Anti-TNF-alpha antibody as well as product and application thereof

Abstract

The invention belongs to the field of antibodies, and particularly relates to an anti-TNF-alpha antibody and a product and application thereof. According to the invention, a canine natural bacteriophage antibody library is utilized, and a specific target TNF-alpha antibody aiming at canine inflammatory bowel disease is screened out. ELISA analysis and BLI affinity detection show that the anti-TNF-alpha antibody can be combined with TNF-alpha. An L929 cytotoxicity experiment shows that the anti-TNF-alpha antibody has the capability of neutralizing the cytotoxicity of the cell factor. By constructing an in-vivo TNBS induced enteritis model, the antibody is found to have a very good treatment effect in treating diseases related to TNF-alpha.

Description

Technical Field

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

[0002] Tumor necrosis factor α (TNF-α) is a proinflammatory cytokine produced by macrophages or monocytes. It exerts its effects by binding to two different receptors, namely TNF receptor 1 (TNFR1) and TNF receptor 2 (TNFR2), which activate signaling pathways involving nuclear factor κB (NF-κB), c-Jun N-terminal kinase (JNK), p42 / p44 mitogen-activated kinase (MAPK), and p38MAPK. TNF α has been identified as a major regulator of inflammatory responses, involving physiological and pathological processes such as inflammation, apoptosis, immune homeostasis, and autoimmunity. It participates in the pathogenesis of a variety of inflammatory and autoimmune diseases, such as infection with pathogenic microorganisms, rheumatoid arthritis, systemic lupus erythematosus, autoimmune skin diseases (including psoriasis), inflammatory bowel disease (ulcerative colitis), autoimmune blood system diseases, ankylosing spondylitis, and non-infectious uveitis. Elevated serum TNF α levels are closely related to the pathophysiology of autoimmune diseases, so neutralizing TNF α has become the main strategy for the treatment of these diseases.

[0003] Prior art CN115974998A proposes a TNFα antagonist peptide and its application. The TNFα antagonist peptide has good stability and has significant anti-inflammatory, immune regulation, and TNFα antagonist effects, and can be used to prepare drugs for the treatment of inflammatory and autoimmune diseases. Prior art CN110066337B discloses an anti-TNFα antibody, which is obtained by constructing a natural rabbit-derived phage antibody library that has not been subjected to antigen immune stimulation, using TNFα recombinant protein as a target, and after 3 rounds of screening and enrichment, a single-chain antibody that specifically binds to TNFα is obtained. The antibody can be used as a specific capture TNFα antibody, a TNFα signaling antibody, and the like. At the same time, a successful natural antibody library can also be directly used for more target screening. At present, the most typical antibody antagonist adalimumab for TNFα has already been successfully launched on the market and applied to the treatment of related diseases.

[0004] However, due to species differences, direct application of human antibodies to dogs often causes severe immune responses, resulting in less than expected efficacy. Therefore, it is particularly important to develop highly effective TNF-α antibodies specific to dogs. Summary of the invention

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

[0006] In a first aspect, the present invention provides an anti-TNF-α antibody, wherein the antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain complementary determining region, and the light chain comprises a light chain complementary determining region.

[0007] The antibody heavy chain complementary determining region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; the antibody light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQID 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 antibody heavy chain complementary determining region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9; the antibody light chain variable region includes 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] EVQLVESGGDLVKPAGSLRLSCVASGFTFSSYAMSWVRQAPGKGLQWV AGISSGGSSTNYADAVKGRFTISRDNAKNTLYLQMNSLRAEDTAVYYCAKDL TLLPRSFDYWGQGTLVTVSS.

[0024] SEQ ID NO.14:

[0025] DIVMTQTPLSLSVSPGEPASISCKASQSLLHSDGKTYLHWFRQKPGQSPQ LLIYGVSNRDTGVPDRFSGSGSGTDFTLKISRVEADDAGVYYCGQGIQSPFTF GQGTKVELK.

[0026] Alternatively, the amino acid sequence of the heavy chain variable region of the anti-TNF-α antibody is SEQ ID NO: 17, and the amino acid sequence of the light chain variable region is SEQ ID NO: 18.

[0027] SEQ ID NO.15:

[0028] EEQLVEFGGDLVKPGGSLRLSCVASGFTFSDYGMSWVRQSPGKGLQEV AYIRGDGGWTYYTDAVKGRFTISRDNAKNTVYLQMDSLRAEDTAVYYCVK APWLAFWGQGTLVTVSS.

[0029] SEQ ID NO.16:

[0030] EIVMTQTPLSLSVSPGEPASISCKASQSLLHSNGNTYLFWYRQKPGQSPQ RLIYKVSNRDPGVPDRISGSGSGTDFTLRISRVEADDAGVYYCGQGIQFPLTFG KGTHLEIK.

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

[0032] Preferably, the anti-TNF-α antibody is a caninized 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:18; 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:17.

[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, IgG B, IgG C and IgG D.

[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 plasmid, phage, virus.

[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 function of the cells is to efficiently express anti-TNF-α antibodies.

[0044] In a sixth aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned 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 associated with TNF-α.

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

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

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

[0049] This patented technology constructs a canine natural phage antibody library and screens out antibodies against specific targets of canine inflammatory bowel disease. That is, the present invention obtains positive clones through the phage library, and obtains high-affinity antibodies through antibody engineering, avoiding the immunogenicity problem caused by conventional monoclonal antibodies. ELISA analysis and BLI affinity detection show that both F6 and G5 can bind to TNF-α. In addition, by conducting toxicity experiments on L929 cells, it was shown that F6 and G5 have the ability to neutralize the cytotoxicity of the 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 the candidate molecule G5 are significantly better than those of the control group, showing significant therapeutic effects, indicating that the candidate molecule can have a good therapeutic effect in inflammatory bowel disease. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0052] Figure 3 This is a diagram for analyzing the antibody blocking activity.

[0053] Figure 4 This is a diagram for analyzing the neutralizing activity of antibodies.

[0054] Figure 5 Figure 3 shows the in vivo therapeutic effect clinical activity score (A), colon density (B), and histopathological score (C). DETAILED DESCRIPTION

[0055] The present invention is described below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention so that the technical solution of the present invention is easier to understand and grasp. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are all commercially available unless otherwise specified.

[0056] Example 1 Preparation and screening of antibodies

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

[0058] SEQ ID NO.17:

[0059] VKSSRTPSDKPVAHVVANPEAEGQLQWLSRRANALLANGVELTDNQLI VPSDGLYLIYSQVLFKGQGCPSTHVLLTHTISRFAVSYQTKVNLLSAIKSPCQR ETPEGTEAKPWYEPIYLGGVFQLEKGDRLSAEINLPNYLDFAESGQVYFGIIAL.

[0060] (1) Immunotube solid phase panning: First, coat the immunotubes by adding 1 mL of a mixture of 50 μg / mL HZ-P02 protein and PBS buffer to the ELISA plate tubes and incubate at 4°C overnight. Then, wash the immunotubes three times with 5 mL of PBST. Block the tubes with 5 mL of 5% skim milk / PBST at 30°C for 1 h. Wash the tubes once with 5 mL of PBS. Add 1 mL of 1×10 12 pfu of library phage, incubate at 30°C for 2h; wash 4-6 times with 5mL PBST (the number of washes can be increased in the subsequent rounds according to the enrichment process); add 1mL of Gly-HCl (pH=2.2) to each tube to elute the phage, and incubate at room temperature for about 6-8min with shaking; add 120-130μL of Tris-HCl (pH=9.6) to neutralize the solution to pH=7.0-8.0; dilute the eluted phage, infect Escherichia coli TG1 in the logarithmic phase, and plate to determine the titer.

[0061] (2) Amplification of eluted phages: Take the eluted phages and add them to the logarithmic phase Escherichia coli TG1 culture medium. After standing at 37°C for 30 minutes, culture at 220 rpm for 30 minutes-1 hour. Add antibiotic Amp to the culture medium and culture at 37°C and 220 rpm until the culture OD is about 0.4-0.6. Add helper phages to the culture medium. After standing at 37°C for 30 minutes, culture at 220 rpm for 45 minutes-1 hour. Centrifuge the culture medium at 3000-5000 rpm and discard the supernatant. Resuspend the cells with an equal volume of 2YT-Amp-Kan culture medium. Culture overnight at 30°C and 220 rpm. The next day, centrifuge the culture medium at 8000 rpm and 4°C for 20 minutes, 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 for 1-2 hours; centrifuge at 8000rpm, 4°C for 30 minutes, and discard the supernatant. Resuspend the precipitate with about 1mL PBS. Centrifuge at 12000rpm for 5 minutes, and transfer the supernatant to a new centrifuge tube; dilute the amplified phage, infect TG1 in the logarithmic phase, and plate to determine the titer.

[0062] (3) Second to fourth rounds of immunotube panning: Use the amplified phage for the second round of panning, following the same steps as (1) and (2). Repeat this process for 3 to 4 rounds.

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

[0064] (5) Monoclonal phage ELISA screening: Select the appropriate round number, dilute the eluted phage to the appropriate concentration, infect TG1 in the logarithmic phase, and plate; the next day, pick 96 monoclones from the plate, inoculate 96 deep-well plates and shake culture at 37°C and 250rpm until the bacterial solution OD = 0.4-0.6; add helper phage to the 96 deep-well plate culture medium. Stand at 37°C for 30min, shake culture at 250rpm, and shake culture at 37°C for 45min-1h; centrifuge the 96 deep-well plate at 4000rpm for 5min, and discard the supernatant. Resuspend the bacterial solution in each well with 2YT-Amp-Kan culture medium, shake culture at 30°C and 250rpm overnight; the next day, centrifuge the 96 deep-well plate at 4000rpm for 10-15min, and take the supernatant for ELISA experiment; coat the immune plate: antigen HZP02 (4μg / mL, buffer PBS), 100μL, overnight at 4°C. Control wells were coated with 100 μL NC-His; washed 3 times with 300 μL PBST; blocked with 300 μL 5% skim milk / PBST at 30°C for 1 h. Washed 2-3 times with 300 μL PBST; added 100 μL phage supernatant to each well. Incubated at 30°C for 1 h. Washed 4-6 times with 300 μL PBST; added 100 μL secondary antibody (anti-M13) diluent, incubated at 30°C for 1 h, washed 4-6 times with 300 μL PBST; added 100 μL colorimetric solution TMB for 3-8 min in the dark, added 100 μL 2M HCl to terminate the reaction, and read with an ELISA reader (450 nm-620 nm).

[0065] (6) Secondary verification of positive clones: After sequencing the positive clones obtained from the monoclonal screening, the double peak sequences and repeated sequences were removed to obtain the final positive clones. The positive clones were verified by secondary ELISA testing according to step (4) to ensure the authenticity of the positive results.

[0066] (7) Based on the degree of enrichment of the polyclonal phage ELISA results, the products after the second round of panning were selected for monoclonal screening.

[0067] (8) Based on the results of monoclonal phage ELISA, the sequence was secondary verified, and two positive clones specific to HZ-P02 were screened out, and two gene sequences were obtained.

[0068] (9) The positive clones F6 and G5 obtained by phage display were subjected to antibody engineering to obtain two monoclonal antibodies. Figure 1 As shown, the screened F6 and G5 antibodies contain complete heavy chains and complete light chains, indicating that they have strong specificity and good affinity.

[0069] Example 2 Antibody affinity detection

[0070] First, the antibody is biotinylated, and then the biotinylated antibody is used as a solid to bind to the biosensor using a streptavidin probe (Gator TM Streptavidin Probes) to solidify the biotinylated antibody. Then different recombinant proteins are used as analytes to measure their binding kinetics with the biotinylated antibody and calculate affinity. The results are as follows Figure 2 As shown, F6, G5, and BMK molecules were combined with inflammatory cytokines respectively. Both F6 and G5 were able to bind to inflammatory cytokines, and their affinity was significantly stronger than that of BMK molecules. The affinity of G5 molecules reached 5.482E-12 (KD / M), showing excellent affinity.

[0071] Example 3 Effects of Antibodies on Cytotoxicity Induced by Inflammatory Cytokines

[0072] The present invention conducted ELISA and cell binding competition analysis on the ligand receptors of secretory and membrane-type proinflammatory cytokines as well as F6 and G5. The results are as follows: Figure 3 As shown, when the candidate molecules F6 and G5 bind to the inflammatory cytokine ligands, their receptor-ligand binding will become relatively weaker, indicating that the candidate molecules F6 and G5 can block the binding of proinflammatory cytokines to receptors, thereby blocking the downstream inflammatory signaling pathways and slowing down the occurrence of inflammation.

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

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

[0075] The present invention constructs an in vivo TNBS-induced enteritis model and divides the model into four groups: G1 (Control), G2 (TNBS+Vehicle), G3 (TNBS+G5 3 mg / mL) and G4 (TNBS+G5 5 mg / mL). Figure 5 As shown, in the enteritis model, compared with the control group, inflammation was significantly downregulated after adding 3 mg / mL and 5 mg / mL of G5 antibody, and the higher the concentration of G5 antibody added, 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 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 the embodiment is not intended to limit the scope of the present invention. It should be pointed out that any equivalent implementation or change that does not deviate from the present invention should be included in the scope of the technical solution of the present invention. Therefore, the protection scope of the patent of the present invention shall be based on the attached requirements.

Claims

1. An anti-TNF-α antibody that binds to canine TNF-α, characterized in that: The anti-TNF-α antibody comprises a heavy chain and a light chain, wherein the heavy chain comprises a heavy chain complementary determining region, and the light chain comprises a light chain complementary determining region, wherein: The antibody heavy chain complementary determining region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3; the antibody light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; Or the antibody heavy chain complementary determining region includes HCDR1, HCDR2 and HCDR3 as shown in SEQ ID NO:7, SEQ ID NO:8 and SEQ ID NO:9; the antibody light chain variable region includes LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO:10, SEQ ID NO:11 and SEQID NO:

12.

2. The anti-TNF-α antibody according to claim 1, characterized in that 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, characterized in that The anti-TNF-α antibody is a caninized antibody.

4. The anti-TNF-α antibody according to any one of claims 1 to 3, 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:18; 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:

17.

5. The anti-TNF-α antibody according to any one of claims 1 to 4, characterized in that The anti-TNF-α antibody binds to canine TNF-α and blocks the binding of canine TNF-α to canine TNFR1 and TNFR2.

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

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

8. A nucleic acid, characterized in that The nucleic acid encodes the anti-TNF-α antibody according to any one of claims 1-6.

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

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

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

12. Use of the anti-TNF-α antibody of claims 1 to 6, the antibody preparation of claim 7, the nucleic acid of claim 8, the expression vector of claim 9, the host cell of claim 10 or the pharmaceutical composition of claim 11 in the preparation of a drug for treating diseases associated with TNF-α.

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