Single-domain antibodies against tnfα and uses thereof

Abstract

The application belongs to the field of immunology and relates to a single-domain antibody against TNFα and use thereof. The single-domain antibody is composed of a heavy chain, the heavy chain comprising a heavy chain CDR1 shown in any one of SEQ ID NO:48-SEQ ID NO:55, a heavy chain CDR2 shown in any one of SEQ ID NO:56-SEQ ID NO:63 and a heavy chain CDR3 shown in any one of SEQ ID NO:64-SEQ ID NO:71. Compared with the prior art, the application has the beneficial effect that the single-domain antibody specific to TNFα is screened by using biological genetic engineering technology, the antibody has good affinity, can block the release of cytokines by specific cells, has good binding activity through prokaryotic expression and eukaryotic expression, and has certain drugability.

Description

Technical Field

[0001] The present invention relates to single-domain antibodies capable of specifically binding to TNFα, pharmaceutical compositions containing the single-domain antibody as an active ingredient, and therapeutic uses thereof Background Art

[0002] Psoriasis is a relatively common chronic inflammatory skin disease, commonly known as psoriasis. The disease is prone to relapse or aggravation in winter, and mostly remits in spring and autumn, with a global prevalence of about 2% to 3%. About one-third of psoriasis patients suffer from psoriatic arthritis (PsA), which is often accompanied by joint swelling and pain, stiffness and movement disorder. Some cases may involve the spine, and severe cases can lead to disability, which seriously affects the physical and mental health of patients. Psoriasis is classified according to its clinical characteristics, mainly into the following types: vulgaris, arthritic, pustular and erythrodermic. More than 90% of psoriasis is vulgaris, and other types of the disease are mostly caused by sudden drug withdrawal when patients use topical irritating drugs, overuse glucocorticoids and immunosuppressants during treatment.

[0003] The pathogenesis of psoriasis is still unclear. Currently, the main therapeutic regimens for PsA include non-steroidal anti-inflammatory drugs, glucocorticoids, anti-rheumatic drugs, azathioprine, retinoic acids, as well as physical therapy, traditional Chinese medicine therapy and other methods to alleviate symptoms and control the disease. Due to the poor efficacy and adverse reactions of traditional therapies, for example, long-term use of hormones will lead to various side effects (elevated blood pressure, elevated blood sugar, osteoporosis, digestive tract ulcer, skin atrophy, etc.), clinical practice has gradually inclined to research on biological agents. Tumor necrosis factor inhibitors are often selected as the first-line biological therapy for PsA patients, and anti-IL-17 biological agents are also available.

[0004] Psoriasis takes excessive proliferation of keratinocytes, infiltration of inflammatory cells and formation of new blood vessels as the three key elements of its histopathological changes. Patients have functional abnormalities such as multiple immune cells, immune molecules and intracellular signal transduction systems. The disease has a certain incubation period, and taking antimalarial drugs, antipsychotic lithium preparations, antihypertensive β-blockers and angiotensin-converting enzyme inhibitors during this period can all induce the occurrence of the disease, which seriously affects the quality of life and even the physical and mental health of patients.

[0005] The pathogenesis of psoriasis is still unclear. It is currently considered as an autoimmune disorder disease under a polygenic genetic background. Its onset is related to immunity mediated by T lymphocytes, mainly CD4+Th1 lymphocytes. The pathogenic process includes the activation of initial T lymphocytes into memory-effector T lymphocytes, which enter the circulation, migrate to the skin, accumulate at the lesion site, and secrete a variety of cytokines to exert various biological functions and cause the disease.

[0006] TNF-α (also known as TNFα) is a cytokine with broad biological activity. TNF-α accounts for 70%–95% of the total activity of the TNF family, which includes TNF-α, TNF-β, and TNF-γ. Under abnormal conditions, especially elevated levels, it can lead to immunopathological responses such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, and Crohn's disease. Studies have found that TNF-α levels in the skin lesions, serum, synovial membrane of joints, or cell culture supernatant of psoriasis patients are significantly higher than in normal controls. These levels decrease to varying degrees after treatment, and the severity of psoriasis is positively correlated with TNF-α levels, suggesting that TNF-α plays an important role in the pathogenesis of psoriasis.

[0007] TNF-α is mainly secreted by macrophages. Other cell types, such as lymphocytes, smooth muscle cells, and fibroblasts, can also produce and release TNF-α under certain conditions. When the immunopathological mechanism of psoriasis is activated, both Th1 and Th2 cells in T lymphocytes can produce TNF-α, enabling it to exert its pathogenic biological effects. TNF-α is known to kill or inhibit tumor cells and has anti-infective effects. It can also participate in inflammatory responses and promote cell proliferation and differentiation, the latter two of which play important roles in the pathogenesis of psoriasis.

[0008] Psoriasis, due to its stubborn and difficult-to-treat nature, is considered a major research topic in the field of dermatology worldwide and is one of the key skin diseases targeted for prevention and treatment globally. With the availability of a series of new biological drugs, psoriasis patients have more and better medication options. In the field of autoimmune diseases, TNFα antagonists and interleukin (IL) drugs are currently hot topics in research.

[0009] Currently, there is a lack of single-domain antibody products against TNFα with strong affinity and pharmaceutical value in existing technologies. Therefore, providing a single-domain antibody against TNFα with high stability and good efficacy is one of the technical problems that urgently needs to be solved in this field. Summary of the Invention

[0010] The purpose of this invention is to provide a single-domain antibody that can specifically bind to TNFα and its uses.

[0011] A first aspect of the present invention provides a single-domain antibody against TNFα, said single-domain antibody being composed of heavy chains, the heavy chains including heavy chain CDR1 shown in any one of SEQ ID NO:48-SEQ ID NO:55, heavy chain CDR2 shown in any one of SEQ ID NO:56-SEQ ID NO:63, and heavy chain CDR3 shown in any one of SEQ ID NO:64-SEQ ID NO:71.

[0012] Preferably, the amino acid sequences of the heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3 are one of the following (1)-(9): (1) CDR1 shown in SEQ ID NO:55, CDR2 shown in SEQ ID NO:57, and CDR3 shown in SEQ ID NO:65; (2) CDR1 shown in SEQ ID NO:50, CDR2 shown in SEQ ID NO:60, and CDR3 shown in SEQ ID NO:70; (3) CDR1 shown in SEQ ID NO:49, CDR2 shown in SEQ ID NO:62, and CDR3 shown in SEQ ID NO:64; (4) CDR1 shown in SEQ ID NO:54, CDR2 shown in SEQ ID NO:58, and CDR3 shown in SEQ ID NO:68; (5) CDR1 shown in SEQ ID NO:53, CDR2 shown in SEQ ID NO:59, and CDR3 shown in SEQ ID NO:67; (6) CDR1 shown in SEQ ID NO:50, CDR2 shown in SEQ ID NO:61, and CDR3 shown in SEQ ID NO:70; (7) CDR1 shown in SEQ ID NO:52, CDR2 shown in SEQ ID NO:63, and CDR3 shown in SEQ ID NO:66; (8) CDR1 shown in SEQ ID NO:51, CDR2 shown in SEQ ID NO:61, and CDR3 shown in SEQ ID NO:69; (9) CDR1 shown in SEQ ID NO:48, CDR2 shown in SEQ ID NO:56, and CDR3 shown in SEQ ID NO:71.

[0013] The above CDR combination (1) corresponds to SEQ ID NO.1, CDR combination (2) corresponds to SEQ ID NO.2, CDR combination (3) corresponds to SEQ ID NO.3 and SEQ ID NO.4, and CDR combinations (4)-(9) correspond to SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.10 respectively.

[0014] All of the above sequences can be replaced with sequences that have "at least 80% homology" or sequences that replace only one or a few amino acids; preferably "at least 85% homology", more preferably "at least 90% homology", even more preferably "at least 95% homology", and most preferably "at least 98% homology".

[0015] In one embodiment, in any one or more CDRs of the heavy chain CDR1, CDR2, and CDR3, one to five arbitrary amino acid residues may be substituted with their conserved amino acids. Specifically, in the heavy chain CDR1, one to five amino acid residues may be substituted with their conserved amino acids; in the heavy chain CDR2, one to five amino acid residues may be substituted with their conserved amino acids; and in the heavy chain CDR3, one to five amino acid residues may be substituted with their conserved amino acids.

[0016] The "single-domain antibody against TNFα" of this invention includes not only complete antibodies but also fragments, derivatives, and analogs of said antibodies. As used herein, the terms "fragment," "derivative," and "analyte" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this invention. The polypeptide fragments, derivatives, or analogs of this invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with an Fc tag). Based on the teachings herein, these fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0017] In a preferred embodiment, the antibody sequence further includes a framework region FR; the framework region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the framework region FR are as follows: The FR1 or a variant of FR1 shown in any of SEQ ID NO:21-27, wherein the variant of FR1 contains a substitution of up to 5 amino acids in the FR1; The FR2 or a variant thereof shown in any one of SEQ ID NO:28-36, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR2; FR3 or a variant thereof shown in any one of SEQ ID NO:37-44, wherein the variant thereof contains a substitution of up to 5 amino acids in the FR3; The FR4 or a variant thereof shown in any one of SEQ ID NO:45-47, wherein the variant thereof contains substitutions of up to 5 amino acids.

[0018] A second aspect of the present invention is to provide the amino acid sequences of single-domain antibodies against TNFα, wherein the amino acid sequences of the single-domain antibodies are shown in SEQ ID NO. 1-10, or the single-domain antibodies have at least 80% (more preferably at least 95%) sequence homology with the amino acid sequences of SEQ ID NO. 1-10.

[0019] In one embodiment, the anti-TNFα single-domain antibody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with an amino acid sequence selected from SEQ ID NO: 1-10, and is capable of specifically binding to the TNFα protein.

[0020] A third aspect of the present invention is to provide a bispecific antibody comprising a first antigen-binding portion and a second antigen-binding portion. The first antigen-binding portion is a single-domain antibody, wherein the amino acid sequence of the single-domain antibody is as shown in SEQ ID NO. 1-10 or has at least 80% (preferably at least 95%) homology with SEQ ID NO. 1-10. The second antigen-binding portion is another antibody, for example, an antibody against IL-17A, IL-6R, IL-6, IL-23, or IL-23R (which can be a monoclonal antibody, polyclonal antibody, single-domain antibody, or any other form of antibody); the second antigen-binding portion may also be an antibody against other antigens, for application in the diagnosis, prevention, or treatment of diseases or the detection of antigens.

[0021] A fourth aspect of the present invention is the use of any of the aforementioned anti-TNFα single-domain antibodies in the preparation of bispecific antibodies.

[0022] A fifth aspect of the present invention is to provide an Fc fusion antibody or humanized antibody of any of the aforementioned anti-TNFα single-domain antibodies.

[0023] A sixth aspect of the present invention is to provide nucleotide molecules encoding the aforementioned anti-TNFα single-domain antibody, the aforementioned Fc fusion antibody, or the aforementioned humanized antibody, the nucleotide sequences of which are shown in SEQ ID NO: 11-20, or have at least 95% sequence homology with SEQ ID NO: 11-20.

[0024] In one embodiment, the nucleic acid molecule encoding the single-domain antibody against TNFα has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence homology with nucleotide sequences selected from SEQ ID NO: 11-20, and the single-domain antibody against TNFα encoded therein is capable of specifically binding to the TNFα protein.

[0025] A seventh aspect of the present invention is to provide an expression vector comprising a nucleotide molecule encoding a single-domain antibody, an Fc fusion antibody, or a humanized antibody against TNFα, the nucleotide sequences of which are shown in SEQ ID NO: 11-20, respectively.

[0026] In a preferred embodiment, the expression vector used is RJK-V4-3 (integrated into RJK-V4-3 by genetic engineering means of a nucleotide molecule encoding a single-domain antibody against TNFα or its Fc fusion antibody or humanized antibody), and other general-purpose expression vectors may be selected as needed.

[0027] An eighth aspect of the present invention is to provide a host cell capable of expressing the aforementioned anti-TNFα single-domain antibody, or an expression vector comprising the aforementioned thereof. Preferably, the host cell is a bacterial cell, a fungal cell, or a mammalian cell.

[0028] In another preferred embodiment, the host cell includes prokaryotic or eukaryotic cells, including bacteria and fungi.

[0029] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0030] In another preferred embodiment, the prokaryotic cells are selected from the group consisting of Escherichia coli, Bacillus subtilis, lactic acid bacteria, Streptomyces, Proteus mirabilis, or combinations thereof.

[0031] In another preferred embodiment, the eukaryotic cells are selected from the group consisting of Pichia pastoris, Saccharomyces cerevisiae, Schizosomalidomiae, Trichoderma, or combinations thereof.

[0032] In another preferred embodiment, the eukaryotic cells shown are selected from the group consisting of insect cells such as armyworms, plant cells such as tobacco, BHK cells, CHO cells, COS cells, myeloma cells, or combinations thereof.

[0033] In another preferred embodiment, the host cell is a suspension ExpiCHO-S cell.

[0034] In another preferred embodiment, the host cell is a suspension 293F cell.

[0035] A ninth aspect of the present invention is to provide a recombinant protein comprising the aforementioned anti-TNFα single-domain antibody. The recombinant protein may be a single-domain antibody as shown in SEQ ID NO. 1-10, or a single-domain antibody having at least 80% homology with SEQ ID NO. 1-10, or a multi-epitope antibody, a multi-specific antibody, or a multivalent antibody; for example, the multi-epitope antibody may consist of more than one sequence from SEQ ID NO. 1-10; the multivalent antibody may consist of one sequence from SEQ ID NO. 1-10 repeated a certain number of times; the multi-specific antibody includes, but is not limited to, the aforementioned bispecific antibody and trispecific antibody; furthermore, the recombinant protein may be a fragment, derivative, or analog of the aforementioned antibody.

[0036] A tenth aspect of the invention is to provide a pharmaceutical composition comprising the aforementioned anti-TNFα single-domain antibody and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally determined based on the isoelectric point of the antibody (the pH of the aqueous carrier medium must deviate from the isoelectric point of the antibody by approximately 2). The formulated pharmaceutical composition can be administered via conventional routes.

[0037] The pharmaceutical compositions of this invention can be directly bound to TNFα protein molecules, and therefore can be used to treat various conditions related to abnormal TNFα expression, such as rheumatoid arthritis, osteoarthritis, psoriasis, inflammatory bowel disease, and Kraton disease. Furthermore, other therapeutic agents can be used concurrently.

[0038] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described single-domain antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. Furthermore, the pharmaceutical compositions of the present invention can be used in conjunction with other therapeutic agents.

[0039] The eleventh aspect of the present invention is to provide a medicament for treating a condition associated with abnormal TNFα expression, comprising the aforementioned anti-TNFα single-domain antibody as an active ingredient.

[0040] In a preferred embodiment, the condition is an autoimmune disease.

[0041] In a preferred embodiment, the conditions associated with abnormal TNFα expression include psoriasis, rheumatoid arthritis, osteoarthritis, inflammatory bowel disease, or Krant's disease. Psoriasis, in particular.

[0042] A twelfth aspect of the present invention is to provide a kit for detecting TNFα levels, comprising the aforementioned anti-TNFα single-domain antibody. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc.

[0043] In a preferred embodiment, the kit includes an antibody that recognizes TNFα protein, a lysis medium for dissolving the sample, and universal reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit can be an in vitro diagnostic device.

[0044] In a preferred embodiment, the kit further contains a second antibody and an enzyme or fluorescent or radiolabeled marker for detection, as well as a buffer solution.

[0045] In a preferred embodiment, the second antibody in the kit is an antibody (as an anti-antibody) of the aforementioned anti-TNFα single-domain antibody, which can be a single-domain antibody, monoclonal antibody, polyclonal antibody, or any other form of antibody.

[0046] In a thirteenth aspect of the present invention, a method for generating a single-domain antibody against TNFα is provided, comprising the steps of: (a) Culturing the host cells described in the eighth aspect of the invention under conditions suitable for generating single-domain antibodies, thereby obtaining a culture containing the single-domain antibody against TNFα; and (b) Isolating or recovering the single-domain antibody against TNFα from the culture; and (c) Optionally, purify and / or modify the single-domain antibody against TNFα obtained in step (b).

[0047] The fourteenth aspect of the present invention is to provide the use of the aforementioned anti-TNFα single-domain antibody or pharmaceutical composition in the preparation of a medicament for inhibiting TNFα gene expression or an anti-psoriasis medicament.

[0048] The fifteenth aspect of the present invention is to provide the use of the aforementioned anti-TNFα single-domain antibody or the aforementioned pharmaceutical composition in the preparation of a medicament for treating a disease.

[0049] In a preferred embodiment, the disease is a condition associated with abnormal TNFα expression.

[0050] In a preferred embodiment, the disease is an autoimmune disease.

[0051] In a preferred embodiment, the disease is psoriasis.

[0052] Compared with the prior art, the beneficial effects of the present invention are: The single-domain antibody of this invention specifically targets the TNFα protein with the correct spatial structure.

[0053] The single-domain antibody of this invention exhibits good specificity, can inhibit the expression of TNFα protein, and has higher blocking activity than existing drugs. It shows great promise for application in the preparation of drugs for treating autoimmune diseases.

[0054] The single-domain antibody of this invention offers flexible expression system options, allowing expression in prokaryotic systems as well as eukaryotic systems such as yeast cells or mammalian cells. Furthermore, its expression cost in prokaryotic systems is low, which can reduce subsequent production costs.

[0055] The single-domain antibody of the present invention has simple multi-combination modification. It can be obtained by simple tandem through genetic engineering to obtain multivalent and multispecific antibodies. Moreover, it has very low immune heterogeneity and will not produce a strong immune response without humanization modification.

[0056] The single-domain antibody of the present invention has a wider affinity range, ranging from nM to pM before affinity maturation, providing multiple options for antibodies for different applications later. Attached Figure Description

[0057] To more clearly illustrate the technical solution of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0058] Figure 1 The enrichment status of the library targeting TNFα in Example 3; Figure 2 This is a graph showing the dose-response curve of antibody-antigen binding in Example 13; Figure 3 This is a graph showing the experimental results of antibody (eukaryotic sample) neutralizing human TNFα to induce IL-6 release in HeLa cells in Example 14; Figure 4 This is a graph showing the experimental results of antibody (humanized sample) neutralizing human TNFα to induce IL-6 release in HeLa cells in Example 15. Detailed Implementation

[0059] The present invention will be further described in detail below with reference to embodiments, so that those skilled in the art can implement it based on the description.

[0060] Single-domain antibodies (sdAbs, also referred to as nanobodies or VHHs by the developer Ablynx) are well known to those skilled in the art. A single-domain antibody is an antibody whose complementarity-determining region is part of a single-domain polypeptide. Therefore, a single-domain antibody contains a single complementarity-determining region (a single CDR1, a single CDR2, and a single CDR3). Examples of single-domain antibodies include antibodies containing only the heavy chain (which naturally does not contain a light chain), single-domain antibodies derived from conventional antibodies, and engineered antibodies.

[0061] Single-domain antibodies can be derived from any species, including mice, humans, camels, llamas, goats, rabbits, and cattle. For example, naturally occurring VHH molecules can be derived from antibodies provided by camelid species such as camels, dromedaries, llamas, and guanacos. Like complete antibodies, single-domain antibodies can selectively bind to specific antigens. Single-domain antibodies may contain only variable domains of the immunoglobulin chain, which have CDR1, CDR2, and CDR3, as well as a frame region.

[0062] As used herein, the term "sequence homology" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is typically expressed as a percentage. Preferably, homology is determined over the overall length of the sequences being compared. Thus, two copies having completely identical sequences have 100% homology.

[0063] In this invention, sequences with high homology to the CDR1-3 sequences disclosed herein can also yield single-domain antibodies against TNFα. In some embodiments, sequences having "at least 80% homology," or "at least 85% homology," "at least 90% homology," "at least 95% homology," or "at least 98% homology" with the sequences in SEQ ID NO: 1-10 can achieve the purpose of the invention.

[0064] In some embodiments, the inventive objective can also be achieved by replacing only one or a few amino acids compared to the sequences in SEQ ID NO: 1-10, for example, by including 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conserved amino acid substitutions. In practice, when determining the degree of sequence homology between two amino acid sequences or when determining the combination of CDR1, CDR2, and CDR3 in a single-domain antibody, those skilled in the art can consider so-called “conserved” amino acid substitutions. In the case of substitution, the substitution will preferably be a conserved amino acid substitution, which can generally be described as an amino acid residue substituted by another amino acid residue having a similar chemical structure, and the substitution has little or no effect on the function, activity, or other biological properties of the polypeptide. The conserved amino acid substitutions are common in the art. For example, a conserved amino acid substitution is the substitution of one or a few amino acids in the following groups (a)-(d) by another or a few amino acids in the same group: (a) polar negatively charged residues and their non-charged amides: Asp, Asn, Glu, Gln; (b) polar positively charged residues: His, Arg, Lys; (c) aromatic residues: Phe, Trp, Tyr; (d) aliphatic nonpolar or weakly polar residues: Ala, Ser, Thr, Gly, Pro, Met, Leu, Ile, Val, Cys. The particularly preferred conserved amino acid substitutions are as follows: Asp is replaced by Glu; Asn is replaced by Gln or His; Glu is replaced by Asp; Gln is replaced by Asn; His is replaced by Asn or Gln; Arg is replaced by Lys; Lys is replaced by Arg or Gln; Phe is replaced by Met, Leu, or Tyr; Trp is replaced by Tyr; Tyr is replaced by Phe or Trp; Ala is replaced by Gly or Ser; Ser is replaced by Thr; Thr is replaced by Ser; Gly is replaced by Ala or Pro; Met is replaced by Leu, Tyr, or Ile; Leu is replaced by Ile or Val; Ile is replaced by Leu or Val; Val is replaced by Ile or Leu; Cys is replaced by Ser. Furthermore, those skilled in the art will understand that the inventiveness of the single-domain antibody lies in the CDR1-3 regions, while the frame region sequences FR1-4 are not immutable, and the sequences of FR1-4 can adopt conserved sequence variants of the sequences disclosed in this invention.

[0065] As used in this article, the term "Fc fusion antibody" refers to a novel protein produced by fusing the Fc fragment of a target antibody with a biologically active functional protein molecule using genetic engineering techniques.

[0066] The term "humanized antibody" refers to an antibody obtained by fusing the heavy chain variable region of a target antibody (such as an animal antibody) with the constant region of a human antibody, or by transplanting the complementarity-determining region (CDR1-3 sequence) of a target antibody into the variable region of a human antibody, or by mutating the target antibody according to the characteristics of the human antibody backbone region (FR1-4). Humanized antibodies can be produced using synthetic methods or site-directed mutagenesis methods.

[0067] The preferred host cell of the present invention is a bacterial cell, a fungal cell, or a mammalian cell.

[0068] This patent involves preparing a target protein and a truncated form of the target protein using genetic engineering technology. The obtained antigen protein is then used to immunize Bactrian camels in Alashan, Inner Mongolia. After multiple immunizations, peripheral blood lymphocytes or spleen cells of the camels are obtained. Through genetic engineering, the variable region coding sequence of the camel-derived antibody is recombined into a phage display vector. Specific antibodies against the antigen protein are screened using phage display technology, and their ability to bind to the antigen and their application in the treatment of autoimmune diseases are further tested.

[0069] The above technical solution will now be broken down and explained in detail, and described with specific embodiments: Example 1: Preparation of recombinant human TNFα extracellular domain protein: The human recombinant extracellular domain protein used in this patent was expressed and purified by the company itself. The specific design scheme of the human TNFα recombinant extracellular domain protein expression vector is as follows: (1) The coding sequence of TNFα was obtained by searching in NCBI. Its accession number is NM_000594.3, the accession number of the amino acid sequence encoded by this sequence is NP_000585.2, and the Uniprot ID is P01375.

[0070] (2) The amino acid sequence corresponding to NP_000585.2 was analyzed for transmembrane region and extracellular terminus using TMHMM and SMART websites, respectively.

[0071] (3) The analysis results show that the extracellular terminus of TNFα protein is amino acid 57-233.

[0072] (4) The nucleotide sequence encoding amino acids 57-233 of TNFα protein was cloned into the vector pcDNA3.4 using gene synthesis.

[0073] (5) The constructed vector was subjected to Sanger sequencing. After comparing with the original sequence and confirming that there were no errors, the recombinant plasmid was extracted in batches to remove endotoxins. It was then transfected with suspension 293F for expression and purification of the target protein. The purified protein had a purity of up to 90%, which met the needs of animal immunity.

[0074] Example 2: Construction of a single-domain antibody library targeting TNFα protein: One mg of the purified human recombinant TNFα protein obtained in Example 1 was mixed with an equal volume of Freund's complete adjuvant and used to immunize a Bactrian camel from Alashan, Inner Mongolia. The camel was immunized once a week for a total of 7 weeks. Except for the first immunization, the remaining six immunizations were performed by mixing one mg of TNFα protein with an equal volume of Freund's incomplete adjuvant. This immunization process was intended to stimulate the camel to produce antibodies against TNFα protein.

[0075] After animal immunization, 150 mL of peripheral blood lymphocytes were collected from camels, and RNA was extracted from the cells. cDNA was synthesized using the extracted total RNA, and VHH (antibody heavy chain variable region) was amplified using nested PCR with the cDNA as a template.

[0076] Then, the pMECS vector and VHH fragment were digested with restriction endonucleases, and the digested fragments were ligated to the vector. The ligated fragments were electroporated into competent TG1 cells to construct a phage display library of TNFα protein, and the library size was determined to be approximately 1 × 10⁻⁶. 9 Meanwhile, the correct insertion rate of the target fragment in the library was detected by colony PCR.

[0077] The results showed that the correct insertion rate for the constructed TNFα library was 97%.

[0078] Example 3: Screening for single-domain antibodies against TNFα protein: Take 200 μL of the recombinant TG1 cells from Example 2 and culture them in 2×TY medium. During the culture, add 40 μL of helper phage VCSM13 to infect the TG1 cells and culture them overnight to amplify the phage. The next day, precipitate the phage with PEG / NaCl and collect the amplified phage by centrifugation.

[0079] 500 μg of TNFα protein diluted in 100 mM pH 8.3 NaHCO3 was coupled onto an ELISA plate and incubated overnight at 4°C. A negative control well (culture medium control) was also included. The next day, 200 μL of 3% skim milk was added, and the plate was blocked at room temperature for 2 hours. After blocking, 100 μL of the amplified phage library (approximately 2 × 10⁻⁶) was added. 11 (1 phage particle), incubate at room temperature for 1 hour; after 1 hour, wash 15 times with PBS + 0.05% Tween-20 to remove unbound phage.

[0080] Phages specifically bound to TNFα protein were dissociated using trypsin at a final concentration of 25 mg / mL and then used to infect *E. coli* TG1 cells in logarithmic growth phase. After incubation at 37°C for 1 hour, phages were generated and collected for the next round of screening. This screening process was repeated once to gradually enrich the phages. The enrichment profile is shown below. Figure 1 As shown.

[0081] Example 4: Screening for specific positive clones against TNFα using phage enzyme-linked immunosorbent assay (ELISA): Following the screening method described in Example 3 above, three rounds of screening were performed on single-domain antibodies against TNFα protein. The phage enrichment factor against TNFα protein reached 10 or higher. After screening, 384 single colonies were selected from the positive clones and inoculated into 96-well plates containing 100 μg / mL ampicillin in 2×TY medium. A blank control was also set up. After incubation at 37°C to the logarithmic phase, IPTG was added to a final concentration of 1 mM and incubated overnight at 28°C.

[0082] Crude antibody was obtained using the osmotic burst method. TNFα recombinant protein was released into 100 mM NaHCO3 (pH 8.3), and 100 μg of protein was coated overnight at 4°C in an ELISA plate. 100 μL of the obtained crude antibody extract was transferred to an ELISA plate containing the antigen and incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and 100 μL of Mouse Anti-HA tag Antibody (HRP) (Mouse anti-HA horseradish peroxidase labeled antibody, ThermoFisher) diluted 1:2000 was added. The plate was incubated at room temperature for 1 h. Unbound antibody was washed away with PBST, and horseradish peroxidase chromogenic solution was added. The reaction was carried out at 37°C for 15 min, and then stop solution was added. The absorbance was read at 450 nm using an ELISA reader.

[0083] When the OD value of the sample well is more than 5 times that of the control well, it is determined to be a positive clone well. The bacteria in the positive clone well are transferred to LB medium containing 100 μg / mL ampicillin for plasmid extraction and sequencing.

[0084] Gene sequences of each clone were analyzed using the sequence alignment software VectorNTI. Clones with identical CDR1, CDR2, and CDR3 sequences were considered the same clone, while clones with different sequences were considered different clones. This resulted in the acquisition of single-domain antibodies specifically targeting TNFα protein (SEQ ID NO. 1-10 and other single-domain antibodies with sequences not shown, including those not shown). Figure 1-4The examples shown include all single-domain antibodies except 1B2, 1D4, 1E9, 1H10, 2B1, 2B8, 2F3, 4A5, 4C11, and 4C3, such as 1B11 and 1B3.

[0085] The amino acid sequence of the antibody is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, constituting the entire VHH. The obtained single-domain antibody recombinant plasmid can be expressed in a prokaryotic system to ultimately obtain the single-domain antibody protein.

[0086] The CDR and FR sequences of the 10 single-domain antibodies are shown in Tables 1-7. The amino acid sequences of the 10 single-domain antibodies are shown in SEQ ID NO. 1-10, and the DNA sequences encoding the 10 single-domain antibodies are shown in SEQ ID NO. 11-20.

[0087] Table 1. CDR1 sequences of 10 single-domain antibodies Table 2. CDR2 sequences of 9 single-domain antibodies Table 3. CDR3 sequences of 10 single-domain antibodies Table 4. FR1 sequences of 10 single-domain antibodies Table 5. FR2 sequences of 10 single-domain antibodies Table 6. FR3 sequences of 10 single-domain antibodies Table 7. FR4 sequences of 10 single-domain antibodies The amino acid sequences of the single-domain antibodies SEQ ID NO.1-10 correspond one-to-one with the single-domain antibodies 1B2, 1D4, 1E9, 1H10, 2B1, 2B8, 2F3, 4A5, 4C11, and 4C3, respectively.

[0088] The nucleotide sequences of the single-domain antibodies SEQ ID NO.11-20 also correspond one-to-one with the single-domain antibodies 1B2, 1D4, 1E9, 1H10, 2B1, 2B8, 2F3, 4A5, 4C11, and 4C3, respectively.

[0089] Example 5: Purification and expression of a specific single-domain antibody against TNFα protein in the host bacterium *Escherichia coli*. The plasmid (pMECS-VHH) of different clones obtained from the sequencing analysis in Example 4 was electroporated into *Escherichia coli* HB2151 and plated on LB+amp+glucose culture plates containing ampicillin and glucose, and incubated overnight at 37°C. Single colonies were selected and inoculated into 5 mL of LB medium containing ampicillin and incubated overnight on a shaker at 37°C.

[0090] Inoculate 1 mL of overnight cultured bacteria into 330 mL of TB culture medium and incubate at 37°C in a shaker. When the OD600nm value reaches 0.6-0.9, add 1 M IPTG and incubate overnight at 28°C in a shaker. Centrifuge to collect E. coli and obtain crude antibody extract using the osmotic rupture method. The antibodies were purified by nickel column affinity chromatography. The purified single-domain antibodies were the single-domain antibodies of SEQ ID NO.1-10, namely 1B2, 1D4, 1E9, 1H10, 2B1, 2B8, 2F3, 4A5, 4C11, and 4C3.

[0091] Example 6: Humanization of anti-TNFα single-domain antibody The humanization method employs high-throughput screening of antibody framework region mutation libraries constructed based on big data analysis results. The detailed steps are as follows: (1) Sequence analysis of human / camel antibody data: Amino acid preference analysis was performed on 13,873 Nb (Human) sequences downloaded in batches from the NCBI website, and amino acid preference analysis was also performed on 2,000 nanobody sequences from our company to obtain the amino terminus ratio data of each site in the framework region; (2) Human-Camel-Derived Weighted Analysis: The above-mentioned human / camel-derived antibody sequences were uniformly numbered according to the IMGT numbering rules and matched one by one. Combined with the amino acid ratio analysis results of the two species, a weighted analysis was performed with a weight of 90% human and 10% camel. The weighted proportion of amino acids at each site was calculated and sorted from high to low. Based on the final weighted results, only amino acid species with a proportion >10% were retained at each site in the framework region. The final weight of amino acids with a proportion >10% was calculated based on the standard that the proportion after retention is combined to 1, which serves as the basis for the design of subsequent custom amino acid libraries. (3) Design of the custom amino acid library: For each individual site to be mutated, the number of amino acids in the >10% range is defined as n, and the ratio of the highest to the lowest proportion in the >10% range is defined as V. The site to be mutated is then judged by its properties: if V≥3 and n≤2, the site is considered to be a "high concentration site", otherwise it is considered to be a "medium-low concentration site". Based on this method, the custom amino acid library is divided into two "high / medium-low concentration libraries" and the custom amino acid libraries are constructed separately. The final weight in (2) above is the reference basis for the types and proportions of amino acids in the site in the library.

[0092] (4) High-throughput screening of custom amino acid libraries: Humanized antibody libraries were constructed for antibody strains 1B2, 1D4, 1E9, 1H10, 2B1, 2B8, 2F3, 4A5, 4C11, and 4C3 (i.e., SEQ ID NO. 1-10). The constructed libraries were then panned using their corresponding antigens to obtain antibody sequences with high affinity and a high degree of humanization. For example, the following humanized antibodies were obtained: 1B2-VF (humanized antibody sequence of 1B2), 1E9-VF (humanized antibody sequence of 1E9), 2B1-VF (humanized antibody sequence of 2B1), 4A5-VF (humanized antibody sequence of 4A5), 4C3-VF (humanized antibody sequence of 4C3), and 4E10-VF (humanized antibody sequence of 4E10).

[0093] Example 7: Construction of a eukaryotic expression vector for an Fc fusion antibody against a single domain anti-TNFα antibody (1) Subcloning the target sequence obtained in Example 4 into a eukaryotic expression vector: The antibody screened in Example 4 was obtained by Sanger sequencing to obtain its nucleotide sequence; (2) The above nucleotide sequence (SEQ ID NO:11-20) was synthesized into the vector RJK-V4-3 designed and modified by our company by sequence synthesis to obtain a recombinant eukaryotic expression vector. The modification method of the vector is as described in Example 11. (3) Transform the recombinant eukaryotic expression vector constructed in step (2) into DH5α Escherichia coli, culture it for plasmid extraction, and remove endotoxins; (4) The extracted plasmids were then sequenced and identified. (5) After confirming the recombinant vector, prepare it for subsequent eukaryotic cell transfection and expression. After expressing the Fc protein of VHH using the method in Example 8 or 9, purify the antibody using the method in Example 10.

[0094] Example 8: Expression of Fc fusion antibody of single-domain antibody against TNFα protein in suspension ExpiCHO-S cells (1) Three days before transfection, use 2.5×10 5 / mL cell passage and expansion culture ExpiCHO-S TM Cells, the calculated desired cell volume, were transferred to 120 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium, the cell concentration was increased to approximately 4 × 10⁻⁶ cells / mL. 6 -6×10 6 live cells / mL; (2) One day before transfection, ExpiCHO-S TM Cells were diluted to a concentration of 3.5 × 10⁻⁶. 6 Live cells / mL, incubate cells overnight; (3) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 7 × 10⁻⁶ cells / day before transfection. 6 -10×10 6 live cells / mL; (4) Use fresh ExpiCHO preheated to 37°C TM The expression medium was used to dilute the cells to 6 × 10⁶. 6 viable cells / mL. The calculated desired cell volume was transferred to 100 mL (final volume) of fresh, preheated ExpiCHO. TM In a 500 mL shake flask of expression medium; (5) Gently invert to mix ExpiFectamine TM CHO reagent, using 3.7 mL OptiPRO TM Culture medium for diluting ExpiFectamine TM CHO reagent, vortex or mix well; (6) Use 4 mL of refrigerated OptiPRO TM Dilute the plasmid DNA in the culture medium and vortex to mix. (7) Incubate the ExpiFectamine CHO / plasmid DNA (the plasmid DNA is the eukaryotic expression vector of the Fc fusion antibody of the anti-TNFα single domain antibody prepared in Example 7) complex at room temperature for 1-5 minutes, and then gently add it to the prepared cell suspension while gently shaking the flask during the addition process. (8) The cells were cultured with shaking in humidified air at 37°C and 8% CO2. (9) Add 600ul of ExpiFectamine on the first day after transfection (18-22 hours later). TM CHO Enhancer and 24mLExpiCHO feed.

[0095] (10) Collect the supernatant about 8 days after transfection (when cell viability is less than 70%).

[0096] Example 9: Expression of Fc fusion antibody against anti-TNFα protein single-domain antibody in suspension 293F cells. Experimental procedure for recombinant single-domain antibody expression (using a 500mL shake flask as an example): (1) Three days before transfection, use 2.5×10 5 After passage and expansion of 293F cells at / mL, the calculated desired cell volume was transferred to a 500mL shake flask containing 120mL (final volume) of fresh, preheated OPM-293CD05 Medium to achieve a cell concentration of approximately 2×10⁻⁶. 6 -3×10 6 Live cells / mL.

[0097] (2) On the day of transfection, measure cell density and percentage of viable cells. Cell density should reach approximately 2 × 10⁻⁶ cells / day before transfection. 6 -3×10 6 Live cells / mL.

[0098] (3) Dilute the cells to 1×10⁻⁵ using preheated OPM-293CD05 Medium. 6 1 live cells / mL. Calculate the required cell volume and transfer it to a 500 mL shake flask containing 100 mL (final volume) of fresh, preheated culture medium.

[0099] (4) Dilute PEI (1 mg / mL) reagent with 4 mL Opti-MEM medium, and mix by swirling or pipetting; dilute plasmid DNA (the plasmid DNA is the eukaryotic expression vector of the Fc fusion antibody of the anti-TNFα single-domain antibody prepared in Example 7) with 4 mL Opt-MEM medium, mix by swirling, and filter with a 0.22 μm filter. Incubate at room temperature for 5 min.

[0100] (5) Add the diluted PEI reagent to the diluted DNA and mix by inverting. Incubate the PEI / plasmid DNA complex at room temperature for 15-20 minutes, then gently add it to the prepared cell suspension while gently shaking the flask during the addition process.

[0101] (6) Culture the cells at 37°C, 5% CO2, and 120 rpm with shaking.

[0102] (7) Add 5 mL of OPM-CHO PFF05 feed at 24 h and 72 h after transfection.

[0103] (8) Collect the supernatant about 7 days after transfection (when cell viability is less than 70%).

[0104] Example 10: Purification of single-domain antibody against TNFα protein (1) The protein expression supernatant obtained in Example 8 or 9 was filtered with a 0.45 μm disposable filter to remove insoluble impurities; (2) The above filtrate was purified by affinity chromatography using a protein purifier. The agarose packing material coupled with Protein A was used to purify the filtrate by utilizing the ability of human Fc to bind to Protein A. (3) Pass the filtrate through a pre-packed Protein A column at a flow rate of 1 mL / min. In this step, the target protein in the filtrate will bind to the packing material. (4) Wash the impurity proteins bound to the column with low-salt and high-salt buffers; (5) Use a low pH buffer to systemically bind the target protein on the column; (6) Quickly add the eluent to a Tris-HCl solution with a pH of 9.0 to neutralize it; (7) After dialysis of the above-neutralized protein solution, perform SDS-PAGE analysis to confirm that the protein purity is above 95% and the concentration is above 0.5 mg / mL, and then store it at low temperature for later use.

[0105] Example 11: Construction of the single-domain antibody eukaryotic expression vector RJK-V4-3 The aforementioned universal target vector for nanobodies, RJK-V4-3, is a modified version of Invitrogen's commercial vector pCDNA3.4 (vector data link: https: / / assets.thermofisher.com / TFS-Assets / LSG / manuals / pcdna3_4_topo_ta_cloning_kit_man.pdf) by incorporating the Fc region of the human IgG4 heavy chain coding sequence. Specifically, this vector contains the CH2 and CH3 hinge regions of the IgG4 heavy chain. The specific modification scheme is as follows: (1) Select the restriction enzyme sites XbaI and AgeI on pcDNA3.4; (2) Multiple cloning sites (MCS) and 6×His tags were introduced at the 5' and 3' ends of the Fc fragment coding sequence, respectively, by overlapping PCR. (3) The above fragment was amplified by PCR using a pair of primers with XbaI and AgeI restriction sites respectively; (4) The recombinant DNA fragments in pcDNA3.4 and (3) were digested with restriction endonucleases XbaI and AgeI, respectively; (5) The digested vector and the insert fragment were ligated with T4 ligase, and then the ligation product was transformed into E. coli, amplified, sequenced and verified to obtain the recombinant plasmid.

[0106] Example 12: Expression and purification of tool antibody (Tab) targeting human TNFα protein. Tab is Adalimumab (Humira), and its sequence is derived from IMGT.

[0107] The searched sequences were entrusted to General Biosystems (Anhui) Co., Ltd. for codon optimization in mammalian cell expression systems and cloned into the pcDNA3.1 vector. After antibiotic selection, plasmid-positive bacteria were selected for amplification, and plasmids were extracted using a plasmid extraction kit (Macherey Nagel, Cat#740412.50). 100 μg of plasmid (40 μg heavy chain + 60 μg light chain) was added per 100 mL of cells, and PEI was used to transiently express the plasmid in 293F cells (medium: FreeStyle 293Expression medium, Thermo, Cat#12338026+F-68, Thermo, Cat#24040032). 6–24 h after transfection, 5% volume of 10% Peptone (Sigma, Cat#P0521-100G) was added, and the cells were cultured in 8% CO2 at 130 rpm for approximately 7–8 days. When cell viability decreased to 50%, the expression supernatant was collected and purified using a gravity column with Protein A (GE, Cat#17-5438-02). After dialysis with PBS, the concentration was determined using Nanodrop, purity was identified by SEC, and binding capacity was verified by indirect ELISA. The Tab obtained by this method has a concentration of not less than 2 mg / ml and a purity greater than 95%.

[0108] Example 13: Determination of Antibody-Antigen Binding Dose-Reaction Curve This embodiment uses a standard enzyme-linked immunosorbent assay (ELISA) kit and its operating procedure.

[0109] (1) Coat 50 μL of 1 μg / mL TNFα (prepared from Example 1) and incubate overnight at 4°C.

[0110] (2) Wash the plate; add 200 μL of 5% milk and seal at 37°C for 2 hours.

[0111] (3) Dilute VHH to 2 μg / mL, then perform a 5-fold serial dilution of the antibody to a total of 8 concentration gradients. Here, VHH is: the Fc fusion antibody of the single-domain antibody against TNFα protein obtained by eukaryotic expression in Example 9, which was purified in Example 10; (4) Wash the plate; add 50 μL of antibody diluted in step (3), double replicates, and incubate at 37°C for 1 h.

[0112] (5) Wash the plate; add 50 μL of mouse anti-HA tag HRP secondary antibody and incubate at 37°C for 30 min.

[0113] (6) Wash the plate (wash several times); add 50 μL of TMB that has been brought back to room temperature, and react at room temperature in the dark for 15 min.

[0114] (7) Add 50 μL of stop solution (1N HCl) and save the microplate reader reading.

[0115] (8) Plot the curve and calculate EC50, as follows: Figure 2 As shown, all 10 single-domain antibodies of this invention exhibit excellent binding efficacy and specificity to TNFα protein.

[0116] Figure 2 In the diagram, 2A shows the binding affinity of single-domain antibodies 1B11, 1B2, 1B3, 1B4, 1B5, and 1B7 to TNFα protein; 2B shows the binding affinity of single-domain antibodies 1C7, 1C8, 1D11, 1D2, 1D3, and 1D4 to TNFα protein; 2C shows the binding affinity of single-domain antibodies 1E1, 1E11, 1E3, 1E6, 1E9, and 1F1 to TNFα protein; and 2D shows the binding affinity of single-domain antibodies 1G5, 1G7, 1H10, and 1... The binding affinity of H8, 2A12, and 2A4 to TNFα protein is shown in 2E; the binding affinity of single-domain antibodies 2A5, 2A8, 2A9, 2B1, 2B10, and 2B11 to TNFα protein is shown in 2F; the binding affinity of single-domain antibodies 2B6, 2B8, 2C2, 2D10, 2D9, and 2F3 to TNFα protein is shown in 2G; and the binding affinity of single-domain antibodies 4A10, 4A5, 4A9, 4B3, 4C3, and 4C11 to TNFα protein is shown in 2G.

[0117] Example 14: Experiment on antibody (eukaryotic sample) neutralization of human TNFα-induced apoptosis in L929 cells The following operations shall be performed in accordance with methods commonly used by those skilled in the art: (1) Seed 10,000 L929 cells per well in a 96-well plate after being passaged 3 or more times after revival. (2) The serially diluted antibody was mixed with 4*11.82ng / mL of TNFα solution and added to the cell well in equal volume; the antibody here is the Fc fusion antibody of the single domain antibody against TNFα protein prepared in Example 9 (expressed in 293F cells) purified in Example 10; in addition, hIgG and Tab controls were set up respectively; Tab was prepared in Example 12; hIgG refers to the isotype control, an immunoglobulin molecule that does not bind to any target and is obtained by commercial purchase; Figure 3 The horizontal axis represents the specific concentration of the antibody in the gradient dilution; (3) After incubation at 37℃ for 24 hours, cell viability was detected using Cell Titer Glo, and the Luminescence value was read; (4) The EC50 concentration of the antibody to neutralize TNFα-induced apoptosis in L929 cells was calculated based on the detection results, and the results are as follows. Figure 3 As shown.

[0118] Figure 3 In the table, 3A shows the experimental results for Tab and hIgG, 3B shows the experimental results for 1A6, 1B2, and 1D4, 3C shows the experimental results for 1E9, 1H10, and 2B1, 3D shows the experimental results for 2B8, 2F3, and 3A3, and 3E shows the experimental results for 4A5, 4C3, 4C11, and 4E10.

[0119] Example 15: Experiment on antibody (humanized sample) neutralization of human TNFα-induced apoptosis in L929 cells was performed according to methods generally known to those skilled in the art: (1) Seed 10,000 L929 cells per well in a 96-well plate after being passaged 3 or more times after revival. (2) The serially diluted antibody was mixed with 4*11.82ng / mL TNFα solution and added to the cell well in equal volumes. The antibody was humanized antibody 1B2-VF, 1E9-VF, 2B1-VF, 4A5-VF, 4C3-VF or 4E10-VF. The humanized antibody against TNFα was constructed in Example 7 using an Fc fusion antibody eukaryotic expression vector (the nucleotide sequence of the humanized antibody was constructed into the vector RJK-V4-3), and then expressed in Example 9 and purified in Example 10. In addition, hIgG and Tab controls were set up respectively. The Tab was prepared in Example 12. hIgG refers to the isotype control, which is an immunoglobulin molecule that does not bind to any target and is obtained through commercial purchase. Figure 4 The horizontal axis represents the specific concentration of the antibody in the gradient dilution; (3) After incubation at 37℃ for 24 hours, cell viability was detected using Cell Titer Glo, and the Luminescence value was read; (4) The EC50 concentration of the antibody to neutralize TNFα-induced apoptosis in L929 cells was calculated based on the detection results, and the results are as follows. Figure 4 As shown.

[0120] Figure 4 In the diagram, 4A shows the experimental results for Tab and hIgG, 4B shows the experimental results for 1B2-VF, 1E9-VF, and 2B1-VF, and 4C shows the experimental results for 4A5-VF, 4C3-VF, and 4E10-VF.

[0121] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A single-domain antibody against TNFα, characterized in that: The single-domain antibody is composed of heavy chains, including heavy chain CDR1, heavy chain CDR2 and heavy chain CDR3; The amino acid sequences of the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are one of the following: (1) CDR1 shown in SEQ ID NO:52, CDR2 shown in SEQ ID NO:63, and CDR3 shown in SEQ ID NO:66; (2) CDR1 shown in SEQ ID NO:53, CDR2 shown in SEQ ID NO:59, and CDR3 shown in SEQ ID NO:

67.

2. The single-domain antibody against TNFα according to claim 1, characterized in that: The single-domain antibody further includes a frame region FR; the frame region FR includes the amino acid sequences of FR1, FR2, FR3, and FR4; the amino acid sequences of the frame region FR are as follows: FR1 or a variant thereof shown in SEQ ID NO: 24 or 25, wherein the variant thereof contains substitutions of up to 5 amino acids in the FR1; FR2 or a variant thereof shown in SEQ ID NO: 28 or 29, wherein the variant thereof contains substitutions of up to 5 amino acids; FR3 or a variant thereof shown in SEQ ID NO: 38 or 39, wherein the variant thereof contains substitutions of up to 5 amino acids in the FR3; The FR4 or a variant thereof shown in SEQ ID NO:47, wherein the variant thereof contains substitutions of up to 5 amino acids.

3. A single-domain antibody against TNFα, characterized in that: The amino acid sequences of the single-domain antibodies are shown in SEQ ID NO.8 or 6, respectively.

4. A bispecific antibody, characterized in that: The bispecific antibody includes a first antigen-binding portion and a second antigen-binding portion, wherein the first antigen-binding portion is a single-domain antibody against TNFα as described in any one of claims 1-3.

5. Use of the anti-TNFα single-domain antibody according to any one of claims 1-3 in the preparation of bispecific antibodies.

6. The Fc fusion antibody or humanized antibody of the single-domain anti-TNFα antibody according to any one of claims 1-3.

7. A polynucleotide molecule encoding a single-domain antibody against TNFα as described in any one of claims 1-3, characterized in that: Their nucleotide sequences are shown in SEQ ID NO: 18 or 16, respectively.

8. An expression vector, characterized in that: It comprises a single-domain antibody encoding the anti-TNFα as described in any one of claims 1-3, or a polynucleotide molecule encoding the Fc fusion antibody or humanized antibody as described in claim 6, or a polynucleotide molecule as described in claim 7.

9. A host cell, characterized in that: It can express the single-domain antibody against TNFα as described in any one of claims 1-3, or the expression vector comprising the one described in claim 8.

10. The host cell according to claim 9, characterized in that: The cells are eukaryotic or prokaryotic cells.

11. A pharmaceutical composition, characterized in that: It comprises a single-domain antibody against TNFα selected from any one of claims 1-3 and a pharmaceutically acceptable carrier.

12. A pharmaceutical agent for treating conditions associated with abnormal TNFα expression, characterized in that: It contains the anti-TNFα single-domain antibody as the active ingredient according to any one of claims 1-3.

13. A kit for detecting TNFα levels, characterized in that: It contains a single-domain antibody against TNFα as described in any one of claims 1-3.

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