Agonist TNFR2 single-domain antibody and application thereof

Abstract

The invention discloses an agonistic TNFR2 single-domain antibody and application thereof. The agonistic TNFR2 single-domain antibody comprises a CDR region which has certain sequence identity with an amino acid sequence as shown in SEQ ID NO: 2-4. The agonistic TNFR2 single-domain antibody disclosed by the invention can be effectively combined with human TNFR2, and the combination of TNF and TNFR2 is not influenced. Meanwhile, the function and proliferation of regulatory T cells are promoted. The molecular weight of the antibody is only one tenth of that of a traditional antibody, the antibody can effectively penetrate through the blood brain barrier under the condition that the production cost and difficulty are effectively reduced, and compared with the traditional antibody, the antibody has larger potential in treatment of central nervous system diseases.

Description

Technical Field

[0001] The present invention belongs to the field of immunology, and specifically relates to an agonistic TNFR2 single domain antibody and an application thereof. Background Art

[0002] In addition to its functions of preventing pathogen invasion and clearing abnormal cells in the body, the homeostasis of the immune system is also very important for human health. Abnormal activation of immune cells, such as T cells or B cells, may cause the immune system to attack its own cells. For example, some common diseases such as asthma, allergies, organ transplant rejection, rheumatoid arthritis, etc. are caused by the abnormal activation of autoimmune cells. In a healthy human body, although T cells or B cells that respond to self-antigens can also be produced, there is also a group of professional immunosuppressive cells in the human body-regulatory T cells, which have MHCⅡ that recognizes self-antigens and can effectively inhibit the immune response of T cells or B cells that are active against self-antigens. Regulatory T cells have multiple markers, among which Foxp3 is considered to be the most important marker. CD4 + Foxp3 + Regulatory T cells are also one of the most widely studied cell populations. Although their immunosuppressive mechanisms are still unclear, existing studies suggest that CD4 + Foxp3 + Regulatory T cells may suppress immune responses in a variety of ways. 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 and can bind to IL-2 better than effector T cells, thereby inhibiting the survival and activation of effector T cells. At the same time, Treg cells also express the co-inhibitory receptor CTLA-4, which has a significantly stronger affinity for CD80 / CD86 than the co-stimulatory molecule CD28, thereby significantly inhibiting the activation of T cells by antigen-presenting cells. In addition, Treg cells can also express immunosuppressive factors such as IL-10 and IL-35 to inhibit T cell function.

[0003] The regulatory effect of TNF on Treg cells has been widely studied. TNF has two receptors, TNFR1 and TNFR2, of which TNFR2 is expressed at a much higher level on Treg cells than TNFR1. Moreover, TNF-TNFR2 plays a decisive role in the activation, function, proliferation and phenotypic stability of regulatory T cells. The activation, proliferation, vitality and phenotypic stability of regulatory T cells after TNFR2 knockout are significantly inhibited. Animal experiments have confirmed that selective activation of TNFR2 can promote the proliferation of Treg cells and thus inhibit autoimmune reactions, and its therapeutic potential has been confirmed 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 also been used to promote the expansion of Treg cells in vitro and maintain the stability of Treg phenotypes, and they also have application potential in adoptive Treg cell transplantation.

[0004] Although convincing evidence shows that TNFR2 signaling has an immunosuppressive effect, in fact, TNFR2 as a co-stimulatory receptor can also promote the differentiation and function of effector T cells. At the same time, TNFR2 activation can lead to the degradation of TRAF2, making tumor cells more sensitive to TNF-TNFR1-induced cell death. In addition, due to the complex characteristics of TNFR2 agonistic antibodies themselves, they can also promote anti-tumor immune responses through a variety of other potential mechanisms. Therefore, TNFR2 agonistic antibodies currently have potential application value in both autoimmune diseases and tumors.

[0005] Single-domain antibody (Nanobody, Nb), that is, heavy chain single-domain antibody VHH, is a special form of antibody found in alpacas and sharks. The single-domain antibody consisting of only one heavy chain variable region obtained by cloning its variable region is the smallest unit that can bind to antigens with complete functions and stability. Single-domain antibodies have the characteristics of high stability, good water solubility, simple humanization, high targeting, and strong penetration. They play a huge role beyond imagination in immune experiments, diagnosis and treatment. Single-domain antibodies are gradually becoming an emerging force in the diagnosis and treatment of the new generation of antibodies.

[0006] Inflammatory diseases, including autoimmune diseases, have had a wide-ranging impact on human society. Although many types of anti-inflammatory drugs have been approved, existing drugs can only alleviate most chronic inflammatory diseases and cannot restore the body's autoimmune homeostasis. Therefore, most patients with chronic inflammation require long-term medication to suppress the level of inflammation in the body. However, long-term medication can lead to drug resistance and other side effects. Treg cells play an indispensable role in maintaining immune homeostasis in the body, and their functional defects are closely related to a variety of inflammatory diseases. Using drugs to selectively activate Treg cells while systemically inhibiting a signal or pathway may enable them to more effectively suppress inflammation while having fewer side effects. Therefore, this may represent a safer and more effective treatment strategy for inflammatory diseases.

[0007] There is evidence that TNFR2 agonists can be used to treat autoimmune diseases, including transplant 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). They also have certain therapeutic effects on other related diseases, such as nerve damage (14) and skin infections (15).

[0008] Currently, several TNFR2 agonistic antibodies have been developed for the treatment of autoimmune diseases and tumors, but they are mainly in the form of traditional antibodies and fusion proteins. The molecular weight of both is relatively large, which means that their production costs are huge. In addition, traditional antibodies are difficult to pass through the blood-brain barrier due to their large molecular weight, so they are difficult to use in the treatment of central nervous system-related diseases. Single-domain antibodies are a new form of antibody drugs that have emerged in recent years. Their molecular weight is only 1 / 10 of that of traditional antibodies, and they have the same selectivity and affinity as traditional antibodies. Without coupling IgG, single-domain antibodies can be produced in yeast or even bacterial expression systems, so the production cost can be significantly reduced. In addition, single-domain antibodies can enter the blood-brain barrier due to their small molecular weight, so they have advantages over traditional antibodies in the treatment of heatstroke nervous system inflammation and as a diagnostic reagent for brain diseases. Summary of the invention

[0009] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention provides an agonist TNFR2 single domain antibody and its application. The agonist TNFR2 single domain antibody in the present invention can effectively bind to human TNFR2 without affecting the binding of TNF to TNFR2. At the same time, it also has the function and proliferation of promoting regulatory T cells. And the molecular weight of the antibody is only one-tenth of that of traditional antibodies. While effectively reducing the production cost and difficulty, it can also effectively penetrate the blood-brain barrier, and has greater potential in the treatment of central nervous system diseases than traditional antibodies.

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

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

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

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

[0014] In some embodiments of the invention, the agonist TNFR2 single domain antibody or antigen binding fragment thereof 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%, 99% sequence identity to the amino acid sequence shown in GRTSSSAAMG (SEQ ID NO:2), SITWSGGTTY (SEQ ID NO:3) and TGRSFAY (SEQ ID NO:4).

[0015] In some embodiments of the invention, the agonist TNFR2 single domain antibody or antigen-binding fragment thereof 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, said amino acid sequence retaining TNFR2-specific binding activity and comprising the amino acid sequence 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%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1.

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

[0021] The second aspect of the present invention provides a nucleic acid molecule encoding the agonist TNFR2 single domain antibody or antigen-binding fragment thereof according to the first aspect of the present invention.

[0022] In some embodiments of the present invention, the nucleic acid molecule further comprises a non-functional modified sequence or group.

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

[0024] In some embodiments of the invention, the group comprises 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 color developing enzyme may include peroxidase and alkaline phosphatase.

[0027] In some embodiments of the present invention, the fluorescent labeling group may include fluorescent proteins, rhodamines, fluoresceins, cyanine 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 a group capable of magnetic resonance imaging and changing relaxation efficiency.

[0029] In some embodiments of the invention, the isotope comprises a radionuclide.

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

[0031] In some embodiments of the 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] The third aspect of the present invention provides an expression element, which comprises the nucleic acid molecule described in the above aspects.

[0033] In some embodiments of the present invention, the expression vector includes a plasmid, a virus and a bacteriophage.

[0034] In the present invention, the selection of the plasmid, virus and bacteriophage is not limited. In the art, any plasmid, virus and bacteriophage that can achieve the expression of the specific nucleic acid molecule in the present invention can be used as a suitable expression vector. Of course, in the present invention, the expression vector includes but is not limited to the above-mentioned plasmid, virus and bacteriophage.

[0035] The fourth aspect of the present invention provides a transformant, wherein the transformant comprises the nucleic acid molecule and / or expression element described in the above 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) an agonistic TNFR2 single domain antibody or antigen binding fragment thereof as described in the above aspects; and

[0039] b) Vector.

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

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

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

[0043] b) Substances targeting TNF;

[0044] c) cancer treatment drugs; and

[0045] d) Immunomodulators.

[0046] In some embodiments of the present invention, the autoimmune disease therapeutic drug includes, but is not limited to, treating transplant rejection, colitis, arthritis (such as rheumatoid arthritis), demyelinating disease, allergic dermatitis and type 1 diabetes.

[0047] In the present invention, the term "demyelinating disease" refers to the destruction, disintegration and loss of myelin sheath of nerve fibers caused by nervous system 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-related disease (MOGAD), acute disseminated encephalomyelitis (ADEM), and concentric circle sclerosis (BCS).

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

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

[0050] In some embodiments of the present invention, the immunomodulator includes but is not limited to immunopotentiators, such as immune factor (including transfer factor, thymosin, immune RNA, etc.) enhancers or supplements; microbial preparations, such as BCG, Corynebacterium brevis, CpG-DNA; chemically synthesized drugs, such as levamisole, cimetidine, etc.; and immunosuppressants, such as chemically synthesized drugs, such as glucocorticoids such as hydrocortisone and prednisone, alkylating agents such as cyclophosphamide and nitrogen mustard, and antimetabolites such as azathioprine and methotrexate; 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] An effective dose of the agonistic TNFR2 single domain antibody or antigen-binding fragment thereof, or pharmaceutical composition described in the above aspects is provided to a subject in need thereof.

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

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

[0055] In the present invention, the effective dose can be reasonably adjusted according to the actual situation and the judgment of the clinician. For different species, it can be reasonably adjusted based on the drug dose conversion formula or ratio between different species in the art.

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

[0057] An effective dose of the agonistic TNFR2 single domain antibody or antigen-binding fragment thereof, or pharmaceutical composition described in the above aspects is provided to a subject in need thereof.

[0058] In some embodiments of the present invention, the cancer includes but is not limited to colon cancer, lung cancer, liver cancer, breast cancer, melanoma, etc.

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

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

[0061] An effective dose of the agonistic TNFR2 single domain antibody or antigen-binding fragment thereof, or pharmaceutical composition described in the above aspects is provided to a subject in need thereof.

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

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

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

[0065] An effective dose of the agonistic TNFR2 single domain antibody or antigen-binding fragment thereof, or pharmaceutical composition described in the above aspects is provided to a subject in need thereof.

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

[0067] The tenth aspect of the present invention provides the use of the agonist TNFR2 single domain antibody or antigen binding fragment thereof described in the above aspects in the preparation of the following drugs:

[0068] a) Tumor drug sensitizers;

[0069] b) Drugs 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 generally refers to any drug that can achieve: increasing the sensitivity of tumor cells to tumor drugs or therapies, increasing the killing rate of tumor drugs or therapies on tumor cells, and significantly enhancing the efficacy of tumor drugs or therapies.

[0073] In some embodiments of the present invention, the tumor drugs include but are not limited to chemotherapeutic agents, radiotherapy agents, targeted therapy agents, immunotherapy agents, etc.

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

[0075] 1. Combination with 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, non-steroidal 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] The eleventh aspect of the present invention provides use of the agonistic TNFR2 single domain antibody or antigen binding fragment thereof described in the above aspects in the preparation of a drug for treating TNFR2 gene-related diseases.

[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 colon cancer, lung cancer, liver cancer, breast cancer, melanoma, etc.

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

[0081] In some embodiments of the present invention, the autoimmune disease includes, but is not limited to, treatment of transplant rejection, colitis, arthritis (such as rheumatoid arthritis), demyelinating disease, allergic dermatitis and type 1 diabetes.

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

[0083] The beneficial effects of the present invention are:

[0084] 1. The present invention provides an agonist TNFR2 single domain antibody, which can effectively bind to human TNFR2, does not affect 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 a small molecular weight, which is only one tenth of that of conventional TNFR2 antibodies in the prior art, and has lower production cost and difficulty.

[0086] 3. The agonistic TNFR2 single domain antibody of the present invention can effectively penetrate the blood-brain barrier and has greater potential in the treatment of central nervous system diseases than conventional TNFR2 antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0087] Figure 1 It shows the expression of TNFR2 in human CD4 T cells, wherein, dotted line: isotype control; solid line: BV421-anti-TNFR2.

[0088] Figure 2 The binding status of TNFR2 single domain antibody labeled with AF488 to human CD4 T cells and its corresponding bar graph.

[0089] Figure 3 The effect of TNFR2 agonistic single domain antibody on Treg cell proliferation and its corresponding bar graph. DETAILED DESCRIPTION

[0090] In order to make the invention purpose, technical solution and technical effect of the present invention clearer, the present invention is further described in detail below in conjunction with specific implementation methods. It should be understood that the specific implementation methods described in this specification are only for explaining the present invention, not for limiting the present invention.

[0091] Unless otherwise specified, the experimental materials and reagents used are consumables and reagents that can be obtained from conventional commercial channels.

[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 cystathionine beta synthase. BSA: bovine serum albumin. FACS buffer: flow cytometry fluorescence sorting buffer. APC-streptavidin: fluorescein (APC)-labeled streptavidin. FITC: fluorescein isothiocyanate.

[0095] Unless otherwise specified or clear from the context of the present invention, the term "antibody" used in the present invention may include complete antibodies and any antigen-binding fragments thereof (i.e., "antigen-binding fragments"). The heavy chain of the "antibody" in the present 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. VH can be further subdivided into hypervariable 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 a binding domain that interacts with an antigen. The constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors, which may include various cells of the immune system (eg, Treg cells) and the first component (C1q) of the classical complement system, among others.

[0096] In the present 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 encompassed by the term "antigen-binding fragment" of an antibody include: (i) a Fab fragment - a monovalent fragment consisting of the VH, CL, and CH1 domains; (ii) a F(ab')2 fragment - a bivalent fragment comprising two Fab fragments linked by a disulfide bond in the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VH domain of a single arm of an antibody, and (v) a dAb fragment consisting of the VH domain (e.g., Ward et al., (1989) Nature 341: 544-546). An isolated complementarity determining region (CDR) or a combination of two or more isolated CDRs linked by a synthetic linker may comprise an antigen-binding domain of an antibody if it is capable of binding to an antigen.

[0097] Unless otherwise indicated, when used in reference to an antibody, such as in the claims, the word "fragment" refers to the antigen-binding fragment of an antibody, so that "antibody or fragment" has the same meaning as "antibody or antigen-binding fragment thereof."

[0098] In the present invention, the phrases "an antibody that recognizes an antigen" and "an antibody specific for an antigen" are used interchangeably herein with the term "an antibody that specifically binds to an antigen".

[0099] In the present 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. Generally, the equilibrium dissociation constant (KD) of antibody (i) binding is about 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 ionomer resonance instrument, or Scatchard analysis of antibody binding to antigen-positive cells.

[0100] In the present invention, the term "nucleic acid molecule" is intended to include DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, and may be a cDNA.

[0101] In the present invention, "conservative sequence modifications" to the antibody sequences provided by the invention are also included, 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 with the antigen. For example, modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative sequence modifications include conservative amino acid substitutions, in which amino acid residues are replaced by amino acid residues with similar side chains. Families of amino acid residues with 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 non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, it is preferred to replace a predicted nonessential amino acid residue in a TNFR2 antibody with another amino acid residue from the same side chain family. Methods for identifying conservative nucleotide and amino acid substitutions that do not abolish antigen binding are well known in the art. See, e.g., 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 two nucleic acids, or designated sequences thereof, are identical in at least about 80% of the nucleotides (usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides) with appropriate insertions or deletions of nucleotides when optimally aligned and compared. Alternatively, substantial homology exists when these segments will hybridize under selective hybridization conditions to the complement of a strand.

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

[0104] The percent identity between two sequences is a function of the number of identical positions shared by the sequences when they are optimally aligned, taking into account the number of gaps that need to be introduced in order to optimally align the two sequences, the length of each gap (i.e., % homology = number of identical positions / total number of positions x 100). The comparison of sequences and determination of the percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the following non-limiting examples.

[0105] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package, using the NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6. The percent 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 program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent 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, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.

[0106] The nucleic acid molecules and amino acid sequences of the present invention can further be used as a "query sequence" to search public databases to, for example, 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, wordlength = 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, wordlength = 3) to obtain amino acid sequences homologous to the protein molecules described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized 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 form. Nucleic acids that are purified from other cellular components or other contaminants, such as other cellular nucleic acids (e.g., other parts of chromosomes) or proteins by standard techniques, including alkaline / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art, are "isolated" or "rendered substantially pure." See Ausubel et al., eds., Current Protocols in Molecular Biology, Greene Publishing and Wiley Interscience, New York (1987).

[0108] In the present invention, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop to which another DNA segment can be connected. Another type of vector is a viral vector, in which another DNA segment can be connected to the viral genome. Some vectors can replicate autonomously in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors with bacterial replication origins). After being introduced into the host cell, other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell, thereby replicating with the host genome. In addition, some vectors can guide the expression of genes operably connected thereto. These vectors are referred to as "recombinant expression vectors" (or simply "expression vectors" or "expressors") in the present invention. Generally, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most commonly used vector forms. However, other forms of expression vectors are also included, such as viral vectors (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), which have equivalent functions.

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

[0110] In the present invention, "immunomodulator" or "immunomodulator" refers to an agent defined as follows, such as a component that can participate in regulating, controlling or modifying the signal transduction pathway of an immune response. "Regulating", "regulating" or "modifying" an immune response refers to any change in the cells of the immune system or any change in the activity of such cells (e.g., effector T cells). This regulation includes stimulation or inhibition of the immune system, which is 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 in the immune system. Both inhibitory and stimulatory immunomodulators have been identified, some of which may have enhanced functions in the tumor microenvironment. In some embodiments, the immunomodulator is located on the surface of a T cell. "Immunomodulatory target" or "immunomodulatory target" is an immunomodulator targeted for binding, and its activity is changed by the combination of a substance, an agent, a part, a compound or a molecule. Immunomodulatory targets include, for example, receptors ("immunomodulatory receptors") and receptor ligands ("immunomodulatory ligands") on the cell surface.

[0111] In the present invention, the term "administer" refers to the use of any of a variety of methods and delivery systems known to those skilled in the art to introduce a composition comprising a therapeutic agent into the body. Preferred routes of administration for the antibodies of the present invention include intravenous, intraperitoneal, intramuscular, subcutaneous, spinal or other parenteral routes of administration, such as by injection or infusion. As described in the present invention, the phrase "parenteral administration" refers to the mode of administration except enteral and topical administration, usually by injection, including but not limited to intravenous, intraperitoneal, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion and in vivo electroporation. Alternatively, the antibodies of the present invention can be administered by non-parenteral routes, such as local, epidermal or mucosal administration routes, such as intranasal, oral, vaginal, rectal, sublingual or topical administration. It can also be administered, for example, once, repeatedly and / or in one or more extended periods.

[0112] In the present invention, the terms "inhibit" or "block" are used interchangeably and encompass 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 the present invention, "cancer" refers to a broad class of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may lead to the formation of malignant tumors or cells that invade adjacent tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0114] In the present invention, the term "treatment" refers to any type of intervention or process performed on a subject or the administration of an active agent to a subject with the goal of reversing, alleviating, ameliorating, inhibiting or slowing down or preventing the progression, development, severity or recurrence of symptoms, complications, pathologies or biochemical markers associated with a disease. Prevention refers to administration to subjects who do not have the disease to prevent the occurrence of the disease or minimize the impact of the disease (if it occurs).

[0115] In the present 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 "therapeutically effective amount" or "therapeutically effective dose" of a drug or therapeutic agent refers to any amount of the drug that promotes disease regression when used alone or in combination with another therapeutic agent, and the disease regression is demonstrated by the following: a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease symptom-free periods, or the prevention of injury or disability caused by disease affliction. A "prophylactically effective amount" or "prophylactically effective dose" of a drug is an amount of the drug that inhibits the development or recurrence of the disease when administered alone or in combination with another therapeutic agent to a subject at risk of developing the disease or suffering from the recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote the regression of the disease or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to those skilled in the art, such as in human subjects during clinical trials, in animal model systems that can predict efficacy in humans, or by measuring the activity of the agent in an in vitro assay.

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

[0117] Example 1 Alpaca Immunization

[0118] In this example, 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] The alpaca negative serum (venous blood) was collected one day before the immunization. The first immunization was injected with 200 μg of the above-mentioned recombinant human TNFR2-His protein fully emulsified with Freund's adjuvant by multi-point injection in the neck and upper hind legs. Alpaca serum was collected 2 to 3 days after the first immunization, and then the alpaca was immunized twice, three times and four times in the same way on the 17th day, 31st day and 45th day respectively. The alpaca serum after four immunizations was collected on the 52nd day after the first immunization, and the serum was mixed with the alpaca serum at a ratio of 1:10 to 1:10. 7The collected alpaca sera were tested in ELISA plates coated with recombinant human TNFR2-His tag at different dilutions to evaluate the antibody serum titer. When the titer result meets the requirements, that is, >1:10 5 When anti-human TNFR2 antibodies can be detected at a dilution of , further blood samples can be collected to build a library.

[0120] The immunization results are as follows:

[0121] Table 1 Immunization results

[0122]

[0123] Among them, the data shown in bold are the titer results that meet the requirements.

[0124] Example 2 Construction of phage library

[0125] The alpaca venous blood obtained above on the 7th day after the fourth immunization (i.e., the 52nd day after the initial immunization) was taken, and peripheral blood mononuclear cells (PBMC) were obtained therefrom by conventional methods in the art. The total RNA in the PBMC was extracted using a commercially available extraction kit, and the cDNA product was obtained using a commercially available reverse transcription kit. The VHH fragment in the cDNA product was amplified by PCR, and then the target fragment was separated by agarose gel electrophoresis and gel recovered. The recovered product was amplified twice and the target fragment was obtained by agarose gel electrophoresis. The target fragment and the vector were digested overnight by restriction endonucleases, and then the digested fragments were recovered by agarose gel electrophoresis. The recovered product was treated with DNA ligase and then transferred into competent Escherichia coli by electrophoresis. It was spread on a culture plate and the number of transformants was determined the next day.

[0126] The primers used in PCR amplification are:

[0127] Upstream F: 5'-CTGGGTGGTCCTGGCTGCTCTT-3' (SEQ ID NO: 5);

[0128] Downstream R: 5'-GCGCTACGTCTGTTGAACTGTT-3' (SEQ ID NO: 6).

[0129] The transformants obtained in the above steps were used as a bacterial library. The bacterial library was inoculated into a 2YT medium containing 100 g / mL ampicillin to an initial OD600 of 0.1-0.2, and then cultured at 37°C and 230 rpm until OD600>0.8. Helper phage M13K07 was added according to the OD600 value (helper phage number: bacterial number = 20:1). After mixing the two, let stand at 37°C for 30 minutes. Then shake slowly at 37°C and 180 rpm for 30 minutes. Centrifuge at 5000 rpm for 10 minutes, discard the supernatant, and resuspend the precipitate with an equal volume of 2YT (2×Yeast Extract Typeptone medium, 100 μg / mL, purchased from Beijing Solebow Technology Co., Ltd.) + A+K (i.e. Amp: 100 μg / mL, Kan: 50 μg / mL), and culture overnight at 30°C and 220 rpm. Centrifuge the overnight culture at 4°C and 10,000 rpm for 20 min, collect the supernatant, and discard the precipitate. Centrifuge the supernatant again for 20 min and collect the supernatant. Add 20% PEG8000 and 2.5M sodium chloride aqueous solution at 1 / 5 of the supernatant volume. Mix and precipitate on ice for more than 2 hours. Centrifuge at 4°C and 10,000 rpm for 20 min and discard the supernatant. Resuspend the precipitate with 1mL 1×PBS. Add PEG8000 / NaCl with 1 / 5 volume of the suspension for secondary precipitation for 1h. Centrifuge at 4°C and 12,000 rpm for 10 min, discard the supernatant, and obtain the phage library. Resuspend the precipitate with 1×PBS, add 100% glycerol to a final concentration of 50%, mix and dispense into 1.5mL EP tubes, and store at -80°C for later use. When using, take 10μL of the bacteria carrying the phage library and dilute it with 2YT culture medium in a gradient 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 solution (TG1 Escherichia coli chemically competent cells, purchased from Kangti Biotechnology Co., Ltd.), and mix gently. Let stand at 37°C for 15 minutes, then spread on Amp-resistant plates and culture overnight. The next day, calculate the titer of the cloned phage library on the titer plate.

[0130] Example 3: Screening of positive clones

[0131] (1) First screening:

[0132] After thawing, human TNFR2 extracellular segment protein (purchased from Biopsies) was placed on ice and coated with immunotubes (50 μg / tube, coating solution was PBS, 2 mL / tube), and slowly rotated overnight at 4°C. At the same time, 50 μg of 3% BSA was coated in parallel as a control. The liquid in the overnight coated immunotube was discarded, and 2 mL of PBS buffer was added to wash the immunotube 3 times at room temperature, rotating and washing for 5 minutes each time. 2 mL of blocking solution (3% BSA) was added and the tube was rotated and blocked at room temperature for 1 hour. The liquid in the blocked immunotube was discarded, and 2 mL of PBST buffer (1×PBS+0.1% Tween20) was added to wash the immunotube 3 times at room temperature, rotating for 5 minutes each time. The washing solution in the immunotube was discarded, 2 mL of 3% skimmed milk powder was added, and the phage library prepared in the above example was calculated according to the following formula and added as the input phage library for the first round of screening, and incubated at room temperature for 2 hours.

[0133]

[0134] Where, V is the volume of phage added (in μL), T library is the phage titer.

[0135] Discard the liquid in the immunotube after incubation at room temperature, add 2mL PBST buffer (1×PBS+0.1% Tween20) and wash the immunotube 20 times at room temperature, rotating and washing for 5 minutes each time. Discard the liquid in the immunotube, remove the residual liquid as much as possible, add 1mL 0.25mg / mL Trypsin solution, and rotate and elute at room temperature for 30 minutes. Add 10μL 10% 4-2-aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF) to terminate the elution. Transfer the solution in the immunotube to a new 1.5mL centrifuge tube to obtain the first round of phage screening eluate.

[0136] (2) Target phage titer detection:

[0137] The titer of the phage eluate obtained in the first round of screening is tested as follows:

[0138] The TG1 strain was streaked with a single colony on a 2×YT solid medium (no resistance) (overnight at 37°C), and a single colony was picked from the single colony plate and transferred to a new 5 mL 2×YT medium and cultured at 37°C overnight. 500 μL of the overnight culture solution was transferred to 5 mL 2×YT liquid medium and cultured at 37°C and 250 rpm until the OD600 was 0.5-0.55. 10 μL of the first round of phage eluate was taken and diluted 10 times in a 1.5 mL centrifuge tube, with a total of 12 gradient dilutions (i.e., 10 μL of the first round of phage eluate was diluted to 100 μL, and 10 μL of the dilution solution was taken and continued to be diluted to 100 μL, and so on, until 10 was reached.-12 ). After the dilution is completed, each tube is shaken and mixed thoroughly. Then 90 μL of SS320 bacterial solution (SS320 Escherichia coli competent cells, purchased from Kangti Biotechnology Co., Ltd.) is added to each dilution centrifuge tube, mixed and incubated at 37°C for 30 minutes. After the incubation is completed, 5 μL of culture solution is taken out from each dilution centrifuge tube and added dropwise to 2×YT solid culture medium (containing Amp), and inverted and cultured at 37°C overnight. The number of single colonies at the dilution that can clearly distinguish single colonies on the statistical plate, and the number of phage particles per milliliter of phage solution is calculated according to the following formula to obtain the phage library titer.

[0139] T (pfu / mL) = N × D × 400

[0140] Wherein, T is the phage titer (unit: pfu / mL), D is the dilution multiple, and N is the number of single colonies at the corresponding dilution multiple.

[0141] (3) Amplification of target phage:

[0142] After confirming the titer of the first round of screening phage eluate, amplify it. The specific steps are as follows:

[0143] The TG1 strain was streaked with single colonies on 2×YT solid medium (no resistance) (overnight at 37°C), and a single colony was picked from the single colony plate and placed in a new 5 mL 2×YT medium, and cultured at 37°C overnight. 500 μL of the bacterial solution after overnight culture was transferred to 5 mL 2×YT liquid medium, and cultured at 37°C and 250 rpm until OD600 was 0.5-0.55. 500 μL of the phage eluate from the first round of screening was added to the above bacterial solution with OD600 of 0.5-0.55, and cultured at 37°C and 250 rpm for 30 minutes. The cultured bacterial solution was evenly spread on a 2% agarose square medium plate (245 mm×245 mm) containing 100 μg / mL Amp and 2% glucose, and cultured at 37°C overnight. Take the square plate cultured overnight, add 6mL 2×YT liquid culture medium to the surface of the culture plate, gently scrape the colonies on the square plate with a spreading stick and collect the bacterial solution into a 15mL centrifuge tube, which is the amplified bacterial library containing the first round of phage screening. At the same time, use a spectrophotometer to measure the OD600 value of the bacterial solution, which is the OD600 value of the bacterial library in the eluate. Add glycerol with a final concentration of 20% to obtain the bacterial library after the first round of screening. Calculate the corresponding bacterial solution volume of the bacterial library in the eluate according to the following formula, and transfer it to 100mL 2×YT liquid culture medium (containing 100μg / mL Amp) so that the initial OD600 is 0.1.

[0144]

[0145] Wherein, V is the volume of the transferred bacterial solution (unit: μL), and OD600 is the OD600 of the bacterial library in the elution solution obtained in the above step.

[0146] Continue culturing at 37°C and 250 rpm until the OD600 of the bacterial solution reaches 0.5-0.55, then calculate according to the following formula and add helper phage M13K07 so that the number of bacteria: number of phages = 1:20.

[0147]

[0148] Where V is the volume of helper phage added (in mL), T helper-phage is the titer of the helper phage used, and OD600 is the OD600 value of the bacterial solution.

[0149] Continue to culture at 37°C and 250 rpm until the OD600 of the bacterial solution reaches 0.5-0.55, then add Kan at a final concentration of 50 μg / mL and culture overnight at 30°C and 250 rpm.

[0150] (4) Purification of target phage:

[0151] Transfer the bacterial solution after overnight culture in the above step to a new 50mL centrifuge tube and centrifuge at 4000rpm and 4℃ for 10min. Transfer the supernatant after centrifugation to a new 50mL centrifuge tube, add 1 / 4 of the volume of the solution pre-cooled 20% PEG / 2.5M NaCl mixed solution at 4℃, mix thoroughly and place on ice for 30min. Centrifuge at 4000rpm and 4℃ for 20min, discard the supernatant. Add 1mL PBS to resuspend the precipitate, transfer the resuspended solution to a new 1.5mL centrifuge tube, and centrifuge at 13000rpm and 4℃ for 20min. Transfer the supernatant after centrifugation to a new 1.5mL centrifuge tube, add 1 / 4 of the volume of the pre-cooled 20% PEG / 2.5M NaCl mixed solution, mix well and place on ice for 10min. Centrifuge at 13000rpm and 4℃ for 10min, discard the supernatant, and add 1mL PBS to resuspend the precipitate. Centrifuge again at 13000 rpm and 4°C for 2 min, transfer the supernatant to a new 1.5 mL centrifuge tube to obtain the purified phage library for the first round of screening, and package it at 100 μL / tube for long-term storage at -80°C or short-term storage (1-2 weeks) at -20°C.

[0152] The method in the above steps is used to perform titer detection on the purified first round screening phage library.

[0153] (5) Multiple screening:

[0154] The phage library is screened repeatedly for multiple rounds according to the above steps to obtain purified phage libraries of different screening rounds.

[0155] (6) Monoclonal ELISA test:

[0156] The TG1 strain was streaked with single colonies (overnight at 37°C) on 2×YT solid medium (no resistance, purchased from Beijing Solebow Technology Co., Ltd.), and a single colony was picked from the single colony plate and placed in a new 5 mL 2×YT medium and cultured overnight at 37°C. 500 μL of the bacterial solution after overnight culture was transferred to 5 mL 2×YT liquid medium and cultured at 37°C and 250 rpm until OD600 was 0.5-0.55.

[0157] Take 10 μL of the phage eluate after the second round of screening, and dilute it 10 times in a 1.5mL centrifuge tube, with a total of 12 gradients (the dilution method is the same as in the above example). Add 90 μL of the above TG1 bacterial solution with an OD600 value of 0.5-0.55 to each dilution centrifuge tube, mix well, and culture at 37°C, 250rpm for 30 minutes. Spread the culture solution evenly on a solid culture medium plate containing 100 μg / mL Amp, and culture at 37°C overnight. Randomly pick monoclonal colonies from the culture medium plate after overnight culture in a sterile 96-well cell culture plate, add 200 μL 2×YT culture medium (containing 100 μg / mL Amp) to each well, and culture at 37°C overnight. Take 2 μL of the bacterial solution after overnight culture and transfer it to a new 96-well cell culture plate containing 200 μL 2×YT liquid culture medium (containing 100 μg / mL Amp) / well, and culture at 37°C for 3 hours. The amount of helper phage M13K07 added to each well was calculated according to the following formula so that the number of bacteria: number of phages = 1:20.

[0158]

[0159] Where V is the volume of helper phage added (in mL), T helper-phage is the titer of the helper phage used, and OD600 is the OD600 value of the bacterial solution.

[0160] The single clone that can grow in the medium containing 100 μg / mL Amp is defined as a positive clone, and the obtained positive clone is incubated at 37°C for 30 minutes, and Kan with a final concentration of 50 μg / mL is added, and cultured at 30°C overnight. Then centrifuge at 4°C and 4000 rpm for 10 minutes, retain the supernatant, and store at 4°C for later use.

[0161] The ELISA plate was coated with human TNFR2 extracellular segment protein (purchased from Biopsies) at 1 ng / μL, and the coating solution was CBS at pH 9.4, 100 μL / well. BSA was coated in parallel as a control, and the plate was coated overnight at 4°C. The liquid in the overnight coated ELISA plate was discarded, 200 μL PBS buffer was added to each well, and the ELISA plate was washed 3 times at room temperature, each time for 10 minutes. 200 μL blocking solution (3% BSA) was added to each well to block the ELISA plate, and the plate was blocked at room temperature for 1 hour. The blocking solution was discarded, 200 μL PBST buffer was added to each well, and the ELISA plate was washed 3 times at room temperature, each time for 10 minutes. Then 120 μL 3% BSA was added to each well, and then 80 μL of the supernatant prepared by centrifugation in the above steps was added to each well, and incubated at room temperature for 2 hours. The liquid in the ELISA plate was discarded, and 200 μL PBST buffer was added to each well to wash 3 times, each time for 10 minutes. Add 100μL M13 Bacteriophage Antibody (HRP) Mouse Mab (1:100 dilution in blocking solution) to each well and incubate at room temperature for 1h. Discard the liquid in the plate and add 200μL PBST buffer to each well for washing 3 times, 10min each time. Then add 100μL TMB single component colorimetric solution to each well and color for 2-3min in the dark. Add 100μL 1MHCl to each well to terminate the reaction, read the OD450 value with an enzyme reader, and record and save the data.

[0162] (7) Secondary verification of positive clones by ELISA:

[0163] In order to exclude false positive results, clones initially identified as positive were subjected to secondary ELISA verification, and clones that were still positive in the secondary verification were retained for subsequent experiments.

[0164] (8) Identification of positive clones:

[0165] Take the positive clone (5 μL) after secondary verification and inoculate it into 1 mL 2×YT medium (containing 100 μg / mL Amp), and culture it at 37°C and 250 rpm until OD600 reaches 0.8-1.0 (about 6-8 hours). Take 0.5 mL of bacterial solution for sequencing. Use GENtle software to perform sequence alignment analysis on the sequenced sequences, and use GENtle software to translate the obtained antibody sequences into amino acids.

[0166] After identification, the amino acid sequence of the obtained agonistic TNFR2 single domain antibody (named UMR2-Nb2) is:

[0167] DVQLQESGGGLVQTGASLRLSCAVS GRTSSSAAMG WFRQAPGKEREFLA SITWSGGTT YYETSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCTAAP TGRSFAY WGQGTQVTVSS (SEQ ID NO: 1).

[0168] The underlined part is its CDR region.

[0169] Example 4 Binding Activity Assay of Agonistic TNFR2 Single Domain Antibodies

[0170] After the above-mentioned agonist TNFR2 single domain antibody was connected to the His tag, it was diluted to 0.5 μg / mL with PBS and then coated in a 96-well ELISA plate, 100 μL per well. Coating was carried out overnight at 4°C. Then 3% skim milk powder was used to block at room temperature for 60 minutes, washed 4 times with PBS, and the washing solution was completely removed. 10 μg / mL of the extracellular segment human TNFR2 protein (purchased from Biopsies) was diluted tenfold into 5 gradients and added to the wells as the experimental group, and a group of blank wells (containing only diluent) were set up at the same time. After incubation at 37°C for 60 minutes, the washing solution was washed 4 times and the washing solution was completely aspirated. Then 100 μL / well of enzyme-labeled secondary antibody (Peroxidase-conjugated AffiniPureF(ab')2Fragment Goat Anti-Human IgG diluted with 3% skim milk powder, 1:20000 dilution) was added and incubated at 37°C in the dark for 8 minutes. The reaction was terminated by adding 100 μL of 1 M HCl and the samples were read at 450 nm using a microplate reader.

[0171] The results are shown in the following table.

[0172] Table 2 Binding activity assay results of agonistic TNFR2 single domain antibodies

[0173]

[0174] From the above results, it can be found that the agonistic TNFR2 single domain antibodies in the above examples have good binding activity to human TNFR2 protein.

[0175] Example 5 Expression of TNFR2 in human CD4 T cells

[0176] After obtaining fresh human PBMC, PBMC were labeled with Pe-Cy7-anti-CD4 (purchased from BD Pharmingen TM ) and BV421-TNFR2 flow cytometry antibody, incubated at room temperature for 20 minutes, then free Pe-Cy7-anti-CD4 and BV421-TNFR2 flow cytometry antibody were removed by FACS buffer, and the expression of TNFR2 on human CD4 T cells was analyzed using a BD Fortessa flow cytometer.

[0177] The results are as follows Figure 1 shown.

[0178] From the above results, it can be found that human CD4 cells express TNFR2.

[0179] Example 6 Cell Binding Assay of Agonistic TNFR2 Single Domain Antibodies

[0180] The agonist TNFR2 single domain antibody (10 μg / mL) was labeled with FITC molecules using an antibody labeling kit (ThermoFisher, A20181). It was then diluted 10-fold into 4 gradients. Incubated for 0.5 h. The cells were collected into a flow tube and Pe-Cy7-anti-CD4 antibody (purchased from BD Pharmingen) was added. TM )-labeled CD4 T cells 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, and the binding of the single domain antibody to CD4 T cells was analyzed using a BD Fortessa flow cytometer.

[0181] The results are shown in the following table and Figure 2 shown.

[0182] Table 3 TNFR2-TNF binding

[0183]

[0184] From the above results, it can be found that TNFR2 single domain antibody can effectively bind to TNFR2 on human CD4 T cells.

[0185] Example 7 Effect of Agonistic TNFR2 Single Domain Antibody on Promoting Treg Proliferation

[0186] CD4 T cells were isolated from healthy volunteers and analyzed using CellTrace TM Violet cell proliferation kit was used for cell staining. Then different concentrations of the agonist TNFR2 single domain antibody obtained in the above example (10 μg / mL, 4 gradients were obtained by 10-fold dilution) and 10 ng / mL recombinant human IL-2 (purchased from BD Pharmingen) were added in sequence. TM ), the positive control group was treated with 10 ng / mL IL-2 and 20 ng / mL recombinant human TNF (BD Pharmingen TM). After culturing in a 37°C constant temperature incubator for 72 hours, the cells were fixed (cell fixation was performed using the Foxp3 cell fixation kit, purchased from Thermal Fisher). Before fixation, dead cells were labeled using the LIVE / DEAD cell activity detection kit (purchased from Thermal Fisher). After fixation at 4°C in the dark for 12-18 hours, the cells were unfixed. The cells were stained with human Pe-cy7-anti-CD4 antibody and APC-anti-Foxp3 antibody (purchased from Thermal Fisher), and the proportion and proliferation of Treg cells were analyzed using BD Fortessa.

[0187] The results are shown in the following table and Figure 3 shown.

[0188] Table 4 Effects of agonistic TNFR2 single domain antibodies on Treg proliferation

[0189]

[0190] From the above results, it can be found that TNFR2 agonistic single domain antibody can promote the proliferation of regulatory T cells in a dose-dependent manner.

[0191] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

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Claims

1. An agonistic TNFR2 single domain antibody or an antigen-binding fragment thereof, characterized in that: The agonistic TNFR2 single domain antibody or antigen binding fragment thereof specifically binds to TNFR2; The agonistic TNFR2 single domain antibody or antigen binding fragment thereof comprises a CDR region having at least 95% sequence identity with the amino acid sequence 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 according to claim 1, characterized in that: The agonistic TNFR2 single domain antibody or antigen binding fragment thereof comprises a CDR region having at least 99% sequence identity with the amino acid sequence 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 according to claim 1 or 2, characterized in that: The agonistic TNFR2 single domain antibody or antigen binding fragment thereof 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, said amino acid sequence retaining TNFR2-specific binding activity and comprising the amino acid sequence 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 antigen binding fragment thereof according to any one of claims 1 to 3.

5. An expression element, characterized in that The expressor comprises the nucleic acid molecule of claim 4.

6. A transformant, characterized in that: The transformant comprises the nucleic acid molecule according to claim 4 and / or the expression element according to claim 5.

7. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises: a) the agonistic TNFR2 single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3; and b) Vector.

8. The pharmaceutical composition according to claim 7, characterized in that The pharmaceutical composition further comprises a second active substance; preferably, the second active substance comprises: a) Drugs for the treatment of autoimmune diseases; b) Substances targeting TNF; c) cancer treatment drugs; and d) Immunomodulators.

9. Use of the agonistic TNFR2 single domain antibody or antigen binding fragment thereof according to any one of claims 1 to 3 in the preparation of the following drugs; a) Tumor drug sensitizers; b) Drugs for the treatment of autoimmune diseases; c) cancer treatment drugs; and d) Immune cell modulators.

10. Use of the agonistic TNFR2 single domain antibody or antigen-binding fragment thereof according to any one of claims 1 to 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 transplant rejection, colitis, arthritis, demyelinating disease, allergic dermatitis and type I diabetes; Preferably, the degenerative diseases include Alzheimer's disease, Parkinson's syndrome and osteoarthritis; Preferably, the tumor includes intestinal cancer, lung cancer, liver cancer, breast cancer and melanoma; Preferably, the other related diseases include nerve damage and skin infection.

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