An antibody that adsorbs an inflammatory factor and use thereof

Abstract

The application belongs to the technical field of biotechnology, and discloses an antibody for adsorbing inflammatory factors and application thereof. The structural unit of the antibody comprises at least one of a nanobody for resisting human IL-1β, a nanobody for resisting human IL-6 and a nanobody for resisting human TNF-α, and the different nanobodies are connected through flexible peptides. In particular, the amino acid sequence of the antibody is shown as SEQ ID NO: 10-12. The antibody of some examples of the application has good specific affinity to at least one of IL-6, IL-1β and TNF-α, the prepared adsorbent can specifically recognize and combine at least one of IL-6, IL-1β and TNF-α, thereby effectively removing inflammatory mediators in blood, and has less influence on other effective components in blood such as immunoglobulin and albumin. The application of the adsorbent in the field of blood purification is helpful to alleviate and treat diseases caused by inflammatory factor storm.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to an antibody for adsorbing inflammatory factors and application thereof. BACKGROUND

[0002] Inflammatory cytokines are a group of polypeptide cell regulatory substances, including interleukins, interferons, growth factors, cell stimulating factors, tumor necrosis factors, etc. Many diseases show high concentrations of inflammatory factors in the blood stream. Inflammatory factors help stimulate and regulate the immune system to help the body resist infection, but if the body produces too much, it will cause autoimmune diseases. Cytokine storm refers to the rapid and large production of various inflammatory factors such as TNF-a, IL-1, IL-6, IL-12, IFN-a, IFN-b, IFN-g, MCP-1 and IL-8 in body fluid after the body is infected by microorganisms. All injuries to a certain extent can trigger the production and release of a large number of inflammatory mediators and cytokines, and then trigger a systemic inflammatory cascade reaction. Their excessive secretion leads to an imbalance between pro-inflammatory factors and anti-inflammatory factors in the human body, thereby causing dysfunction of distant organs. Therefore, it is particularly important to remove inflammatory factors and reduce the level of inflammatory factors in the body of patients.

[0003] Among the many inflammatory factors, TNF-a is the most important inflammatory mediator. TNF-a can activate the cytokine cascade reaction in the inflammatory response, induce IL-1, IL-6, IL-8 and secondary inflammatory mediators [platelet activating factor (PAF), prostaglandin, nitric oxide (NO) and leukotriene, etc.], thereby triggering an inflammatory chain reaction. TNF-a stimulates the release of neutrophils from the bone marrow, promotes the differentiation and activation of monocytes and macrophages, stimulates the synthesis of acute phase proteins, and activates the common pathways of the coagulation and complement systems. The secretion of TNF-a can release IL-1. Monocytes and tissue macrophages are the main sources of IL-1, and IL-1 has a and β two types, which are structurally related polypeptides. Most IL-1a exists in the form of a precursor in the cytosol of cells, and a small amount exists in the form of a physiologically active form on the cell membrane. IL-1β is split by IL-1β converting enzyme and is easily degraded by proteases, fibrinolysis or other proteases. IL-1 is a strong inducer of granulocyte / macrophage colony-stimulating factor (GM-CSF). The release of a large amount of IL-1 causes activated neutrophils to gather on the vascular wall, stimulates the procoagulant activity of endothelial cells, and increases leukocyte binding. IL-1 can produce many blood and metabolic abnormalities similar to those seen in severe sepsis.

[0004] IL-6 is a multifunctional cytokine. Various cells such as macrophages, T cells, B cells, fibroblasts, endothelial cells, mesangial cells of glomeruli and several tumor cells can produce IL-6. It is mainly produced by monocytes, acts on endothelial cells, increases the expression of adhesion molecules, promotes platelet aggregation, and participates in the acute phase response of infection. IL-6 is rapidly released within 1 hour after injury, and plays the roles of B cell stimulating factor, hybridoma / plasmacytoma growth factor, hepatocyte stimulating factor and cytotoxic T cell differentiation factor. IL-6 and IL-1β together affect the differentiation of thymus cells, and together with TNF-α enhance T cell proliferation and promote the activation of polymorphonuclear leukocytes (PMN). Anti-IL-6 monoclonal antibody can protect experimental animals from lethal E. coli infection. Therefore, IL-6, IL-1β and TNF-α are the three most important inflammatory factors and affect each other.

[0005] Monoclonal antibody drugs targeting IL-6, IL-1β and TNF-α have been approved for marketing, but long-term use of monoclonal antibody drugs can also cause drug resistance. In addition to drug treatment, blood purification is another method for rapidly reducing inflammatory factors, such as the CytoSorb adsorbent in the United States, which can be used for adsorption and removal of inflammatory factors. However, its main principle is based on pore adsorption, which has poor specificity. The technical principle of immunoadsorption (IA) therapy is based on the specific recognition between specific molecules. Generally, highly specific antigens, antibodies or molecules with specific physical and chemical affinity are used as adsorption functional groups (ligands), which are covalently coupled to a carrier medium with good blood compatibility, thereby obtaining an adsorption material with high selectivity for target substances. By connecting the nanobodies targeting IL-6, IL-1β and TNF-α to the carrier, the corresponding inflammatory factors can be specifically adsorbed, and other components such as albumin and immunoglobulin are almost not adsorbed. Nanobodies have small molecular weight, good stability, easy prokaryotic expression and construction of multivalent antibodies, thereby exerting better synergistic effect. SUMMARY

[0006] The purpose of the present application is to overcome at least one deficiency of the prior art and provide an antibody for adsorbing inflammatory factors and application thereof.

[0007] The technical solution adopted by the present application is:

[0008] In a first aspect of the present application, there is provided:

[0009] An antibody for adsorbing inflammatory factors, the structural unit of which comprises at least one of a nanobody against human IL-1β, a nanobody against human IL-6 and a nanobody against human TNF-α, and different nanobodies are connected by a flexible peptide, wherein:

[0010] The complementarity determining regions CDR1-CDR3 of the anti-human IL-1β nanobody are selected from one of the following groups:

[0011] Group 1: the amino acid sequences of CDR1-CDR3 are TSNGA, SDFNWDSDQAKN, NEFAYSRDATVYDQ, respectively;

[0012] Group 2: the amino acid sequences of CDR1-CDR3 are FQSGM, VITYDDSSFKSAKG, SAYYWSKEANLYHD, respectively;

[0013] Group 3: the amino acid sequences of CDR1-CDR3 are SYTSGMG, ALTDDKNRSFYWDSVRN, ARSTGYSRNPTAY, respectively;

[0014] The complementarity determining regions CDR1-CDR3 of the anti-human IL-6 nanobody are selected from one of the following groups:

[0015] Group 4: the amino acid sequences of CDR1-CDR3 are NNAMA, ISRRGGSGSEYYADSVRS, GYDNYDY, respectively;

[0016] Group 5: the amino acid sequences of CDR1-CDR3 are GSFMG, DIMGSRYTNYYVDSVKG, SGLLRRGYDY, respectively;

[0017] Group 6: the amino acid sequences of CDR1-CDR3 are INNPMG, DIMGPGGYYADSVDT, VKSDERPGYDY, respectively;

[0018] The complementarity determining regions CDR1-CDR3 of the anti-human TNF-α nanobody are selected from one of the following groups:

[0019] Group 7: the amino acid sequences of CDR1-CDR3 are EQWMY, VINTNGLITKYPDSAEG, SVLGLQGFT, respectively;

[0020] Group 8: the amino acid sequences of CDR1-CDR3 are DYIMGS, EINNTGAITKYPDSAKG, SPSASERESSALYKY, respectively;

[0021] Group 9: the amino acid sequences of CDR1-CDR3 are DTWIY, EITLITKQPDSAKG, SLLGQSPSAFNYDH, respectively.

[0022] In some examples of antibodies, the amino acid sequence of the anti-human IL-1β nanobody is selected from one of the following:

[0023] In some examples of the antibody, SEQ4: QVQLVESGGGLVQAGDSLRLSCVASGLTFSTSNGAWFRQVPGKDREFVASDFNWDSDQAKNRFTISRDNAKNTVYLQADNLTPTDTAVYYCAANEFAYSRDATVYDQWGQGTRVTVSS

[0024] SEQ5: QVQLVESGGGLVQPGGSLRLSCAASGLTVSFQSGMWFRQVPGKEREFVAVITYDDSSFKSAKGRFTISRDNAKNTVTLQADNLKPTDTAVYYCAASAYYWSKEANLYHDWGQGTRVTVSS

[0025] SEQ6: QVQLVESGGGSVQPGGSLRLSCAASGRTFSSYTSGMGWFRQAPGKEREFVSALTDDKNRSFYWDSVRNRFTISRDDAKNTVYLQMNSLRPEDTAVYYCAAARSTGYSRNPTAYWGQGTQVTVSS.

[0026] In some examples of the antibody, the amino acid sequence of the Nanobody that binds to human IL-6 is selected from one of the following:

[0027] SEQ1: QVQLVESGGGLVHPGDSLRLSCVASGLTSFNNAMAWFRQVPGKEREFVAISRRGGSGSEYYADSVRSRFTISRDNAKNTVYLQADSLKPTDTAVYYCAAGYDNYDYWGQGTRVTVSS

[0028] SEQ2: QVQLVESGGGLVQPGGSLRLSCAASGLTFSGSFMGWFRQVPGKEREFVADIMGSRYTNYYVDSVKGRFTISRDNGKNTVYLQADNLRPTDTAVYYCAASGLLRRGYDYWGQGTRVTVSS

[0029] SEQ3: QVQLVESGGGLVQAGDSLRLSCAASGLTFSINNPMGWFRQVPGKEREFVADIMGPGGYYADSVDTRFTISRDNGKNTVYLQADNLRPTDTAVYYCAAVKSDERPGYDYWGQGTRVTVSS.

[0030] In some examples of the antibodies, the amino acid sequence of the anti-human TNF-α nanobody is selected from one of the following:

[0031] SEQ7: QVQLVESGGGLVQPGGSLKLSCAVSGLTFSEQWMYWFRQVPGKEREFVAVINTNGLITKYPDSAEGRFTISRDNAKNTVYLQADSLKPTDTAVYYCAASVLGLQGFTWGQGTRVTVSS

[0032] SEQ8: QVQLVESGGGLVQAGDSLRLSCAASGLTFSDYIMGSWFRQVPGKEREFVAEINNTGAITKYPDSAKGRFTISRDNGKNTVYLQADNLRPTDTAVYYCAASPSASERESSALYKYWGQGTRVTVSS

[0033] SEQ9: QVQLVESGGGLVQPGGSLRLSCAASGLTVSDTWIYWFRQVPGKEREFVAEITLITKQPDSAKGRFTISRDNAKNTVTLQADNLKPTDTAVYYCAASLLGQSPSAFNYDHWGQGTRVTVSS.

[0034] In some examples of the antibodies, it is a multivalent antibody fused from one anti-human IL-1β nanobody, one anti-human IL-6 nanobody and one anti-human TNF-α nanobody.

[0035] In some examples of the antibodies, the amino acid sequence of the multivalent antibody is as shown in SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12.

[0036] The above features can be combined arbitrarily without conflict.

[0037] In a second aspect of the present application, there is provided:

[0038] Use of the antibodies of the first aspect of the present application, the use comprising:

[0039] Preparation of an adsorbent for an inflammatory factor;

[0040] Preparation of a detection reagent for an inflammatory factor;

[0041] Preparation of a separation or purification reagent for an inflammatory factor;

[0042] The inflammatory factor is IL-1β, IL-6 or TNF-α.

[0043] In some embodiments, the adsorbent is a solid support coupled with the antibody of the first aspect of the application.

[0044] In some embodiments, the solid support is selected from one of agarose gel microspheres, cellulose spheres, resin microspheres and the like.

[0045] In a third aspect of the application, there is provided:

[0046] A gene encoding the antibody of the first aspect of the application.

[0047] In a fourth aspect of the application, there is provided:

[0048] An expression system expressing the antibody of the first aspect of the application, or containing the gene of the third aspect of the application.

[0049] In some embodiments, the expression system is a protein expression system such as E. coli, yeast, insect cells and the like.

[0050] The application has the following advantages:

[0051] The antibody of some embodiments of the application has good specific affinity to at least one of IL-6, IL-1β and TNF-α, and the prepared adsorbent can specifically recognize and bind to at least one of IL-6, IL-1β and TNF-α, thereby effectively removing inflammatory mediators in the blood, while having less effect on other effective components in the blood such as immunoglobulin and albumin. The application of the adsorbent in the field of blood purification is helpful for alleviating and treating diseases caused by inflammatory factor storm.

[0052] The multivalent antibody of some embodiments of the application, in particular the trivalent antibody against human IL-6, IL-1β and TNF-α, can simultaneously specifically recognize and bind to IL-6, IL-1β and TNF-α, thereby effectively removing inflammatory mediators in the blood, while having less effect on other effective components in the blood such as immunoglobulin and albumin. The application of the adsorbent in the field of blood purification is helpful for alleviating and treating diseases caused by inflammatory factor storm. BRIEF DESCRIPTION OF DRAWINGS

[0053] Figure 1 is the activity determination result of anti-IL-6 nanobody.

[0054] Figure 2 is the activity determination of anti-IL-1β nanobody.

[0055] Figure 3 is the activity determination of anti-TNF-α nanobody. DETAILED DESCRIPTION

[0056] The technical solutions of the present application are further illustrated below with examples.

[0057] Example 1: Establishment of phage antibody library binding human IL-6, IL-1β, TNF-α

[0058] The commercial IL-6 (BD company) was used to immunize the llama, and the peripheral blood mononuclear cells (PBMC) were collected. The RNA was extracted and reversely transcribed into cDNA. After amplification by PCR, the cDNA was digested by hindIII and NotI and then connected to the phagemid vector pHIAT-1. The vector was transformed into E. coli TG1 to form the original phage library. The helper phage M13KO7 was added to the TG1 strain grown to the logarithmic phase, and after overnight culture, the supernatant was collected by centrifugation, and the phagemid was precipitated by PEG. After resuspending the phage with PBS and filtering through a 0.45 um filter to remove bacteria, the phage VHH antibody library was obtained, and the capacity of the library was 2.5×10 11 . Similarly, the commercial IL-1β and TNF-α (BD company) were used to immunize the llama, and the phage libraries binding human IL-1β and TNF-α were obtained, and the capacities of the libraries were 3.1×10 12 and 8.6×10 11 , respectively.

[0059] Example 2: Screening of phage library

[0060] The titer of the helper phage M13KO7 was 1×10 11 pfu / ml. The TG1 was infected with the helper phage M13KO7.

[0061] (1) First round of screening: coat human IL-6 on ELISA plate, 5 μg / well, 4°C overnight, wash plate 3 times; dissolve BSA in PBST to a concentration of 3%, 200 μL / well, 37°C block for 2 h, wash plate 3 times; add 100 μL of phage library solution to each well, 37°C incubate for 2 h, wash plate 6 times; add 100 μL of glycine-HCL buffer to each well, 10 min of light shaking at room temperature, aspirate eluent, add 80 μL of Tris-HCl buffer for neutralization. Take 10 μL of eluent to measure titer, the rest is added to 5 mL of TG1 bacteria that are grown to the logarithmic phase, 37°C infection for 30 min, add preheated 2 x YT medium to a total volume of 10 mL, 37°C, 250 rpm, 30 min, add ampicillin to a final concentration of 100 μg / mL, culture for 2 h. Add kanamycin to a final concentration of 70 μg / mL, incubate overnight, 4°C, 4000 rpm, centrifuge for 15 min, collect supernatant, add 5 mL of PEG / NaCl, ice bath for 1 h, 4°C, 9000 rpm, centrifuge for 20 min, discard supernatant. Invert the centrifuge tube on the water-absorbing paper and pat dry. Then resuspend with 1 ml of PBS, 4°C, 14000 rpm, centrifuge for 10 min, remove residual cell debris, and the precipitate is the phage antibody particles.

[0062] 2) Second round of screening: coat human IL-6 on ELISA plate, 1 μg / well, wash plate 3 times; use 3% skim milk powder, wash plate 3 times; add 100 μL of phage library solution to each well, 37°C incubate for 2 h, wash plate 10 times; the rest of the operations are the same as the first round of screening.

[0063] 3) Third round of screening: coat human IL-6 on ELISA plate, 0.1 μg / well, wash plate 3 times; block with 3% BSA, wash plate 3 times; add 100 μL of phage library solution to each well, 37°C incubate for 2 h, wash plate 15 times; the rest of the operations are the same as the first round of screening.

[0064] 4) After three rounds of screening, infect TG1 bacteria with phage, spread on culture dishes, randomly pick 48 single clones from the culture plates, use the original library as a negative control, ELISA detection, select clones with OD 450 values greater than 2.1 times that of the original library as preliminary positive clones. A total of 28 positive clones were screened, sequenced. According to the sequencing results, 10 sequences were selected as candidate sequences after removing duplicate sequences. Anti-IL-1β, TNF-α phage libraries were screened as above, and 10 sequences were also selected for each.

[0065] Example 3: In vitro recombinant expression and antibody purification

[0066] The gene sequence screened in Example 2 was transferred into a PET28 plasmid through enzyme cutting sites Ncol and Xhol, and expressed in E. coli BL21 (DE3). After expansion culture in LB medium containing 70 μg / mL kanamycin, the bacterial bodies were collected, ultrasonically broken (open for 5 s, stop for 10 s, working time 20 min), and centrifuged at 10 000 rpm for 10 min, and the supernatant was collected. The three strains with the highest soluble expression were selected by electrophoresis for the next step of protein purification. The his tag was used to purify the camel single-domain antibody using nickel ion chelation filler, and the elution peak was collected to obtain the camel single-domain antibody.

[0067] Example 4: Determination of the activity of the nanobody

[0068] The adsorption performance was determined by ELISA. Human IL-6 was coated on the plate at 100 ng / well, 4°C overnight, blocked, and then the purified nanobody was diluted to 1000 pg / mL, 500 pg / mL, 250 pg / mL, 125 pg / mL, 62.5 pg / mL, 31.25 pg / mL, and 0 pg / mL with PBS. Anti-His and HRP-labeled antibodies were added at 100 uL per well, and incubated at room temperature for 1 h; TMB color developing solution was used for color development for 10 min, the reaction was terminated, and the absorbance was detected at 450 nm. In the same way, human IL-1β and TNF-α were coated on the ELISA plate, and the activity of the anti-IL-1β and anti-TNF-α nanobody was determined without changing the other operations. The results are shown in Figure 1 、 Figure 2 、 Figure 3 It can be seen from Figure 1 that the activity of SEQ1 is the highest among the three anti-IL-6 nanobodies, but the three antibodies are not much different. It can be seen from Figure 2 that the activity of SEQ4 and SEQ5 is equivalent among the three anti-IL-1β nanobodies, and SEQ6 is lower. It can be seen from Figure 3 that the activity of SEQ7 is the highest among the three anti-TNF-α nanobodies, and the activity of SEQ9 is lower.

[0069] Example 5: Expression and purification of multivalent nanobody

[0070] Three sequences of IL-6, IL-1β, and TNF-α in Examples 3 and 4 were selected to design a fusion protein sequence. SEQ10 was obtained by fusing SEQ1, SEQ4, and SEQ7, SEQ11 was obtained by fusing SEQ2, SEQ5, and SEQ8, and SEQ12 was obtained by fusing SEQ3, SEQ6, and SEQ9. The gene was transferred into E. coli by genetic engineering for expression and purification, and the method was the same as that in Example 3.

[0071] Example 6: Synthesis of immunoadsorbent and determination of adsorption performance

[0072] Take 5 mL of iodine acetyl pre-activated agarose filler, rinse the filler with about 30 mL of coupling solution (50 mM Tris, 5 mM EDTA-Na, pH 8.5), and dry; add 5 mL of antibody solution with a concentration of 10 mg / mL (SEQ10 corresponds to adsorbent 1, SEQ11 corresponds to adsorbent 2, SEQ12 corresponds to adsorbent 3), mix well, take out and transfer to a centrifuge tube, 28°C, 120 rpm shaking incubation for 1 h; after the reaction is completed, drain the solution, rinse the filler with 3 times the column volume of coupling solution (50 mM Tris, 5 mM EDTA-Na, pH 8.5), and dry; add 5 mL of blocking solution (50 mM Tris, 5 mM EDTA-Na, 50 mM L-cysteine, pH 8.5), mix well, take out and transfer to a centrifuge tube, 28°C shaking incubation for 1 h; finally, dry the blocking solution, and wash the filler with 3 times the column volume of pH 8.0, 20 mM PBS, and store with 20% ethanol.

[0073] Take 1 mL of the synthesized adsorbent filler and load it into a chromatography column, equilibrate with 5 mL of PBS solution, then add 500 pg / mL of IL-6, 1000 pg / mL of IL-1β, and 10000 pg / mL of TNF-α to 5 mL of human plasma,

[0074] Load, then equilibrate with 5 mL of PBS solution, collect the flow-through solution, and measure the content of each inflammatory factor in the plasma stock solution and the flow-through solution using IL-6, IL-1β, and TNF-α kits (BD company), and calculate the clearance rate. Take the blank agarose GESepharose 6FF as a control. The results are shown in Tables 1-3.

[0075] Table 1 Adsorption performance determination of adsorbent 1

[0076]

[0077] Table 2 Adsorption performance determination of adsorbent 2

[0078]

[0079] Table 3 Adsorption performance determination of adsorbent 3

[0080]

[0081] From the data in Tables 1-3, it can be seen that the adsorption effects of the three adsorbents on IL-6 and IL-1β inflammatory factors are similar, with little difference. Adsorbent 1 has better adsorption effect on TNF-α, higher than adsorbent 2 and adsorbent 3.

[0082] The amino acid sequences and numbering involved in the present invention are as follows, in each sequence, the complementarity determining regions are marked with underlines.

[0083] IL-6

[0084] SEQ1, SEQ ID NO: 1:

[0085] QVQLVESGGGLVHPGDSLSCVASGLTFS NNAMA WFRQVPGKEREFVA ISRRGGSGSEYYADSVRS RFTISRDNAKNTVYLQADSLKPTDTAVYYCAA GYDNYDY WGQGTRVTVSS

[0086] SEQ2, SEQ ID NO: 2:

[0087] QVQLVESGGGLVQPGGSLSCAASGLTFS GGGGSGGGGSGGGGS WFRQVPGKEREFVA TSNGA RFTISRDNGKNTVYLQADNLRPTDTAVYYCAA SDFNWDSDQAKN WGQGTRVTVSS

[0088] SEQ3, SEQ ID NO: 3:

[0089] QVQLVESGGGLVQAGDSLSCAASGLTFS NEFAYSRDATVYDQ WFRQVPGKEREFVA GGGGSGGGGSGGGGS RFTISRDNGKNTVYLQADNLRPTDTAVYYCAA EQWMY WGQGTRVTVSS

[0090] IL-1β

[0091] SEQ4, SEQ ID NO: 4:

[0092] QVQLVESGGGLVQAGDSLSCVASGLTFS VINTNGLITKYPDSAEG WFRQVPGKDREFVA SVLGLQGFT RFTISRDNAKNTVYLQADNLTPTDTAVYYCAA GSFMG WGQGTRVTVSS

[0093] SEQ5, SEQ ID NO: 5:

[0094] QVQLVESGGGLVQPGGSLSCAASGLTVS DIMGSRYTNYYVDSVKGWFRQVPGKEREFVA SGLLRRGYDY RFTISRDNAKNTVTLQADNLKPTDTAVYYCAA GGGGSGGGGSGGGGS WGQGTRVTVSS

[0095] SEQ6, SEQ ID NO: 6:

[0096] QVQLVESGGGSVQPGGSLRLSCAASGRTFS FQSGM WFRQAPGKEREFVS VITYDDSSFKSAKG RFTISRDDAKNTVYLQMNSLRPEDTAVYYCAA SAYYWSKEANLYHD WGQGTQVTVSS

[0097] TNF -a

[0098] SEQ7, SEQ ID NO: 7:

[0099] QVQLVESGGGLVQPGGSLKLSCAVSGLTFS GGGGSGGGGSGGGGS WFRQVPGKEREFVA YIMGS RFTISRDNAKNTVYLQADSLKPTDTAVYYCAA EINNTGAITKYPDSAKG WGQGTRVTVSS

[0100] SEQ8, SEQ ID NO: 8:

[0101] QVQLVESGGGLVQAGDSLRLSCAASGLTFS SPSASERES WFRQVPGKEREFVA SALYKY RFTISRDNGKNTVYLQADNLRPTDTAVYYCAA INNPMG WGQGTRVTVSS

[0102] SEQ9, SEQ ID NO: 9:

[0103] QVQLVESGGGLVQPGGSLRLSCAASGLTVS DIMGPGGYYADSVDT WFRQVPGKEREFVA VKSDERPGYDY RFTISRDNAKNTVTLQADNLKPTDTAVYYCAA GGGGSGGGGSGGGGS WGQGTRVTVSS

[0104] SEQ10, SEQ ID NO: 10:

[0105] QVQLVESGGGLVHPGDSLRLSCVASGLTSFSYTSGMG WFRQVPGKEREFVA ALTDDKNRSFYWDSVRN RFTISRDNAKNTVYLQADSLKPTDTAVYYCAA ARSTGYSRNPTAY WGQGTRVTVSS GGGGSGGGGSGGGGS QVQLVESGGGLVQAGDSLRLSCVASGLTFS DTWIY WFRQVPGKDREFVA EITLITKQPDSAKG RFTISRDNAKNTVYLQADNLTPTDTAVYYCAA SLLGQSPSA WGQGTRVTVSS FNYDH QVQLVESGGGLVQPGGSLKLSCAVSGLTFS NNAMA WFRQVPGKEREFVA ISRRGGSGSEYYADSVRS RFTISRDNAKNTVYLQADSLKPTDTAVYYCAA GYDNYDY WGQGTRVTVSS

[0106] SEQ11, SEQ ID NO: 11:

[0107] QVQLVESGGGLVQPGGSLRLSCAASGLTFS GSFMG WFRQVPGKEREFVA DIMGSRYTNYYVDSVKG RFTISRDNGKNTVYLQADNLRPTDTAVYYCAA SGLLRRGYDY WGQGTRVTVSS INNPMG QVQLVESGGGLVQPGGSLRLSCAASGLTVS DIMGPGGYYADSVDT WFRQVPGKEREFVA VKSDERPGYDY RFTISRDNAKNTVTLQADNLKPTDTAVYYCAA TSNGA WGQGTRVTVSS SDFNWDSDQAKN QVQLVESGGGLVQAGDSLRLSCAASGLTFS D NEFAYSRDATVYDQ WFRQVPGKEREFVA FQSGM RFTISRDNGKNTVYLQADNLRPTDTAVYYCAA VITYDDSSFKSAKG SAYYWSKEANLYHD WGQGTRVTVSS

[0108] SEQ12, SEQ ID NO: 12:

[0109] QVQLVESGGGLVQAGDSLRLSCAASGLTFS SYTSGMG WFRQVPGKEREFVAALTDDKNRSFYWDSVRN RFTISRDNGKNTVYLQADNLRPTDTAVYYCAA ARSTGYSRNPTAY WGQGTRVTVSS EQWMY QVQLVESGGGSVQPGGSLRLSCAASGRTFS VINTNGLITKYPDSAEG WFRQAPGKEREFVS SVLGLQGFT RFTISRDDAKNTVYLQMNSLRPEDTAVYYCAA DYIMGS WGQGTQVTVSS EINNTGAITKYPDSAKG QVQLVESGGGLVQPGGSLRLSCAASGLTVS SPSASERESSALYKY WFRQVPGKEREFVA DTWIY RFTISRDNAKNTVTLQADNLKPTDTAVYYCAA EITLITKQPDSAKG SLLGQSPSAFNYDH WGQGTRVTVSS

[0110] SEQ 13, SEQ ID NO: 13: ​

[0111] SEQ 14, SEQ ID NO: 14: ​

[0112] SEQ 15, SEQ ID NO: 15: ​

[0113] SEQ 16, SEQ ID NO: 16: ​

[0114] SEQ 17, SEQ ID NO: 17: ​

[0115] SEQ 18, SEQ ID NO: 18: ​

[0116] SEQ 19, SEQ ID NO: 19: ​

[0117] SEQ 20, SEQ ID NO: 20: ​

[0118] SEQ 21, SEQ ID NO: 21: ​

[0119] SEQ 22, SEQ ID NO: 22: ​

[0120] SEQ 23, SEQ ID NO: 23: ​

[0121] SEQ24, SEQ ID NO: 24: ​

[0122] SEQ25, SEQ ID NO: 25: ​

[0123] SEQ26, SEQ ID NO: 26: ​

[0124] SEQ27, SEQ ID NO: 27: ​

[0125] SEQ28, SEQ ID NO: 28: ​

[0126] SEQ29, SEQ ID NO: 29: ​

[0127] SEQ30, SEQ ID NO: 30: ​

[0128] SEQ31, SEQ ID NO: 31: ​

[0129] SEQ32, SEQ ID NO: 32: ​

[0130] SEQ33, SEQ ID NO: 33: ​

[0131] SEQ34, SEQ ID NO: 34: ​

[0132] SEQ35, SEQ ID NO: 35: ​

[0133] SEQ36, SEQ ID NO: 36: ​

[0134] SEQ37, SEQ ID NO: 37: ​

[0135] SEQ38, SEQ ID NO: 38: ​

[0136] SEQ39, SEQ ID NO: 39: ​

[0137] The above is a further detailed description of the present application, which cannot be considered as a limitation of the specific implementation of the present application. For those skilled in the art to which the present application belongs, simple deductions or replacements without departing from the concept of the present application are within the protection scope of the present application.

Claims

1. An antibody adsorbing inflammatory factors, whose structural unit comprises at least one of a nanobody against human IL-1β, a nanobody against human IL-6 and a nanobody against human TNF-α, the different nanobodies being linked by a flexible peptide, characterized in that, Wherein: The complementarity determining regions CDR1~CDR3 of the anti-human IL-1β nanobody are selected from one of the following groups: Group 1: the amino acid sequences of CDR1~CDR3 are respectively: TSNGA, SDFNWDSDQAKN, NEFAYSRDATVYDQ; Group 2: the amino acid sequences of CDR1~CDR3 are respectively: FQSGM, VITYDDSSFKSAKG, SAYYWSKEANLYHD; Group 3: the amino acid sequences of CDR1~CDR3 are respectively: SYTSGMG, ALTDDKNRSFYWDSVRN, ARSTGYSRNPTAY; The complementarity determining regions CDR1~CDR3 of the anti-human IL-6 nanobody are selected from one of the following groups: Group 4: the amino acid sequences of CDR1~CDR3 are respectively: NNAMA, ISRRGGSGSEYYADSVRS, GYDNYDY; Group 5: the amino acid sequences of CDR1~CDR3 are respectively: GSFMG, DIMGSRYTNYYVDSVKG, SGLLRRGYDY; Group 6: the amino acid sequences of CDR1~CDR3 are respectively: INNPMG, DIMGPGGYYADSVDT, VKSDERPGYDY; The complementarity determining regions CDR1~CDR3 of the anti-human TNF-α nanobody are selected from one of the following groups: Group 7: the amino acid sequences of CDR1~CDR3 are respectively: EQWMY, VINTNGLITKYPDSAEG, SVLGLQGFT; Group 8: the amino acid sequences of CDR1~CDR3 are respectively: DYIMGS, EINNTGAITKYPDSAKG, SPSASERESSALYKY; Group 9: the amino acid sequences of CDR1~CDR3 are respectively: DTWIY, EITLITKQPDSAKG, SLLGQSPSAFNYDH.

2. The antibody of claim 1, wherein the antibody is an antibody that adsorbs an inflammatory factor. The amino acid sequence of the anti-human IL-1β nanobody is selected from one of the following: SEQ4: QVQLVESGGGLVQAGDSLRLSCVASGLTFSTSNGAWFRQVPGKDREFVASDFNWDSDQAKNRFTISRDNAKNTVYLQADNLTPTDTAVYYCAANEFAYSRDATVYDQWGQGTRVTVSS SEQ5: QVQLVESGGGLVQPGGSLRLSCAASGLTVSFQSGMWFRQVPGKEREFVAVITYDDSSFKSAKGRFTISRDNAKNTVTLQADNLKPTDTAVYYCAASAYYWSKEANLYHDWGQGTRVTVSS SEQ6: QVQLVESGGGS VQPGGSLRLSCAASGRTFSSYTSGMGWFRQAPGKEREFVSALTDDKNRSFYWDSVRNRFTISRDDAKNTVYLQMNSLRPEDTAVYYCAAARSTGYSRNPTAYWGQGTQVTVSS.

3. The antibody of claim 1, wherein the antibody is an antibody that adsorbs an inflammatory factor. The amino acid sequence of the anti-human IL-6 Nanobody is selected from one of the following: SEQ1: QVQLVESGGGLVHPGDSLRLSCVASGLTSFNNAMAWFRQVPGKEREFVAISRRGGSGSEYYADSVRSRFTISRDNAKNTVYLQADSLKPTDTAVYYCAAGYDNYDYWGQGTRVTVSS SEQ2: QVQLVESGGGLVQPGGSLRLSCAASGLTFSGSFMGWFRQVPGKEREFVADIMGSRYTNYYVDSVKGRFTISRDNGKNTVYLQADNLRPTDTAVYYCAASGLLRRGYDYWGQGTRVTVSS SEQ3: QVQLVESGGGLVQAGDSLRLSCAASGLTFSINNPMGWFRQVPGKEREFVADIMGPGGYYADSVDTRFTISRDNGKNTVYLQADNLRPTDTAVYYCAAVKSDERPGYDYWGQGTRVTVSS.

4. The antibody of claim 1, wherein the antibody is capable of adsorbing an inflammatory factor. The amino acid sequence of the anti-human TNF-a Nanobody is selected from one of the following: SEQ7: QVQLVESGGGLVQPGGSLKLSCAVSGLTFSEQWMYWFRQVPGKEREFVAVINTNGLITKYPDSAEGRFTISRDNAKNTVYLQADSLKPTDTAVYYCAASVLGLQGFTWGQGTRVTVSS SEQ8: QVQLVESGGGLVQAGDSLRLSCAASGLTFSDYIMGSWFRQVPGKEREFVAEINNTGAITKYPDSAKGRFTISRDNGKNTVYLQADNLRPTDTAVYYCAASPSASERESSALYKYWGQGTRVTVSS SEQ9: QVQLVESGGGLVQPGGSLRLSCAASGLTVSDTWIYWFRQVPGKEREFVAEITLITKQPDSAKGRFTISRDNAKNTVTLQADNLKPTDTAVYYCAASLLGQSPSAFNYDHWGQGTRVTVSS.

5. The antibody of any one of claims 1 to 4, wherein the antibody is capable of adsorbing an inflammatory factor. It is a multivalent antibody resulting from the fusion of one anti-human IL-1 b Nanobody, one anti-human IL-6 Nanobody and one anti-human TNF-a Nanobody.

6. The antibody of claim 5, wherein the antibody is an antibody that adsorbs an inflammatory factor. The amino acid sequence of the multivalent antibody is shown in SEQ ID NO: 10, SEQ ID NO: 11, or SEQ ID NO:

12.

7. Use of the antibody adsorbing an inflammatory factor according to any one of claims 1 to 6, for: preparing an adsorbent for an inflammatory factor; preparing a test reagent for an inflammatory factor; preparing a separation or purification reagent for an inflammatory factor; the inflammatory factor is IL-1β, IL-6, or TNF-α.

8. Use according to claim 7, characterized in that, the adsorbent is a solid support to which the antibody adsorbing an inflammatory factor according to any one of claims 1 to 6 is coupled.

9. A gene encoding the antibody adsorbing an inflammatory factor according to any one of claims 1 to 6.

10. An expression system expressing the antibody adsorbing an inflammatory factor according to any one of claims 1 to 6, or containing the gene according to claim 9.

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