes, which acts by binding to two different receptors, namely TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2). These two receptors activate signaling pathways involving nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK), p42 / p44 mitogen-activated protein kinase (MAPK), and p38 MAPK. TNFα has been identified as a major regulator of the inflammatory response and is involved in physiological and pathological processes such as inflammation, apoptosis, immune homeostasis, and autoimmunity. It participates in the pathogenesis of various inflammatory and autoimmune diseases, including pathogenic microorganism infections, rheumatoid arthritis, systemic lupus erythematosus, autoimmune skin diseases (including psoriasis), inflammatory bowel disease (ulcerative colitis), autoimmune hematological diseases, ankylosing spondylitis, and non-infectious uveitis. Elevated serum TNFα levels are closely related to the pathophysiology of autoimmune diseases, and thus neutralizing TNFα has become a major strategy for treating these diseases.

[0003] The prior art CN115974998A proposes a TNFα antagonist peptide and its application. The TNFα antagonist peptide has good stability and significant anti-inflammatory, immune-regulating, and TNFα antagonistic effects, and can be used to prepare drugs for treating inflammatory and autoimmune diseases. The prior art CN110066337B discloses an anti-TNFα antibody, which is obtained by constructing a natural rabbit-derived phage antibody library without antigen immunostimulation, using TNFα recombinant protein as a target, and enriching through 3 rounds of screening to obtain a single-chain antibody that specifically binds to TNFα. This antibody can be used as a specific capture TNFα antibody, TNFα signal antibody, etc. At the same time, the successfully constructed natural antibody library can also be directly applied to the screening of more targets. Currently, the most typical antibody antagonist against TNFα, adalimumab, has already been successfully marketed and is used in the treatment of related diseases.

[0004] However, due to species differences, directly applying human antibodies to dogs often causes severe immune reactions, resulting in the drug efficacy not meeting expectations. Therefore, it is particularly important to develop highly efficient TNF-α antibodies specific for dogs. Summary of the Invention

[0005] In view of the above deficiencies, the present invention provides a novel canine anti-TNF-α monoclonal antibody. In vitro and in vivo experimental results show that this antibody has extremely high affinity for canine TNF-α and exhibits significant neutralizing ability, and the following technical solutions are proposed:

[0006] In a first aspect, the present invention provides an anti-TNF-α antibody, the antibody comprising a heavy chain and a light chain, the heavy chain comprising heavy chain complementarity determining regions, and the light chain comprising light chain complementarity determining regions.

[0007] The heavy chain complementarity determining regions of the antibody comprise HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; the light chain complementarity determining regions of the antibody comprise LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.

[0008] SEQ ID NO.1: GFTFSSYA.

[0009] SEQ ID NO.2: ISSGGSST.

[0010] SEQ ID NO.3: AKDLTLLPRSFDY.

[0011] SEQ ID NO.4: QSLLHSDGKTY.

[0012] SEQ ID NO.5: GVS.

[0013] SEQ ID NO.6: GQGIQSPFT.

[0014] Or the heavy chain complementarity determining regions of the antibody comprise HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9; the light chain complementarity determining regions of the antibody comprise LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11 and SEQ ID NO:12.

[0015] SEQ ID NO.7: GFTFSDYG.

[0016] SEQ ID NO.8: IRGDGGWT.

[0017] SEQ ID NO.9: VKAPWLAF.

[0018] SEQ ID NO.10: QSLLHSNGNTY.

[0019] SEQ ID NO.11: KVS.

[0020] SEQ ID NO.12: GQGIQFPLT.

[0021] Specifically, the amino acid sequence of the heavy chain variable region of the anti-TNF-α antibody is SEQ ID NO:13, and the amino acid sequence of the light chain variable region is SEQ ID NO:14.

[0022] SEQ ID NO.13:

[0023] EVQLVESGGDLVKPAGSLRLSCVASGFTFSSYAMSWVRQAPGKGLQWVAGISSGGSSTNYADAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAKDLTLLPRSFDYWGQGTLVTVSS.

[0024] SEQ ID NO.14:

[0025] DIVMTQTPLSLSVSPGEPASISCKASQSLLHSDGKTYLHWFRQKPGQSPQLLIYGVSNRDTGVPDRFSGSGSGTDFTLKISRVEADDAGVYYCGQGIQSPFTFGQGTKVELK.

[0026] Alternatively, the amino acid sequence of the heavy chain variable region of the anti-TNF-α antibody is SEQ ID NO:15, and the amino acid sequence of the light chain variable region is SEQ ID NO:16.

[0027] SEQ ID NO.15:

[0028] EEQLVEFGGDLVKPGGSLRLSCVASGFTFSDYGMSWVRQSPGKGLQEVAYIRGDGGWTYYTDAVKGRFTISRDNAKNTVYLQMDSLRAEDTAVYYCVKAPWLAFWGQGTLVTVSS.

[0029] SEQ ID NO.16:

[0030] EIVMTQTPLSLSVSPGEPASISCKASQSLLHSNGNTYLFWYRQKPGQSPQRLIYKVSNRDPGVPDRISGSGSGTDFTLRISRVEADDAGVYYCGQGIQFPLTFGKGTHLEIK.

[0031] Specifically, the anti-TNF-α antibody is a monoclonal antibody.

[0032] Preferably, the anti-TNF-α antibody is a canineized antibody.

[0033] Specifically, the anti-TNF-α antibody comprises a variable light chain sequence and a variable heavy chain sequence, wherein:

[0034] The variable light chain sequence has at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 14; the variable heavy chain sequence has at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 13;

[0035] Or the variable light chain sequence has at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 16; the variable heavy chain sequence has at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO: 15.

[0036] More specifically, the anti-TNF-α antibody binds to canine TNF-α and blocks the binding of canine TNF-α to canine TNFR1 and TNFR2.

[0037] Specifically, the anti-TNF-α antibody comprises a canine heavy chain constant region selected from the constant regions of IgGA, IgGB, IgGC and IgGD.

[0038] In a second aspect, the present invention provides an antibody preparation expressing the aforementioned antibody.

[0039] In a third aspect, the present invention provides a nucleic acid expressing the aforementioned anti-TNF-α antibody.

[0040] In a fourth aspect, the present invention provides an expression vector comprising the aforementioned nucleic acid sequence.

[0041] The expression vector may include, but is not limited to, plasmids, phages, viruses.

[0042] In a fifth aspect, the present invention provides a cell expressing the aforementioned anti-TNF-α antibody or nucleotide sequence or expression vector.

[0043] The role of the cell is that it can highly express the anti-TNF-α antibody.

[0044] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the aforementioned antibody, antibody preparation, nucleic acid molecule, expression vector and host cell.

[0045] In a seventh aspect, the present invention provides the use of the aforementioned anti-TNF-α antibody, nucleic acid, expression vector, host cell or pharmaceutical composition in the preparation of a drug for treating diseases related to TNF-α.

[0046] Specifically, the application is achieved through antibody-antigen binding.

[0047] The anti-TNF-α antibodies described above are also known as F6 and G5.

[0048] Compared with the prior art, the beneficial technical effects of the present invention are as follows:

[0049] The present patent technology constructs a canine natural phage antibody library and screens out antibodies against specific targets for canine inflammatory bowel disease. That is, the present invention obtains positive clones through the phage library, and through antibody engineering transformation, high-affinity antibodies are obtained, avoiding the immunogenicity problems caused by conventional monoclonal antibodies. ELISA analysis and BLI affinity detection show that both F6 and G5 can bind to TNF-α. In addition, through toxicity experiments on L929 cells, it is shown that F6 and G5 have the ability to neutralize the cytotoxicity of this cytokine. The present invention also constructs an in vivo TNBS-induced enteritis model and finds that the clinical activity score, colon weight / length ratio, and histopathological score of candidate molecule G5 are significantly better than those of the control group, showing significant curative effects, indicating that this candidate molecule can have good therapeutic effects in inflammatory bowel disease. Description of the Drawings

[0050] Figure 1 They are respectively the structural reference diagrams of F6 and G5.

[0051] Figure 2 A is the antibody affinity analysis diagram, Figure 2 B is the OD value of antibodies at different concentrations.

[0052] Figure 3 It is the antibody blocking activity analysis diagram.

[0053] Figure 4 It is the antibody neutralization activity analysis diagram.

[0054] Figure 5 They are the clinical activity score diagram (A), colon density diagram (B), and histopathological score diagram (C) of the in vivo treatment effect. Detailed Embodiments

[0055] The present invention will be described below in conjunction with specific embodiments. The following embodiments are not used to limit the present invention, but only to illustrate the present invention, so that the technical solutions of the present invention can be more easily understood and grasped. The experimental methods described in the following embodiments are all conventional methods unless otherwise specified; the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0056] Example 1 Preparation and Screening of Antibodies

[0057] The antibodies obtained in the present invention are mainly screened from a natural phage library. The antigen used during the process is canine TNF-α, and its sequence is as shown in SEQ ID NO.17; the negative control is NCHis (purchased from PuJian Biotechnology); the library information is a canine natural library, and the library capacity is 1.06×10 10 . The acquisition methods are, in sequence, 3 to 4 rounds of library panning, polyclonal ELISA verification, monoclonal ELISA verification, validated ELISA verification for positive clones, and sequencing. The specific methods are as follows:

[0058] SEQ ID NO.17:

[0059] VKSSSRTPSDKPVAHVVANPEAEGQLQWLSRRANALLANGVELTDNQLI VPSDGLYLIYSQVLFKGQGCPSTHVLLTHTISRFAVSYQTKVNLLSAIKSPCQR ETPEGTEAKPWYEPIYLGGVFQLEKGDRLSAEINLPNYLDFAESGQVYFGIIAL。

[0060] (1) Immune tube solid-phase panning: First, coat the immune tubes, that is, add 1 mL of a mixture of 50 μg / mL HZ-P02 protein and buffer PBS into the enzyme-labeled plate tubes and incubate overnight at 4°C; then wash the immune tubes 3 times with 5 mL of PBST; block with 5 mL of 5% skim milk / PBST at 30°C for 1 h; then wash once with 5 mL of PBS; add 1 mL of 1×10 12 pfu of library phage into each tube, incubate at 30°C for 2 h; wash 4 - 6 times with 5 mL of PBST (the number of washing times can be increased according to the enrichment degree in the subsequent rounds); add 1 mL of Gly-HCl (pH = 2.2) to each tube to elute the phage, and incubate with shaking at room temperature for about 6 - 8 min; add 120 - 130 μL of Tris-HCl (pH = 9.6) neutralization solution to adjust the pH to 7.0 - 8.0; after diluting the eluted phage, infect the logarithmic-phase Escherichia coli TG1 and plate to determine the titer.

[0061] (2) Amplification of eluted phages: Pipette the eluted phages and add them to the logarithmic-phase Escherichia coli TG1 bacterial solution. After standing at 37°C for 30 min, culture at 220 rpm for 30 min - 1 h; add the antibiotic Amp to the medium and culture at 37°C and 220 rpm until the OD of the bacterial solution is about 0.4 - 0.6; add helper phages to the bacterial solution. After standing at 37°C for 30 min, culture at 220 rpm for 45 min - 1 h; centrifuge the bacterial solution at 3000 - 5000 rpm and discard the supernatant. Resuspend the bacterial cells with an equal volume of 2YT - Amp - Kan medium. Culture overnight at 30°C and 220 rpm; the next day, centrifuge the bacterial solution at 8000 rpm, 4°C for 20 min, and transfer the supernatant to a new centrifuge tube. Add 1 / 4 volume of 5×PEG / NaCl solution. After thorough mixing, place on ice or at 4°C and let stand for 1 - 2 h; centrifuge at 8000 rpm, 4°C for 30 min and discard the supernatant. Resuspend the precipitate with about 1 mL of PBS. Centrifuge at 12000 rpm for 5 min and transfer the supernatant to a new centrifuge tube; dilute the amplified phages and infect the logarithmic-phase TG1, plate and measure the titer.

[0062] (3) Panning of immunotubes from the second to the fourth round: Use the amplified phages for the second-round panning, and the steps are the same as (1)(2). Repeat the panning 3 to 4 times.

[0063] (4) Polyclonal phage ELISA detection: Coat the immunoplates (antigen TNF-α); after washing 3 times with 300 μL of PBST, add 300 μL of 5% skim milk / PBST and block at 30°C for 1 h; then wash 2 - 3 times with 300 μL of PBST; dilute the phages amplified in each round with PBS, with the dilution factor increasing 3-fold. The initial concentration is 1×10 12 pfu / mL. Add 100 μL of the diluted amplified phages to each well. Incubate at 30°C for 1 h, wash 4 - 6 times with 300 μL of PBST; add 100 μL of the secondary antibody (anti-phage M13) (purchased from Invitrogen) dilution and incubate at 30°C for 1 h, wash 4 - 6 times with 300 μL of PBST; add 100 μL of the chromogenic solution TMB and develop color in the dark for 3 - 8 min, add 100 μL of 2M HCl to terminate the reaction, and read the absorbance with an ELISA reader (450 nm - 620 nm).

[0064] (5) Monoclonal phage ELISA screening: Select an appropriate number of rounds, dilute the eluted phages to an appropriate concentration, infect logarithmic-phase TG1, and plate them; the next day, pick 96 monoclonal colonies from the plate, inoculate them into a 96-deep well plate, and culture them at 37 °C with shaking at 250 rpm until the OD of the bacterial solution is 0.4 - 0.6; add helper phages to the medium in the 96-deep well plate. Incubate statically at 37 °C for 30 min, then culture with shaking at 250 rpm at 37 °C for 45 min - 1 h; centrifuge the 96-deep well plate at 4000 rpm for 5 min and discard the supernatant. Resuspend the bacterial solution in each well with 2YT-Amp-Kan medium and culture overnight at 30 °C with shaking at 250 rpm; the next day, centrifuge the 96-deep well plate at 4000 rpm for 10 - 15 min and take the supernatant for ELISA experiment; Coat the immunosorbent plate: Antigen HZP02 (4 μg / mL, buffer PBS), 100 μL, overnight at 4 °C. Coat 100 μL of NC-His in the control wells; Wash 3 times with 300 μL of PBST; Block with 300 μL of 5% skim milk / PBST at 30 °C for 1 h. Wash 2 - 3 times with 300 μL of PBST; Add 100 μL of phage supernatant to each well. Incubate at 30 °C for 1 h. Wash 4 - 6 times with 300 μL of PBST; Add 100 μL of secondary antibody (anti-M13) dilution, incubate at 30 °C for 1 h, and wash 4 - 6 times with 300 μL of PBST; Add 100 μL of chromogenic solution TMB and develop color in the dark for 3 - 8 min, add 100 μL of 2M HCl to terminate the reaction, and read the absorbance with an ELISA reader (450 nm - 620 nm).

[0065] (6) Secondary verification of positive clones: After sequencing the positive clones obtained by monoclonal screening, remove the double-peak sequences and repetitive sequences to obtain the final positive clones. The positive clones are subjected to secondary ELISA detection and verification according to step (4) to ensure the authenticity of the positive results.

[0066] (7) According to the enrichment degree of the results of polyclonal phage ELISA, select the products after the second round of panning for monoclonal screening.

[0067] (8) Conduct secondary verification of the sequences according to the results of monoclonal phage ELISA, and screen out 2 positive clones specific to HZ-P02 to obtain 2 gene sequences.

[0068] (9) Perform antibody engineering modification on the positive clones F6 and G5 obtained by phage display to obtain 2 monoclonal antibodies. The results are as Figure 1 shown. The screened antibodies F6 and G5 contain complete heavy chains and complete light chains, indicating strong specificity and good affinity.

[0069] Example 2 Affinity Detection of Antibodies

[0070] First, the antibody was biotinylated, and then the biotinylated antibody was used as a solidified substance to bind to the biotinylated antibody immobilized on the biosensor using streptavidin probes (Gator TM Streptavidin Probes). Then, different recombinant proteins were used as analytes to measure their binding kinetic data with the biotinylated antibody and calculate the affinity. The results are as Figure 2 shown. When the F6, G5, and BMK molecules were respectively bound to inflammatory cytokines, both F6 and G5 could bind to the inflammatory cytokines, and their affinities were significantly stronger than that of the BMK molecule. The affinity of the G5 molecule reached 5.482E-12 (KD / M), showing excellent affinity.

[0071] Example 3 Effect of antibodies on the cytotoxicity induced by inflammatory cytokines

[0072] In the present invention, ELISA and cell-binding competition assays were performed on the ligand receptors of secreted and membrane-bound pro-inflammatory cytokines, as well as F6 and G5. The results are as Figure 3 shown. When the candidate molecules F6 and G5 bind to the inflammatory cytokine ligands, the binding of their receptor-ligand will become relatively weaker, indicating that the candidate molecules F6 and G5 can block the binding of pro-inflammatory cytokines to the receptors, thereby blocking the downstream inflammatory signaling pathway and slowing down the occurrence of inflammation.

[0073] Subsequently, L929 cells were cultured first. When the number of cells reached a certain level, F6, G5, and BMK antibodies were added. After a period of time, the cytotoxic effects of the cells were observed, and the readings of the microplate reader (450 nm) were measured. The experimental results Figure 4 showed that G5 and F6 could significantly inhibit the cytotoxic effects induced by this cytokine, indicating that F6 and G5 have the ability to neutralize the cytotoxicity of this cytokine.

[0074] Example 4 Evaluation of the in vivo therapeutic effect of antibodies

[0075] In the present invention, an in vivo TNBS-induced enteritis model was constructed and divided into four groups: G1 (Control), G2 (TNBS + Vehicle), G3 (TNBS + G5 3 mg / mL), and G4 (TNBS + G5 5 mg / mL). The experimental results are as Figure 5 shown. In the enteritis model, compared with the control group, after adding 3 mg / mL and 5 mg / mL of G5 antibody, the inflammation was significantly downregulated, and the higher the concentration of the added G5 antibody, the more obvious the decrease in the inflammatory effect; at the same time, the clinical activity score, colon density, and histopathological score of the candidate molecule G5 were all significantly better than those of the control group.

[0076] The above detailed description is a specific description of one of the feasible embodiments of the present invention, and this embodiment is not intended to limit the patent scope of the present invention. It should be noted that all equivalent implementations or changes made without departing from the present invention should be included within the scope of the technical solution of the present invention. Therefore, the protection scope of the present invention shall be subject to the appended claims.

Claims

1. An anti-TNF-α antibody that binds to canine TNF-α, characterized in that, The anti-TNF-α antibody described above comprises a heavy chain and a light chain. The heavy chain comprises heavy chain complementarity determining regions, and the light chain comprises light chain complementarity determining regions, wherein: The heavy chain complementarity determining regions of the antibody comprise HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 respectively; the light chain complementarity determining regions of the antibody comprise LCDR1 and LCDR3 as shown in SEQ ID NO:4 and SEQ ID NO:6 respectively, and LCDR2 with the amino acid sequence of GVS; or the heavy chain complementarity determining regions of the antibody comprise HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9 respectively; the light chain complementarity determining regions of the antibody comprise LCDR1 and LCDR3 as shown in SEQ ID NO:10 and SEQ ID NO:12 respectively, and LCDR2 with the amino acid sequence of KVS.

2. The anti-TNF-α antibody according to claim 1, wherein, The amino acid sequence of the heavy chain variable region of the anti-TNF-α antibody is SEQ ID NO:13, and the amino acid sequence of the light chain variable region is SEQ ID NO:14; or the amino acid sequence of the heavy chain variable region of the anti-TNF-α antibody is SEQ ID NO:15, and the amino acid sequence of the light chain variable region is SEQ ID NO:

16.

3. The anti-TNF-α antibody according to claim 1, wherein The anti-TNF-α antibody is a canineized antibody.

4. The anti-TNF-α antibody according to any one of claims 1, characterized in that, The anti-TNF-α antibody comprises a variable light chain sequence and a variable heavy chain sequence, wherein: The variable light chain sequence has at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:14; the variable heavy chain sequence has at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:13; or the variable light chain sequence has at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:16; the variable heavy chain sequence has at least 85%, at least 90%, at least 95% or at least 98% sequence identity with the amino acid sequence of SEQ ID NO:

15.

5. The anti-TNF-α antibody according to any one of claims 1-4, characterized in that, The anti-TNF-α antibody comprises a canine heavy chain constant region selected from the constant regions of IgG-A, IgG-B, IgG-C and IgG-D.

6. An antibody preparation, characterized in that, The antibody preparation comprises the anti-TNF-α antibody according to any one of claims 1-5.

7. A nucleic acid, characterized in that, The nucleic acid encodes the anti-TNF-α antibody according to any one of claims 1-5.

8. An expression vector, characterized in that, The expression vector comprises the nucleic acid according to claim 7.

9. A host cell, characterized in that, The host cell comprises the expression vector according to claim 8.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the anti-TNF-α antibody according to any one of claims 1-5, the antibody preparation according to claim 6, the nucleic acid according to claim 7, the expression vector according to claim 8 and the host cell according to claim 9.

11. Use of the anti-TNF-α antibody according to any one of claims 1-5, the antibody preparation according to claim 6, the nucleic acid according to claim 7, the expression vector according to claim 8, the host cell according to claim 9, or the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating TNF-α-related inflammation.

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