Agonistic TNFR2 single-domain antibody and use thereof

Abstract

Disclosed are an agonistic TNFR2 single-domain antibody and a use thereof, said antibody comprising a CDR region having a certain sequence identity with the amino acid sequences shown in SEQ ID NO:2-4. The agonistic TNFR2 single-domain antibody can effectively bind to human TNFR2 without affecting the binding of TNF to TNFR2. At the same time, it also has the function of promoting regulatory T-cell activity and proliferation. Moreover, the molecular weight of the antibody is only one-tenth that of a conventional antibody, allowing effective penetration of the blood-brain barrier while significantly reducing production cost and complexity, thereby exhibiting greater potential than conventional antibodies in the treatment of central nervous system diseases.

Description

A single-domain antibody that stimulates TNFR2 and its application Technical Field

[0001] This invention belongs to the field of immunology, specifically relating to an agonistic TNFR2 single-domain antibody and its applications. Background Technology

[0002] Besides preventing pathogen invasion and clearing abnormal cells, the immune system's homeostasis is crucial for human health. Abnormal activation of immune cells, such as T cells or B cells, can lead to the immune system attacking its own cells. Common diseases like asthma, allergies, organ transplant rejection, and rheumatoid arthritis are caused by this abnormal activation of autoimmune cells. While healthy individuals can produce T cells or B cells that respond to self-antigens, the body also possesses a group of specialized immunosuppressive cells—regulatory T cells—which recognize self-antigens via MHC II and effectively suppress the immune response of active T cells or B cells to self-antigens. Regulatory T cells have several biomarkers, with Foxp3 considered the most important. CD4 + FoxP3 + Regulatory T cells are also one of the most extensively studied cell groups. Although their immunosuppressive mechanisms remain unclear, current research suggests that CD4+... + FoxP3 + Regulatory T cells may suppress immune responses through various mechanisms. For example, IL-2 deprivation is one of the potential mechanisms by which Treg cells exert their immunosuppressive function. Because Treg cells express higher levels of CD25, they can bind IL-2 better than effector T cells, thereby inhibiting the survival and activation of effector T cells. Simultaneously, Treg cells also express the co-inhibitory receptor CTLA-4, which has a significantly stronger affinity for CD80 / CD86 than for the co-stimulatory molecule CD28, thus significantly inhibiting the activation of T cells by antigen-presenting cells. Furthermore, Treg cells can also express immunosuppressive factors such as IL-10 and IL-35 to suppress T cell function.

[0003] The regulatory role of TNF on Treg cells has been extensively studied. TNF has two receptors, TNFR1 and TNFR2, with TNFR2 expression levels on Treg cells being significantly higher than TNFR1. Furthermore, TNF-TNFR2 plays a decisive role in the activation, function, proliferation, and phenotypic stability of regulatory T cells. TNFR2 knockout significantly inhibits the activation, proliferation, viability, and phenotypic stability of regulatory T cells. Animal experiments have confirmed that selective activation of TNFR2 can promote Treg cell proliferation and thus suppress autoimmune responses, and its therapeutic potential has been demonstrated in autoimmune diseases such as graft rejection, colitis, arthritis, and demyelinating diseases. Recent studies have also shown that TNFR2 agonists have therapeutic potential for degenerative diseases such as Alzheimer's disease. In addition, TNFR2 agonists have been used in vitro to promote Treg cell expansion and maintain Treg phenotypic stability, and also show potential application in adoptive Treg cell transplantation.

[0004] Although compelling evidence suggests that TNFR2 signaling has immunosuppressive effects, TNFR2, as a co-stimulatory receptor, can also promote the differentiation and function of effector T cells. Furthermore, TNFR2 activation can lead to the degradation of TRAF2, thereby making tumor cells more sensitive to TNF-TNFR1-induced cell death. In addition, due to the complex nature of TNFR2 agonist antibodies, they can also promote anti-tumor immune responses through various other potential mechanisms. Therefore, TNFR2 agonist antibodies currently have potential applications in both autoimmune diseases and tumors.

[0005] Nanobody (Nb), also known as heavy chain single-domain antibody (VHH), is a unique form of antibody found in alpacas and sharks. Cloning its variable region yields a single-domain antibody consisting of only one heavy chain variable region, which is currently the smallest stable, fully functional antigen-binding unit available. Nanobody antibodies possess characteristics such as high stability, good water solubility, simple humanization, high targeting, and strong penetration, playing an incredibly significant role in immunological experiments, diagnosis, and treatment. They are gradually becoming an emerging force in next-generation antibody diagnostics and therapy.

[0006] Inflammatory diseases, including autoimmune diseases, have a wide-ranging impact on human society. Although various types of anti-inflammatory drugs have been approved, existing medications only provide relief for most chronic inflammatory diseases and cannot restore the body's immune homeostasis. Therefore, most patients with chronic inflammation require long-term medication to suppress inflammation levels. However, long-term medication can lead to drug resistance and other side effects. Treg cells play an indispensable role in maintaining immune homeostasis, and their functional defects are closely related to various inflammatory diseases. Selectively activating Treg cells while systematically inhibiting a specific signaling pathway may enable them to more effectively suppress inflammation with fewer side effects, thus potentially representing a safer and more effective treatment strategy for inflammatory diseases.

[0007] Evidence suggests that TNFR2 agonists can be used to treat autoimmune diseases, including graft rejection (1), rheumatoid arthritis (2), demyelinating diseases (3,4), allergic dermatitis (5,6), type 1 diabetes (7,8), degenerative diseases including Alzheimer's disease (9), Parkinson's syndrome (10), osteoarthritis (11), and various solid tumors (12,13), and also have some efficacy against other related diseases, such as nerve damage (14) and skin infections (15).

[0008] Several TNFR2 agonist antibodies have been developed for the treatment of autoimmune diseases and tumors; however, they are mainly in the form of traditional antibodies and fusion proteins. Both have relatively large molecular weights, meaning their production costs are substantial. Furthermore, traditional antibodies, due to their large molecular weight, have difficulty crossing the blood-brain barrier, thus limiting their use in treating central nervous system diseases. Single-domain antibodies, on the other hand, are a new form of antibody drug that has emerged in recent years. Their molecular weight is only one-tenth that of traditional antibodies, while possessing comparable selectivity and affinity. Without IgG conjugation, single-domain antibodies can be produced in yeast or even bacterial expression systems, significantly reducing production costs. Moreover, their smaller molecular weight allows them to cross the blood-brain barrier, giving them an advantage over traditional antibodies in the treatment of heatstroke-related neurological inflammation and as diagnostic reagents for brain diseases. Summary of the Invention

[0009] This invention aims to at least solve one of the technical problems existing in the prior art. To this end, this invention provides an agonistic TNFR2 single-domain antibody and its applications. The agonistic TNFR2 single-domain antibody of this invention can effectively bind to human TNFR2 without affecting the binding of TNF to TNFR2. Simultaneously, it also promotes the function and proliferation of regulatory T cells. Furthermore, the molecular weight of this antibody is only one-tenth that of traditional antibodies, effectively reducing production costs and difficulties, and it can also effectively cross the blood-brain barrier, showing greater potential in the treatment of central nervous system diseases compared to traditional antibodies.

[0010] In a first aspect, the present invention provides an agonist TNFR2 single-domain antibody or an antigen-binding fragment thereof, said agonist TNFR2 single-domain antibody or antigen-binding fragment thereof specifically binding to TNFR2.

[0011] In some embodiments of the present invention, the agonist TNFR2 single-domain antibody or its antigen-binding fragment comprises a CDR region having at least 75%, 80%, 85%, 90%, or 95% sequence identity with the amino acid sequences shown in GRTSSSAAMG (SEQ ID NO:2), SITWSGGTTY (SEQ ID NO:3), and TGRSFAY (SEQ ID NO:4).

[0012] In some embodiments of the present invention, the agonistic TNFR2 single-domain antibody or its antigen-binding fragment comprises a CDR region having at least 95% sequence identity with the amino acid sequences shown in GRTSSSAAMG (SEQ ID NO:2), SITWSGGTTY (SEQ ID NO:3), and TGRSFAY (SEQ ID NO:4).

[0013] In some embodiments of the present invention, the agonist TNFR2 single-domain antibody or its antigen-binding fragment comprises a CDR region having at least 99% sequence identity with the amino acid sequences shown in GRTSSSAAMG (SEQ ID NO:2), SITWSGGTTY (SEQ ID NO:3), and TGRSFAY (SEQ ID NO:4).

[0014] In some embodiments of the present invention, the agonistic TNFR2 single-domain antibody or its antigen-binding fragment comprises a CDR region having 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequences shown in GRTSSSAAMG (SEQ ID NO:2), SITWSGGTTY (SEQ ID NO:3), and TGRSFAY (SEQ ID NO:4).

[0015] In some embodiments of the present invention, the agonist TNFR2 single-domain antibody or its antigen-binding fragment comprises:

[0016] a) The amino acid sequence shown in SEQ ID NO: 1; or

[0017] b) An amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid sequence retains TNFR2 specific binding activity and contains the amino acid sequences shown in SEQ ID NO: 2-4.

[0018] In some embodiments of the present invention, the amino acid sequence described in b) has at least 95% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0019] In some embodiments of the present invention, the amino acid sequence described in b) has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0020] In some embodiments of the present invention, the mutation (deletion, substitution, modification and / or addition) of a specific amino acid sequence in the variant sequence is performed within the range of maintaining the original function and / or desired function of the amino acid, or may be interpreted as not excluding the above-mentioned mutations.

[0021] In a second aspect, the present invention provides a nucleic acid molecule that encodes the agonistic TNFR2 single-domain antibody or an antigen-binding fragment thereof described in the first aspect of the present invention.

[0022] In some embodiments of the present invention, the nucleic acid molecule further includes non-functional modifying sequences or groups.

[0023] In some embodiments of the present invention, the non-functional modified sequence includes a tag sequence, a fluorescent protein sequence, and an resistance sequence.

[0024] In some embodiments of the present invention, the group includes a reporter group.

[0025] In some embodiments of the present invention, the reporter group includes a chromogenic enzyme, a fluorescent labeling group, a chemiluminescent labeling group, an isotope, or a magnetic functional group.

[0026] In some embodiments of the present invention, the chromogenic enzyme may include peroxidase or alkaline phosphatase.

[0027] In some embodiments of the present invention, the fluorescent labeling group may include fluorescent protein groups, rhodamine groups, fluorescein groups, anthocyanin dyes, cyanine dyes, AlexaFluor dyes, and / or quantum dot fluorescent groups.

[0028] In some embodiments of the present invention, the magnetic functional group may include groups that enable magnetic resonance imaging and alter relaxation efficiency.

[0029] In some embodiments of the present invention, the isotope includes a radioactive nuclide.

[0030] In some embodiments of the present invention, the modified nucleic acid molecule has at least 90% sequence identity compared with the unmodified nucleic acid molecule.

[0031] In some embodiments of the present invention, the sequence identity is 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0032] A third aspect of the invention provides an expressor comprising the nucleic acid molecules described in the foregoing aspects.

[0033] In some embodiments of the present invention, the expression vector includes plasmids, viruses, and bacteriophages.

[0034] In this invention, the selection of plasmids, viruses, and bacteriophages is not limited. In the art, any plasmid, virus, or bacteriophage capable of expressing a specific nucleic acid molecule as described in this invention can be used as a suitable expression vector. Of course, in this invention, the expression vectors include, but are not limited to, the aforementioned plasmids, viruses, and bacteriophages.

[0035] A fourth aspect of the present invention provides a transformant comprising the nucleic acid molecule and / or expression unit described in the foregoing aspects.

[0036] In some embodiments of the present invention, the transformants include microorganisms, plant cells, and animal cells.

[0037] A fifth aspect of the present invention provides a pharmaceutical composition comprising:

[0038] a) The agonistic TNFR2 single-domain antibody or its antigen-binding fragment described above; and

[0039] b) Carrier.

[0040] In some embodiments of the present invention, the pharmaceutical composition further includes a second active substance.

[0041] In some embodiments of the present invention, the second active substance includes:

[0042] a) Medications for the treatment of autoimmune diseases;

[0043] b) Substances that target TNF;

[0044] c) Cancer treatment drugs; and

[0045] d) Immunomodulators.

[0046] In some embodiments of the present invention, the autoimmune disease treatment drugs include, but are not limited to: treatment of graft rejection, colitis, arthritis (such as rheumatoid arthritis), demyelinating diseases, allergic dermatitis, and type 1 diabetes.

[0047] In this invention, the term "demyelinating disease" refers broadly to the destruction, disintegration, and loss of myelin sheaths in nerve fibers caused by neurological lesions. Specifically, it refers to diseases such as idiopathic inflammatory demyelinating disease of the central nervous system (DDS), multiple sclerosis (MS), neuromyelitis optica spectrum disorder (NMOSD), anti-myelin oligodendrocyte glycoprotein immunoglobulin G antibody-associated disease (MOGAD), acute disseminated encephalomyelitis (ADEM), and concentric sclerosis (BCS).

[0048] In some embodiments of the present invention, the TNF-targeting substance includes, but is not limited to, antibodies, inhibitors, or drug molecules that target TNF.

[0049] In some embodiments of the present invention, the cancer treatment drugs include, but are not limited to, chemotherapy drugs, targeted drugs, hormonal drugs, monoclonal antibodies, immunotherapy drugs, and biological factor modulators (biological response modulators).

[0050] In some embodiments of the present invention, the immunomodulators include, but are not limited to, immunostimulants, such as immune factor (including transfer factor, thymosin, immune ribonucleic acid, etc.) enhancers or supplements; microbial preparations, such as BCG, Corynebacterium pumilus, CpG-DNA; chemically synthesized drugs, such as levamisole, cimetidine, etc.; and immunosuppressants, such as chemically synthesized drugs, such as glucocorticoids like hydrocortisone and prednisone, alkylating agents like cyclophosphamide and nitrogen mustard, and antimetabolites like azathioprine and methotrexate; and fungal metabolites, such as cyclosporine, rapamycin, FK-506, etc.

[0051] A sixth aspect of the present invention provides a method for treating an autoimmune disease, comprising:

[0052] Provide effective doses of the agonistic TNFR2 single-domain antibody or its antigen-binding fragment, or pharmaceutical composition described above, to subjects in need.

[0053] In some embodiments of the present invention, the autoimmune diseases include, but are not limited to, graft rejection, colitis, arthritis (such as rheumatoid arthritis), demyelinating diseases, allergic dermatitis, and type 1 diabetes.

[0054] In some embodiments of the present invention, the effective dose is approximately 0.01-100 mg / kg body weight.

[0055] In this invention, the effective dose can be reasonably adjusted according to the actual situation and the judgment of the clinician. When dealing with different species, the dose can be reasonably adjusted based on the drug dose conversion formula or ratio between different species in the field.

[0056] A seventh aspect of the present invention provides a method for treating cancer, comprising:

[0057] Provide effective doses of the agonistic TNFR2 single-domain antibody or its antigen-binding fragment, or pharmaceutical composition described above, to subjects in need.

[0058] In some embodiments of the present invention, the cancers include, but are not limited to, colorectal cancer, lung cancer, liver cancer, breast cancer, melanoma, etc.

[0059] In some embodiments of the present invention, the effective dose is approximately 0.01-100 mg / kg body weight.

[0060] An eighth aspect of the present invention provides a method for treating degenerative diseases, comprising:

[0061] Provide effective doses of the agonistic TNFR2 single-domain antibody or its antigen-binding fragment, or pharmaceutical composition described above, to subjects in need.

[0062] In some embodiments of the present invention, the degenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's syndrome, and osteoarthritis.

[0063] In some embodiments of the present invention, the effective dose is approximately 0.01-100 mg / kg body weight.

[0064] A ninth aspect of the present invention provides a method for treating TNFR2-related diseases, comprising:

[0065] Provide effective doses of the agonistic TNFR2 single-domain antibody or its antigen-binding fragment, or pharmaceutical composition described above, to subjects in need.

[0066] In some embodiments of the present invention, the TNFR2-related diseases include nerve damage and skin infections.

[0067] A tenth aspect of the present invention provides the use of the agonistic TNFR2 single-domain antibody or its antigen-binding fragment described above in the preparation of the following pharmaceutical products:

[0068] a) Tumor drug sensitizers;

[0069] b) Medications for the treatment of autoimmune diseases;

[0070] c) Cancer treatment drugs; and

[0071] d) Immune cell modulators.

[0072] In some embodiments of the present invention, the tumor drug sensitizer refers to any drug that can: increase the sensitivity of tumor cells to tumor drugs or therapies, increase the killing rate of tumor drugs or therapies on tumor cells, or significantly enhance the efficacy of tumor drugs or therapies.

[0073] In some embodiments of the present invention, the tumor drug includes, but is not limited to, chemotherapy agents, radiotherapy agents, targeted therapy agents, and immunotherapy agents.

[0074] In some embodiments of the present invention, the therapy includes, but is not limited to, treatments achieved using the following combined strategies:

[0075] 1. Combination chemotherapy agents, radiotherapy agents, targeted therapy agents, and immunotherapy agents (including but not limited to anti-PD-1 / PD-L1 drugs, anti-CTLA-4 drugs, and anti-LAG-3 drugs) for the treatment of tumors.

[0076] 2. Combination anti-inflammatory drugs, including steroidal anti-inflammatory drugs, nonsteroidal anti-inflammatory drugs, targeted drugs, and biologics (including but not limited to anti-TNF biologics, anti-IL-6 biologics, etc.) for the treatment of autoimmune inflammation.

[0077] In an eleventh aspect of the present invention, the use of the agonistic TNFR2 single-domain antibody or its antigen-binding fragment described above in the preparation of a medicament for treating TNFR2 gene-related diseases is provided.

[0078] In some embodiments of the present invention, the TNFR2 gene-related diseases include: autoimmune diseases, degenerative diseases, tumors, and other related diseases.

[0079] In some embodiments of the present invention, the tumor includes, but is not limited to, colorectal cancer, lung cancer, liver cancer, breast cancer, melanoma, etc.

[0080] In some embodiments of the present invention, the degenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's syndrome, and osteoarthritis.

[0081] In some embodiments of the present invention, the autoimmune diseases include, but are not limited to: treatment of graft rejection, colitis, arthritis (such as rheumatoid arthritis), demyelinating diseases, allergic dermatitis, and type 1 diabetes.

[0082] In some embodiments of the invention, the other related diseases include nerve damage and skin infections.

[0083] The beneficial effects of this invention are:

[0084] 1. This invention provides an agonistic TNFR2 single-domain antibody that can effectively bind to human TNFR2 without affecting the binding of TNF to TNFR2, and effectively promotes the function and proliferation of regulatory T cells.

[0085] 2. Compared with conventional TNFR2 antibodies in the prior art, the agonist TNFR2 single-domain antibody of the present invention has the advantage of small molecular weight, which is only one-tenth of that of conventional TNFR2 antibodies in the prior art, resulting in lower production cost and difficulty.

[0086] 3. The agonistic TNFR2 single-domain antibody in this invention can effectively cross the blood-brain barrier and has greater potential in the treatment of central nervous system diseases compared with conventional TNFR2 antibodies. Attached Figure Description

[0087] Figure 1 shows the expression of TNFR2 in human CD4T cells, where the dashed line represents the isotype control and the solid line represents BV421-anti-TNFR2.

[0088] Figure 2 shows the binding of TNFR2 single-domain antibody labeled with AF488 to human CD4T cells and the corresponding bar chart.

[0089] Figures 3A-3B show the effect of TNFR2 agonist single-domain antibody on Treg cell proliferation (Figure 3A) and its corresponding bar chart (Figure 3B). Detailed Implementation

[0090] To make the objectives, technical solutions, and effects of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described in this specification are merely for explaining the invention and are not intended to limit the invention.

[0091] Unless otherwise specified, all experimental materials and reagents used are commercially available consumables and reagents.

[0092] Materials and Definitions

[0093] PBSTB: PBS buffer (pH 7.4) containing 0.05% Tween and 1% BSA (bovine serum albumin).

[0094] Amp: Ampicillin. Kan: Kanamycin. PEG: Polyethylene Glycol. CBS: Rat Cystatin β Synthase. BSA: Bovine Serum Albumin. FACS Buffer: Flow Cytometry Fluorescence Sorting Buffer. APC-streptavidin: Fluorescein (APC)-labeled streptavidin. FITC: Fluorescein Isothiocyanate.

[0095] Unless otherwise stated or clearly understood from the context of this invention, the term "antibody" as used herein may include the complete antibody and any antigen-binding fragment thereof (i.e., "antigen-binding fragment"). The heavy chain of an "antibody" in this invention consists of a heavy chain variable region (VH) and a heavy chain constant region. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region consists of three domains, CH1, CH2, and CH3. The VH can be further subdivided into highly variable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH consists of three CDRs and four framework regions (FRs), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain variable region contains binding domains that interact with the antigen. The constant region of an antibody can mediate the binding of immunoglobulins to host tissues or factors, which may include various cells of the immune system (e.g., Treg cells) and the first component (C1q) of the classical complement system.

[0096] In this invention, the term "antigen-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., TNFR2). Examples of binding fragments covered by the term "antigen-binding fragment" of an antibody include: (i) Fab fragments—monovalent fragments consisting of VH, CL, and CH1 domains; (ii) F(ab′)2 fragments—bivalent fragments comprising two Fab fragments linked by disulfide bonds through hinge regions; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of the VH domain of a single arm of the antibody; and (v) dAb fragments consisting of VH domains (e.g., Ward et al., (1989) Nature 341: 544-546). If antigen binding is possible, a separate complementarity-determining region (CDR) or a combination of two or more separate CDRs linked by a synthetic linker may contain the antigen-binding domain of the antibody.

[0097] Unless otherwise stated, when referring to an antibody, as in the claims, the word "fraction" means an antigen-binding fragment of the antibody, such that "antibody or fragment" has the same meaning as "antibody or antigen-binding fragment thereof".

[0098] In this invention, the phrases “antibody that recognizes an antigen” and “antibody that is specific to an antigen” are used interchangeably with the term “antibody that binds specifically to an antigen”.

[0099] In this invention, the terms "specific binding" and "selective binding" refer to the binding of an antibody to an epitope on a predetermined antigen without binding to other antigens. Typically, the equilibrium dissociation constant (KD) for antibody (i) binding is approximately less than 10. -7 M, for example, is approximately less than 10⁻⁸ M, 10⁻⁹ M, or 10⁻¹⁰ M or even lower, and can be determined by methods such as surface plasmon resonance (SPR) technology using an plasma resonance analyzer, or by Scatchard analysis of antibody binding to antigen-positive cells.

[0100] In this invention, the term "nucleic acid molecule" is intended to include both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, and can be cDNA.

[0101] This invention also includes "conserved sequence modifications" to the antibody sequences provided by this invention, i.e., nucleotide and amino acid sequence modifications that do not eliminate the binding effect of the antibody encoded by the nucleotide sequence or containing the amino acid sequence to the antigen. For example, modifications can be introduced using standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conserved sequence modifications include conserved amino acid substitutions, wherein amino acid residues are replaced by amino acid residues having similar side chains. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferable to replace the predicted non-essential amino acid residues in the TNFR2 antibody with another amino acid residue from the same side chain family. Methods for identifying conserved substitutions of nucleotides and amino acids that do not eliminate antigen binding are well known in the art. See, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12(10): 879-884 (1999); and Burks et al., Proc. Natl. Acad. Sci. USA 94: 412-417 (1997).

[0102] For nucleic acid molecules, the term "sequence identity" means that, when optimally aligned and compared, two nucleic acids or their designated sequences are identical in at least about 80% of their nucleotides (typically at least about 90% to 95%, and more preferably at least about 98% to 99.5% of their nucleotides) and have appropriate nucleotide insertions or deletions. Alternatively, substantial homology exists when these segments will hybridize with complementary sequences of the strands under selective hybridization conditions.

[0103] For amino acid sequences, the term “sequence identity” means that, when optimally aligned and compared, two amino acid sequences or their designated sequences are identical in at least about 80% of the amino acids (generally at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the amino acids) and have appropriate amino acid insertions or deletions.

[0104] The percentage of identity between two sequences is a function of the number of gaps that need to be introduced for optimal alignment of the two sequences, the length of each gap, and the number of common positions shared by the sequences when they are optimally aligned (i.e., homology % = number of common positions / total number of positions × 100). The comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms, as described in the following non-limiting examples.

[0105] The percentage of identity between two nucleotide sequences can be determined using the GAP procedure in the GCG software package, which uses the NWSgapdna.CMP matrix and vacancy weights of 40, 50, 60, 70, or 80, and length weights of 1, 2, 3, 4, 5, or 6. The percentage of identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)), which has been incorporated into the ALIGN procedure (version 2.0), using a PAM120 weighted residue table, a vacancy length penalty of 12, and a vacancy penalty of 4. In addition, the percentage of identity between two amino acid sequences can be determined using the Needleman and Wunsch (J.Mol.Biol.(48):444-453(1970)) algorithm (which has been incorporated into the GAP program in the GCG software package), using a Blossum 62 matrix or a PAM250 matrix, with vacancy weights of 16, 14, 12, 10, 8, 6 or 4, and length weights of 1, 2, 3, 4, 5 or 6.

[0106] The nucleic acid molecules and amino acid sequences in this invention can be further used as “query sequences” to search public databases for, for example, to identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul et al. (1990) J.Mol.Biol.215:403-10. BLAST nucleotide searches can be performed using the NBLAST program (score = 100, word length = 12) to obtain nucleotide sequences homologous to the nucleic acid molecules described herein. BLAST protein searches can be performed using the XBLAST program (score = 50, word length = 3) to obtain amino acid sequences homologous to the protein molecules described herein. For vacancy alignments for comparative purposes, Gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res.25(17):3389-3402. When using BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0107] Nucleic acid molecules can be present in intact cells, in cell lysates, or in partially purified or substantially pure forms. Nucleic acids purified from other cellular components or other contaminants (e.g., other cellular nucleic acids (e.g., other parts of chromosomes)) or proteins using standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and others well known in the art, are “isolated” or “present substantially pure.” See Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0108] In this invention, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid," which is a circular double-stranded DNA loop into which an additional DNA segment can be linked. Another type of vector is a viral vector, in which an additional DNA segment can be linked to a viral genome. Some vectors are capable of autonomous replication in the host cells to which they are introduced (e.g., bacterial vectors with bacterial origins of replication and attachable mammalian vectors). After introduction into a host cell, other vectors (e.g., non-attached mammalian vectors) can be integrated into the host cell's genome, thereby replicating along with the host genome. Furthermore, some vectors are capable of directing the expression of genes operatively linked to them. These vectors are referred to in this invention as "recombinant expression vectors" (or simply "expression vectors" or "expression units"). Typically, expression vectors useful in recombinant DNA technology are in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably because plasmids are the most commonly used form of vector. However, other forms of expression vectors, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), are also included, which have equivalent functions.

[0109] In this invention, the term "transformant" includes "recombinant host cell" (or simply "host cell"), which is intended to refer to a cell containing nucleic acids not naturally present in that cell, and may be a cell in which a recombinant expression vector has been introduced. It should be understood that such terms are intended not only to refer to the specific subject cell, but also to the progeny of that cell. Because certain modifications may occur in the progeny due to mutations or environmental influences, such progeny may actually differ from the parent cell, but are still included within the scope of the term "transformant" as used in this invention.

[0110] In this invention, "immunomodulator" or "immunomodulator" refers to an agent as defined below, such as a component that can participate in regulating, modulating, or modifying signaling pathways of an immune response. "Regulating," "modifying," or "modifying" an immune response refers to any alteration of the cells of the immune system or any alteration in the activity of such cells (e.g., effector T cells). Such regulation includes stimulation or inhibition of the immune system, manifested as an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells, or any other changes that may occur within the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have an enhancing function in the tumor microenvironment. In some embodiments, the immunomodulator is located on the surface of T cells. An "immunomodulatory target" or "immunomodulatory target" is an immunomodulator that is targeted for binding, the activity of which is altered by binding to a substance, agent, part, compound, or molecule. Immunomodulatory targets include, for example, receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands") on the cell surface.

[0111] In this invention, the term "administration" refers to the introduction of a composition comprising a therapeutic agent into the body using any of a variety of methods and delivery systems known to those skilled in the art. Preferred routes of administration for the antibodies described in this invention include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal, or other parenteral administration routes, such as by injection or infusion. As described in this invention, the phrase "parenteral administration" refers to administration methods other than enteral and local administration, typically by injection, including but not limited to intravenous, intraperitoneal, intramuscular, intraarticular, intrathecal, intralymphatic, intralesional, intracystic, intraorbital, intracardiac, intradermal, tracheal, subcutaneous, subepidermal, intra-articular, subcystic, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. Alternatively, the antibodies described in this invention may be administered via non-parenteral routes, such as local, epidermal, or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual, or local administration. Administration may also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0112] In this invention, the terms “inhibition” or “blocking” are used interchangeably and cover partial and complete inhibition / blocking of at least about 50%, such as at least about 60%, 70%, 80%, 90%, 95%, 99%, or 100%.

[0113] In this invention, "cancer" refers to a large class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division can lead to the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant parts of the body via the lymphatic system or bloodstream.

[0114] In this invention, the term "treatment" refers to any type of intervention or procedure performed on a subject or the administration of an active pharmaceutical agent to a subject with the aim of reversing, alleviating, improving, inhibiting, slowing, or preventing the progression, development, severity, or recurrence of disease-related symptoms, complications, signs, or biochemical markers. Prevention refers to administration to a subject who does not have the disease to prevent the occurrence of the disease or minimize the effects of the disease (if it occurs).

[0115] In this invention, the term "effective dose" or "effective amount" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutic effective dose" or "therapeutic effective amount" of a drug or therapeutic agent refers to any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression demonstrated by a reduction in the severity of disease symptoms, an increase in the frequency and duration of symptom-free periods, or prevention of injury or disability due to disease suffering. A "preventive effective dose" or "preventive effective amount" of a drug is an amount of drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing or relapsing into a disease, inhibits the development or relapse of the disease. The ability of a therapeutic or preventive agent to promote disease regression or inhibit the development or relapse of a disease can be assessed using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal models where efficacy in humans can be predicted, or by measuring the activity of the agent in an in vitro assay.

[0116] In this invention, the terms "patient" and "subject" refer to any human or non-human animal receiving preventive or therapeutic treatment.

[0117] Example 1: Alpaca Immunization

[0118] In this embodiment, recombinant human TNFR2 protein with a His tag (TN2-H5227, purchased from ACROBiosystems, hereinafter referred to as recombinant human TNFR2-His protein) was used as an immunogen to immunize alpacas.

[0119] Alpaca negative serum (venous blood) was collected one day before immunization. The initial immunization involved multiple injections of 200 μg of the recombinant human TNFR2-His protein, fully emulsified with Freund's adjuvant. Alpaca serum was collected 2–3 days after the initial immunization, followed by secondary, tertiary, and quaternary immunizations on days 17, 31, and 45, respectively. Alpaca serum after the four immunizations was collected on day 52 after the initial immunization and analyzed at a dilution of 1:10 to 1:10. 7Antibody serum titers were assessed in collected alpaca serum at different dilutions in ELISA plates coated with recombinant human TNFR2-His tags. A titer of >1:10 was considered satisfactory. 5 When anti-human TNFR2 antibodies can be detected at a certain dilution, further blood collection and library construction can be carried out.

[0120] The immunization results are as follows:

[0121] Table 1 Immunization Results

[0122] The data shown in bold are all valence results that meet the requirements.

[0123] Example 2: Construction of phage library

[0124] Alpaca venous blood was collected on day 7 after the fourth immunization (i.e., day 52 after the initial immunization), and peripheral blood mononuclear cells (PBMCs) were obtained from it using standard methods in the art. Total RNA was extracted from the PBMCs using a commercially available extraction kit, and cDNA products were obtained using a commercially available reverse transcription kit. The VHH fragment in the cDNA product was amplified by PCR, and the target fragment was then separated and recovered by agarose gel electrophoresis. The recovered product was amplified again, and the target fragment was obtained by agarose gel electrophoresis. The target fragment and the vector were digested with restriction endonucleases overnight, and the digested fragments were recovered by agarose gel electrophoresis. The recovered product was treated with DNA ligase and then transformed into competent E. coli by electroporation. The transformed cells were plated and the number of transformants was determined the next day.

[0125] The primers used in PCR amplification are:

[0126] Upstream F: 5'-CTGGGTGGTCCTGGCTGCTCTT-3' (SEQ ID NO: 5);

[0127] Downstream R: 5'-GCGCTACGTCTGTTGAACTGTT-3' (SEQ ID NO: 6).

[0128] The transformants obtained in the above steps were used as bacterial libraries. These libraries were inoculated into 2YT medium containing 100 g / mL ampicillin, with an initial OD600 of 0.1-0.2, and then cultured at 37°C and 230 rpm until OD600 > 0.8. Helper phage M13K07 (helper phage to bacteria ratio = 20:1) was added according to the OD600 value. The mixture was incubated at 37°C for 30 min. Then, it was gently shaken at 37°C and 180 rpm for 30 min. After centrifugation at 5000 rpm for 10 min, the supernatant was discarded, and the pellet was resuspended in an equal volume of 2YT (2×Yeast Extract Typeptone medium, 100 μg / mL, purchased from Beijing Solarbio Science & Technology Co., Ltd.) + A + K (i.e., Amp: 100 μg / mL, Kan: 50 μg / mL), and cultured overnight at 30°C and 220 rpm. Overnight culture was centrifuged at 4°C and 10,000 rpm for 20 min, the supernatant was collected, and the precipitate was discarded. The supernatant was centrifuged again for 20 min, and the supernatant was collected. 1 / 5 of the supernatant volume was added to an aqueous solution containing 20% ​​PEG8000 and 2.5 M sodium chloride. After mixing, the mixture was incubated on ice for at least 2 hours. The mixture was then centrifuged at 4°C and 10,000 rpm for 20 min, and the supernatant was discarded. The precipitate was resuspended in 1 mL of 1×PBS. A second precipitation was performed by adding 1 / 5 of the suspension volume of PEG8000 / NaCl for 1 hour. The mixture was centrifuged at 4°C and 12,000 rpm for 10 min, and the supernatant was discarded to obtain the phage library. The precipitate was resuspended in 1×PBS, and 100% glycerol was added to a final concentration of 50%. After mixing, the mixture was aliquoted into 1.5 mL EP tubes and stored at -80°C for later use. When using, 10 μL of bacterial cells carrying the phage library was serially diluted with 2YT medium, starting from 10... -4 and 10 -5 Take 10 μL of the mixture from each concentration tube and add it to 90 μL of TG1 bacterial culture (TG1 E. coli chemocompetent cells, purchased from Kangti Biotechnology Co., Ltd.), and mix gently. Incubate at 37°C for 15 min, then spread on Amp-resistant plates and incubate overnight. The next day, calculate the titer of the clonal phage library on the titer plate.

[0129] Example 3: Screening of positive clones

[0130] (1) First screening:

[0131] After thawing the human TNFR2 extracellular fragment protein (purchased from Bispecies) on ice, it was coated onto immunotubes (50 μg / tube, 2 mL / tube of PBS coating solution) and incubated overnight at 4°C with slow rotation. Simultaneously, 50 μg of 3% BSA was coated as a control. The liquid in the overnight coated immunotubes was discarded, and the tubes were washed three times with 2 mL of PBS buffer at room temperature, rotating for 5 min each time. 2 mL of blocking buffer (3% BSA) was added, and the tubes were blocked by rotation at room temperature for 1 h. The liquid in the blocked immunotubes was discarded, and the tubes were washed three times with 2 mL of PBST buffer (1×PBS + 0.1% Tween 20) at room temperature, rotating for 5 min each time. The washing solution was discarded, and 2 mL of 3% skim milk powder was added. The phage library prepared in the above examples was calculated according to the following formula and added as the input phage library for the first round of screening. The tubes were incubated by rotation at room temperature for 2 h.

[0132] Where V is the volume of added phage (in μL), and T library This refers to the phage titer.

[0133] Discard the liquid in the immunofluorescence tube after incubation at room temperature. Add 2 mL of PBST buffer (1×PBS + 0.1% Tween 20) and wash the tube 20 times at room temperature, rotating for 5 min each time. Discard the liquid in the tube, removing as much residual liquid as possible. Add 1 mL of 0.25 mg / mL Trypsin solution and elute by rotating for 30 min at room temperature. Add 10 μL of 10% 4-2-aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF) to stop elution. Transfer the solution from the immunofluorescence tube to a new 1.5 mL centrifuge tube to obtain the first round of phage selection eluent.

[0134] (2) Target phage titer detection:

[0135] The titer of the phage eluent obtained from the first round of screening was determined using the following method:

[0136] The TG1 strain was streaked with single colonies on 2×YT solid medium (without antibiotics) overnight at 37°C. A single colony was picked from the streak plate and transferred to a fresh 5 mL of 2×YT medium, and incubated overnight at 37°C. 500 μL of the overnight culture was transferred to 5 mL of 2×YT liquid medium and incubated at 37°C and 250 rpm until the OD600 reached 0.5-0.55. 10 μL of the first-round selection phage eluent was serially diluted 10-fold in 1.5 mL centrifuge tubes, for a total of 12 dilutions (i.e., 10 μL of the first-round selection phage eluent was diluted to 100 μL, then 10 μL of the diluted solution was further diluted to 100 μL, and so on, until a 10-fold serial dilution was achieved). -12After dilution, thoroughly shake each tube to mix. Then add 90 μL of SS320 bacterial culture (SS320 E. coli competent cells, purchased from Kangti Biotechnology Co., Ltd.) to each dilution centrifuge tube, mix well, and incubate at 37°C for 30 min. After incubation, take 5 μL of culture medium from each dilution centrifuge tube and add it dropwise to 2×YT solid medium (containing Amp), and incubate overnight at 37°C upside down. The number of single colonies at different dilutions can be clearly distinguished on the statistical plate, and the number of phage particles per milliliter of phage solution can be calculated according to the following formula to obtain the phage library titer. T(pfu / mL)=N×D×400

[0137] Where T is the phage titer (unit: pfu / mL), D is the dilution factor, and N is the number of single colonies at the corresponding dilution factor.

[0138] (3) Amplification of the target phage:

[0139] After confirming the titer of the phage elution buffer from the first round of screening, amplification was performed. The specific steps are as follows:

[0140] Strawberry strain TG1 was streaked onto 2×YT solid medium (without antibiotics) overnight at 37°C. A single colony was picked from the streak plate and transferred to 5 mL of fresh 2×YT medium, and incubated overnight at 37°C. 500 μL of the overnight culture was transferred to 5 mL of 2×YT liquid medium and incubated at 37°C and 250 rpm until the OD600 reached 0.5–0.55. 500 μL of the first round of phage elution buffer was added to the bacterial culture with an OD600 of 0.5–0.55, and incubated at 37°C and 250 rpm for 30 min. The cultured bacterial culture was then evenly spread onto 2% agarose square plates (245 mm × 245 mm) containing 100 μg / mL Amp and 2% glucose, and incubated overnight at 37°C. Take a square plate that has been cultured overnight, add 6 mL of 2×YT liquid medium to the surface of the plate, gently scrape off the colonies with a spreader, and collect the bacterial solution into a 15 mL centrifuge tube. This is the amplified bacterial library containing the first round of selection phages. Simultaneously, measure the OD600 value of the bacterial solution using a spectrophotometer; this is the OD600 value of the bacterial library in the elution buffer. Add glycerol to a final concentration of 20% to obtain the bacterial library after the first round of selection. Calculate the corresponding bacterial volume of the bacterial library in the elution buffer using the formula below, and transfer it to 100 mL of 2×YT liquid medium (containing 100 μg / mL Amp) to make the initial OD600 0.1.

[0141] Where V is the volume of the transferred bacterial solution (in μL), and OD600 is the OD600 of the bacterial library in the elution solution obtained in the above steps.

[0142] Continue culturing at 37℃ and 250rpm until the bacterial OD600 reaches 0.5-0.55. Then, calculate and add helper phage M13K07 according to the following formula to make the ratio of bacteria to phage 1:20.

[0143] Where V is the volume of helper phage added (in mL), and T helper-phage The titer of the helper phage used is given, and OD600 is the OD600 value of the bacterial culture.

[0144] Continue culturing at 37℃ and 250rpm until the bacterial OD600 reaches 0.5-0.55, then add Kan to a final concentration of 50μg / mL and incubate overnight at 30℃ and 250rpm.

[0145] (4) Purification of the target phage:

[0146] Transfer the overnight cultured bacterial culture from the above steps to a new 50 mL centrifuge tube and centrifuge at 4000 rpm and 4°C for 10 min. Transfer the supernatant from the centrifugation to a new 50 mL centrifuge tube, add 1 / 4 volume of pre-chilled 20% PEG / 2.5M NaCl solution at 4°C, mix thoroughly, and incubate on ice for 30 min. Centrifuge at 4000 rpm and 4°C for 20 min, and discard the supernatant. Resuspend the precipitate in 1 mL of PBS, transfer the resuspended solution to a new 1.5 mL centrifuge tube, and centrifuge at 13000 rpm and 4°C for 20 min. Transfer the supernatant from the centrifugation to a new 1.5 mL centrifuge tube, add 1 / 4 volume of pre-chilled 20% PEG / 2.5M NaCl solution, mix well, and incubate on ice for 10 min. Centrifuge at 13000 rpm and 4°C for 10 min, discard the supernatant, and resuspend the precipitate in 1 mL of PBS. Centrifuge again at 13000 rpm and 4℃ for 2 min, and transfer the supernatant to a new 1.5 mL centrifuge tube to obtain the purified first-round screening phage library. Aliquot the library into 100 μL tubes and store at -80℃ for long-term storage or at -20℃ for short-term storage (1-2 weeks).

[0147] The titer of the purified first-round screening phage library was determined using the methods described above.

[0148] (5) Multiple screening:

[0149] Repeat the above steps to screen the phage library multiple times to obtain purified phage libraries from different screening rounds.

[0150] (6) Monoclonal ELISA detection:

[0151] The TG1 strain was streaked with single colonies on 2×YT solid medium (antibiotic-free, purchased from Beijing Solarbio Science & Technology Co., Ltd.) overnight at 37°C. A single colony was picked from the streak plate and transferred to a fresh 5 mL of 2×YT medium, and incubated overnight at 37°C. 500 μL of the overnight culture was then transferred to 5 mL of 2×YT liquid medium and incubated at 37°C and 250 rpm until the OD600 reached 0.5–0.55.

[0152] Take 10 μL of the phage eluent from the second round of screening and perform 10-fold serial dilutions in 1.5 mL centrifuge tubes, for a total of 12 dilutions (dilution method as described in the previous example). Add 90 μL of the TG1 bacterial suspension with an OD600 value of 0.5-0.55 to each dilution centrifuge tube, mix well, and incubate at 37°C and 250 rpm for 30 min. Spread the culture evenly onto solid medium plates containing 100 μg / mL Amp and incubate overnight at 37°C. Randomly pick single colonies from the overnight culture plates and place them into sterile 96-well cell culture plates. Add 200 μL of 2×YT medium (containing 100 μg / mL Amp) to each well and incubate statically at 37°C overnight. Transfer 2 μL of the overnight culture to a new 96-well cell culture plate containing 200 μL of 2×YT liquid medium (containing 100 μg / mL Amp) per well and incubate statically at 37°C for 3 h. The amount of helper phage M13K07 added to each well should be calculated according to the following formula, so that the ratio of bacteria to phages is 1:20.

[0153] Where V is the volume of helper phage added (in mL), and T helper-phage The titer of the helper phage used is given, and OD600 is the OD600 value of the bacterial culture.

[0154] Single clones capable of growing on medium containing 100 μg / mL Amp were defined as positive clones. The obtained positive clones were incubated at 37°C for 30 min, then Kan was added to a final concentration of 50 μg / mL, and the mixture was incubated overnight at 30°C. The clones were then centrifuged at 4°C and 4000 rpm for 10 min, and the supernatant was retained and stored at 4°C for later use.

[0155] Human TNFR2 extracellular fragment protein (purchased from Bismuth Substances) 1 ng / μL, coated with CBS at pH 9.4 (100 μL / well) to coat the microplate. Simultaneously, BSA was coated as a control, and the plates were incubated overnight at 4°C. The liquid in the overnight coated microplate was discarded, and 200 μL of PBS buffer was added to each well. The plates were washed three times at room temperature for 10 min each time. 200 μL of blocking buffer (3% BSA) was added to each well to block the microplate, and the plates were blocked at room temperature for 1 h. The blocking buffer was discarded, and 200 μL of PBST buffer was added to each well. The plates were washed three times at room temperature for 10 min each time. Then, 120 μL of 3% BSA was added to each well, followed by 80 μL of the supernatant from the previous steps, which was centrifuged and stored for later use. The plates were incubated at room temperature for 2 h. The liquid in the microplate was discarded, and 200 μL of PBST buffer was added to each well to wash three times for 10 min each time. Add 100 μL of M13 Bacteriophage Antibody (HRP) Mouse Mab (diluted 1:100 in blocking buffer) to each well and incubate at room temperature for 1 hour. Discard the liquid in the plate, and wash each well three times with 200 μL of PBST buffer for 10 minutes each time. Then add 100 μL of TMB single-component chromogenic solution to each well and incubate in the dark for 2-3 minutes. Stop the reaction by adding 100 μL of 1M HCl to each well, read the OD450 value using a microplate reader, and record and save the data.

[0156] (7) Secondary ELISA verification of positive clones:

[0157] To rule out false positives, clones initially identified as positive were subjected to a second ELISA test. Clones that still showed positive results in the second test were retained for subsequent experiments.

[0158] (8) Identification of positive clones:

[0159] After two rounds of validation, 5 μL of the positive clone was inoculated into 1 mL of 2×YT medium (containing 100 μg / mL Amp) and incubated at 37°C and 250 rpm until the OD600 reached 0.8–1.0 (approximately 6–8 h). 0.5 mL of the bacterial culture was then sequenced. The sequenced sequences were aligned using GENtle software, and the antibody sequences were translated into amino acids using GENtle software.

[0160] The amino acid sequence of the obtained TNFR2-stimulating single-domain antibody (named UMR2-Nb2) was identified as follows:

[0161] The crossed-out section is its CDR area.

[0162] Example 4: Assay of Binding Activity of Agonistant TNFR2 Single-Domain Antibody

[0163] After linking the above-mentioned agonistic TNFR2 single-domain antibody to a His tag, it was diluted to 0.5 μg / mL with PBS and then coated into 96-well microplates, 100 μL per well. Coating was carried out overnight at 4°C. The plates were then blocked with 3% skim milk at room temperature for 60 minutes, washed four times with PBS to completely remove the washing buffer. 10 μg / mL extracellular human TNFR2 protein (purchased from Bismuth Substances) was diluted 10-fold into five gradients and added to the wells as the experimental group. A blank group (containing only diluent) was also included. After incubation at 37°C for 60 minutes, the plates were washed four times with washing buffer and the washing buffer was completely removed. Then, 100 μL / well of enzyme-labeled secondary antibody (Peroxidase-conjugated AffiniPureF(ab')2Fragment Goat Anti-Human IgG diluted with 3% skim milk, 1:20000 dilution) was added, and the plates were incubated at 37°C in the dark for 8 minutes. The reaction was terminated by adding 100 μL of 1M HCl, and the reading was taken at 450 nm using an ELISA reader.

[0164] The results are shown in the table below.

[0165] Table 2. Results of binding activity assay of agonistic TNFR2 single-domain antibodies

[0166] The results above show that the agonist TNFR2 single-domain antibody in the above embodiments has good binding activity to human TNFR2 protein.

[0167] Example 5: Expression of TNFR2 in human CD4 T cells

[0168] After obtaining fresh PBMCs, mark them with Pe-Cy7-anti-CD4 (purchased from BD Pharmaceuticals). TM Pe-Cy7-anti-CD4 and BV421-TNFR2 flow cytometry antibody were incubated at room temperature for 20 min, and then the free Pe-Cy7-anti-CD4 and BV421-TNFR2 flow cytometry antibody were removed with FACS buffer. The expression of TNFR2 on human CD4T cells was analyzed using a BD Fortessa flow cytometer.

[0169] The results are shown in Figure 1.

[0170] The results above show that human CD4 cells express TNFR2.

[0171] Example 6: Cell binding assay of agonistic TNFR2 single-domain antibody

[0172] FITC molecules were labeled with an agonist TNFR2 single-domain antibody (10 μg / mL) using an antibody labeling kit (ThermoFisher, A20181). The label was then diluted 10-fold to four different gradients and incubated for 0.5 h. Cells were then collected into flow cytometry tubes, and Pe-Cy7-anti-CD4 antibody (purchased from BD Pharmaceuticals) was added. TM CD4T cells labeled with ) were incubated at room temperature for 20 min, and then the free single-domain antibody and CD4 antibody (i.e. Pe-Cy7-anti-CD4 antibody) were removed by FACS buffer. The binding of single-domain antibody to CD4T cells was analyzed by BD Fortessa flow cytometry.

[0173] The results are shown in the table below and Figure 2.

[0174] Table 3. TNFR2-TNF binding status

[0175] The results above show that TNFR2 single-domain antibodies can effectively bind to TNFR2 on human CD4T cells.

[0176] Example 7: The promoting effect of agonistic TNFR2 single-domain antibody on Treg proliferation

[0177] CD4 T cells were isolated from healthy volunteers and used CellTrace TM Cell staining was performed using the Violet cell proliferation kit. Then, different concentrations of the agonist TNFR2 single-domain antibody obtained in the above examples (10 μg / mL, diluted 10-fold to obtain four gradients) and 10 ng / mL recombinant human IL-2 (purchased from BD Pharmaceuticals) were added sequentially. TM The positive control group consisted of treatments containing 10 ng / mL IL-2 and 20 ng / mL recombinant human TNF (BD Pharmingen). TM After culturing at 37°C for 72 hours, cells were fixed using a Foxp3 cell fixation kit (Thermal Fisher). Dead cells were labeled using the LIVE / DEAD cell viability assay kit (Thermal Fisher) before fixation. Cells were then unfixed after 12-18 hours at 4°C in the dark. Cells were stained with human Pe-cy7-anti-CD4 antibody and APC-anti-Foxp3 antibody (Thermal Fisher), and the proportion and proliferation of Treg cells were analyzed using BD Fortessa.

[0178] The results are shown in the table below and Figures 3A-B.

[0179] Table 4. Effects of TNFR2-stimulating single-domain antibodies on Treg proliferation.

[0180] The results above show that TNFR2 agonist single-domain antibodies can promote the proliferation of regulatory T cells in a dose-dependent manner.

[0181] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

[0182] References

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Claims

1. An agonistic TNFR2 single-domain antibody or an antigen-binding fragment thereof, characterized in that, The agonist TNFR2 single-domain antibody or its antigen-binding fragment specifically binds to TNFR2; The agonistic TNFR2 single-domain antibody or its antigen-binding fragment contains a CDR region having at least 95% sequence identity with the amino acid sequences shown in GRTSSSAAMG (SEQ ID NO:2), SITWSGGTTY (SEQ ID NO:3), and TGRSFAY (SEQ ID NO:4).

2. The agonistic TNFR2 single-domain antibody or antigen-binding fragment thereof of claim 1, wherein, The agonistic TNFR2 single-domain antibody or its antigen-binding fragment contains a CDR region having at least 99% sequence identity with the amino acid sequences shown in GRTSSSAAMG (SEQ ID NO:2), SITWSGGTTY (SEQ ID NO:3), and TGRSFAY (SEQ ID NO:4).

3. The agonistic TNFR2 single-domain antibody or antigen-binding fragment thereof of claim 1 or 2, characterized in that, The agonist TNFR2 single-domain antibody or its antigen-binding fragment comprises: a) The amino acid sequence shown in SEQ ID NO: 1; or b) An amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1, wherein the amino acid sequence retains TNFR2 specific binding activity and contains the amino acid sequences shown in SEQ ID NO: 2-4.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the agonistic TNFR2 single-domain antibody or its antigen-binding fragment as described in any one of claims 1-3.

5. An expression product, characterized by, The expresser comprises the nucleic acid molecule of claim 4.

6. A transformant characterized in that, The transformant contains the nucleic acid molecule of claim 4 and / or the expression unit of claim 5.

7. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: a) the agonistic TNFR2 single-domain antibody or its antigen-binding fragment as described in any one of claims 1-3; and b) Carrier.

8. The pharmaceutical composition of claim 7, wherein, The pharmaceutical composition further includes a second active substance; preferably, the second active substance comprises: a) Medications for the treatment of autoimmune diseases; b) Substances that target TNF; c) Cancer treatment drugs; and d) Immunomodulators.

9. The use of the agonistic TNFR2 single-domain antibody or its antigen-binding fragment as described in any one of claims 1-3 in the preparation of the following pharmaceutical products; a) Tumor drug sensitizers; b) Medications for the treatment of autoimmune diseases; c) Cancer treatment drugs; and d) Immune cell modulators.

10. The use of the agonistic TNFR2 single-domain antibody or its antigen-binding fragment as described in any one of claims 1-3 in the preparation of a medicament for treating TNFR2 gene-related diseases; The TNFR2 gene related diseases include: Autoimmune diseases, degenerative diseases, tumors, and other related diseases; Preferably, the autoimmune diseases include graft rejection, colitis, arthritis, demyelinating diseases, allergic dermatitis, and type 1 diabetes. Preferably, the degenerative diseases include Alzheimer's disease, Parkinson's syndrome, and osteoarthritis; Preferably, the tumor includes colorectal cancer, lung cancer, liver cancer, breast cancer, and melanoma; Preferably, the other related diseases include nerve damage and skin infections.

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