Novel antibody molecules and their pharmaceutical uses

Abstract

The present invention belongs to the technical field of cellular immunology, and specifically relates to a novel antibody molecule and its pharmaceutical use. The antibody provided by the present invention comprises a first single-domain antibody and a second single-domain antibody; the amino acid sequence of the first single-domain antibody comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; the amino acid sequence of the second single-domain antibody comprises HCDR4 as shown in SEQ ID NO: 4, HCDR5 as shown in SEQ ID NO: 5, and HCDR6 as shown in SEQ ID NO: 6. The antibody of the present invention has strong binding ability, strong blocking ability and stability.

Description

Technical Field

[0001] The present invention belongs to the technical field of cellular immunology, and in particular relates to novel antibody molecules and pharmaceutical uses thereof. Background Art

[0002] The IL-17 cytokine family includes IL-17A, IL-17B, IL-17C, IL-17D, IL-17E and IL-17F, while the IL-17 receptor family includes IL-17RA, IL-17RB, IL-17RC, IL-17RD and IL-17RE. Among them, IL-17A and its receptor IL-17RA have been studied the most. IL-17A is a proinflammatory cytokine involved in the development of certain autoimmune diseases and verification-related diseases. IL-17A can promote the secretion of inflammatory factors such as IL-8, IL-6, and tumor necrosis factor (TNF)-α, and promote the production of endothelial cell adhesion molecules, leading to a significant inflammatory response. Helper T cell 17 (Th17) is a surface antigen differentiation cluster 4 positive (CD4+) T cell that can produce IL-17, and is mainly involved in the occurrence of autoimmune diseases by secreting interleukin (IL)-17A.

[0003] Monoclonal antibodies are produced by B cells. They exist on the cell surface in the form of receptors and can only bind to one antigenic determinant and react specifically, so that the same lymphoid system activated by the reaction can only produce immunoglobulins against this antigenic determinant. Monoclonal antibodies are mainly composed of antigen binding domain (Fab) and crystallizable region (Fc). Fab can regulate related signaling pathways after recognizing tumor antigens, while the Fc segment mediates antibody-dependent cell-mediated cytotoxicity, antibody-dependent cell-mediated phagocytosis, complement-dependent cytotoxicity and other effects after interacting with cells expressing Fc receptors, participating in anti-tumor immune response. Compared with immunosuppressants or anti-inflammatory drugs, neutralizing monoclonal antibodies against IL-17 have higher specificity and selectivity and less side effects and toxicity in the treatment of autoimmune diseases.

[0004] Chinese patent CN109745559A discloses a liquid preparation of a monoclonal antibody against human IL-17A. The liquid preparation of the monoclonal antibody against human IL-17A provided by the invention includes the monoclonal antibody against human IL-17A, a surfactant, a sugar and an amino acid, which is stable during manufacture, storage and administration, has low aggregates, and is suitable for administration at high concentrations (e.g., for subcutaneous administration). The stable liquid preparation of the monoclonal antibody against human IL-17A of the invention can be effectively used in the preparation of drugs for treating immune-mediated inflammatory reactions.

[0005] Chinese patent CN110582512A discloses a monoclonal antibody against both IL-17A and IL-17F and its use, wherein the antibody binds to both IL-17A and IL-17F and inhibits the activities of both IL-17A and IL-17F, wherein the antibody comprises CDR1H, CDR2H, CDR3H; and CDR1L, CDR2L and CDR3L. The invention also provides the use of the antibody in the preparation of a drug for treating a disease associated with IL-17A and / or IL-17F in a subject, and a pharmaceutical composition comprising the antibody.

[0006] Currently, monoclonal antibodies are still widely used in the treatment of immune diseases, but they have certain toxicity and side effects that may affect the health of the subjects. In addition, due to their structure, the preparation of monoclonal antibodies is relatively complex and costly.

[0007] Single-domain antibodies (sdAb) are different from traditional monoclonal antibodies. Single-domain antibodies have no light chains, are small in size, oval in shape, and have a relative molecular mass of about 15kDa. Single-domain antibodies contain 4 framework regions, which form the immunoglobulin core structure and 3 complementary determining regions that can bind to antigens. The N-terminus of the CDR1 region of single-domain antibodies is very variable, and the CDR3 region is longer, containing an average of 16 amino acids, which increases the diversity of VHH. Single-domain antibodies can recognize and bind to special antibody epitopes, and single-domain antibodies can also deeply recognize hidden epitopes wrapped by antigens, reaching areas that conventional antibodies cannot reach. Single-domain antibodies have strong temperature and acid-base tolerance, and have obvious advantages over traditional antibodies in their tolerance under extreme conditions.

[0008] There are few studies on IL-17A single-domain antibodies in the prior art. Therefore, developing a single-domain antibody drug with high stability, strong affinity and low production cost is a technical problem that needs to be solved urgently.

[0009] The inventors are committed to the development of anti-IL-17A antibodies. Based on the 9 single-domain antibodies screened out (which have been applied for separately), they conducted further technical development and obtained 12 single-domain antibody combinations with relatively improved affinity, blocking effect and stability. Based on the relevant provisions of the Patent Law on unity, they requested protection for the 12 different single-domain antibody combinations respectively.

[0010] The present invention is a patent application for one of the above 12 single domain antibody combination antibodies.

[0011] Based on the above, the inventor has also developed gene-modified stem cell technology and subsequent application technology, and based on the relevant provisions of the Patent Law on unity, requested protection for three different gene-modified stem cells and three different applications respectively.

[0012] To facilitate understanding of the technical solution of the present invention, it is optional to refer to other patent application documents of this project. Summary of the invention

[0013] Terms and Claims of the Present Invention:

[0014] 1. As used herein, the term "amino acid" includes natural amino acids, synthetic amino acids, and amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids. Natural amino acids are amino acids encoded by a genetic code. Amino acid analogs refer to amino acids that have the same basic chemical structure as naturally occurring amino acids. Amino acids may be referred to herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0015] 2. As used herein, the term "antibody" refers to a polypeptide or fragment thereof that specifically binds to and recognizes an antigen, including the framework region of an immunoglobulin gene. The use of the term antibody is intended to include whole antibodies and antigen-binding fragments thereof. The term antibody includes monospecific antibodies, bispecific antibodies, and multispecific antibodies, as long as they exhibit the desired biological activity or function.

[0016] 3. As used herein, the terms "single domain antibody (VHH)" and "single domain antibody" (sdAb, or nanobody) have the same meaning, referring to cloning the variable region of the antibody heavy chain to construct a single domain antibody consisting of only one heavy chain variable region, which is the smallest antigen-binding fragment with complete functions. Usually, an antibody naturally lacking the light chain and heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

[0017] 4. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Antigenic determinants typically contain chemically active surface groups of molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. For example, an epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 continuous or non-continuous amino acids in a unique spatial conformation, which can be a "linear" epitope or a "conformational" epitope.

[0018] 5. As used herein, the term "amino acid sequence" refers to the order in which amino acids are linked to form a peptide chain (or polypeptide). The amino acid sequence can only be read in one direction.

[0019] 6. As used herein, the term "nucleic acid molecule" may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The full-length sequence of the nucleotide molecule or its fragment in the present invention can usually be obtained by PCR amplification or artificial synthesis.

[0020] 7. As used herein, the term "nucleotide sequence" refers to the order of bases in DNA or RNA, i.e., the order of A, T, G, C in DNA, or the order of A, U, G, C in mRNA, and also includes the order of bases in rRNA, tRNA, and mRNA.

[0021] 8. As used herein, the term "framework region" refers to the skeleton region. There are about 110 amino acid sequences near the N-terminus of the H and L chains of immunoglobulins that vary greatly, while the amino acid sequences of other parts are relatively constant. Based on this, the light chain and heavy chain can be divided into variable regions (V) and constant regions (C). The variable region includes the hypervariable region HVR or complementary determining region CDR and the FR skeleton region.

[0022] 9. As used herein, the term "humanized" antibody refers to the Fr region of the variable region (VH or VHH) of the antibody, the constant region (i.e., CH and CL regions) or all of the antibody encoded by human antibody genes. Humanized antibodies include chimeric antibodies, modified antibodies, and fully humanized antibodies.

[0023] 10. As used herein, the term "Fc region" refers to the C-terminal region of an immunoglobulin, which is a functional structural unit consisting of only CH2 and CH3 in the heavy chain constant domain. Fc has no ability to bind to antigens, but it has the property of extending half-life and has a constant amino acid sequence.

[0024] 11. As used herein, the term "variable" means that certain parts of the variable region of an antibody are different in sequence, which forms the binding and specificity of each specific antibody to its specific antigen. However, the variability is not evenly distributed throughout the variable region of an antibody. It is concentrated in three segments called complementary determining regions or hypervariable regions in the variable region of the light chain and heavy chain. Herein, "variable region" and "complementarity determining region" are used interchangeably.

[0025] 12. As used herein, the term "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide after being introduced into a suitable host cell.

[0026] 13. The terms "hypervariable region", "hypervariable region", "complementarity determining region", "HVR" or "CDR" as used herein refer to regions in the variable domain region of an antibody that are highly variable in sequence and / or form structurally defined loops ("hypervariable loops"). Typically, a natural four-chain antibody comprises six HVRs or CDRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). Based on the Chothia definition rule, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3) are located at amino acid residues L26-L32 (L1), L50-L52 (L2), L91-L96 (L3), H26-H32 (H1), H52-H56 (H2) and H96-H101 (H3) (Chothia et al., J. Mol. Biol. 196: 901-917 (1987)). Based on the Kabat definition rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3) are located at amino acid residues L24-L34 (L1), L50-L56 (L2), L89-L97 (L3), H31-H35 (H1), H50-H65 (H2) and H95-H102 (H3) (Kabat et al., Sequences of Proteins of Immunological Interest, the fifth edtion, Public Health Service, National Institutes of Health, Bethesda, MD (1991)). Based on the IMGT definition rules, exemplary CDRs (LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3) are located at amino acid residues L27-L32 (L1), L50-L51 (L2), L89-L97 (L3), H26-H33 (H1), H51-H56 (H2) and H93-H102 (H3) (Honjo, T. and Alt, FW (1995) Immunoglobulin genes. Academic Press pp. 3-443). It is well known to those skilled in the art that the CDRs of antibodies can be defined in the art by a variety of methods, such as the Kabat definition rules based on sequence variability, the Chothia definition rules based on the position of the structural loop region and the reference tool for antibody humanization design based on CDR transplantation (see J Mol Biol. 273: 927-48, 1997).It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass the complementarity determining region defined by any of the above-mentioned known schemes described in the present invention. Although the scope of protection claimed in the present disclosure is based on the sequence shown in the IMGT definition rules, the amino acid sequences corresponding to other CDR definition rules should also fall within the scope of protection of the present invention.

[0027] 14. The term "EC50" as used herein refers to the concentration for 50% of maximal effect, i.e., the concentration that can induce 50% of the maximal effect (herein, "IL-17A binding ability").

[0028] 15. The term "IC50" as used herein refers to half maximal inhibitory concentration, i.e., the concentration that can cause 50% maximal inhibitory effect (in this article, "the ability to inhibit the binding of IL-17A and its receptor IL-17RA").

[0029] The technical solution of the present invention is as follows:

[0030] In a first aspect, the present invention provides an antibody comprising a first single domain antibody and a second single domain antibody;

[0031] The amino acid sequence of the first single-domain antibody comprises HCDR1 as shown in SEQ ID NO: 1, HCDR2 as shown in SEQ ID NO: 2, and HCDR3 as shown in SEQ ID NO: 3; and / or; an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs: 1-3;

[0032] The amino acid sequence of the second single-domain antibody comprises HCDR4 as shown in SEQ ID NO:4, HCDR5 as shown in SEQ ID NO:5, HCDR6 as shown in SEQ ID NO:6; and / or; an amino acid sequence having at least 80% sequence identity with the amino acid sequence shown in SEQ ID NO:4-6.

[0033] Preferably, the amino acid sequence of the antibody comprises: an amino acid sequence obtained by at least one of addition, deletion, modification and / or substitution on the amino acid sequence shown in SEQ ID NO: 1-6.

[0034] Preferably, the amino acid sequence of the antibody comprises: an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid differences compared to the amino acid sequence shown in SEQ ID NO: 1-6.

[0035] SEQ ID NO: 1 is: ESLLRLYA.

[0036] SEQ ID NO: 2 is: HTTSDTT.

[0037] SEQ ID NO: 3 is: HVTSMRDSQNY.

[0038] SEQ ID NO:4 is: GFSIHIYA.

[0039] SEQ ID NO:5 is: ITRGGVT.

[0040] SEQ ID NO:6 is: NVGGTNGGY.

[0041] In some embodiments, the substitution of one or more amino acids can be a conservative substitution of one or more amino acids. Such conservative substitutions are preferably substitutions of one amino acid in the following groups (a) to (e) by another amino acid residue in the same group: (a) small aliphatic, non-polar or weakly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, non-polar residues: Met, Leu, He, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0042] In some specific embodiments, conservative substitutions are as follows: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0043] Preferably, the first single-domain antibody or the second single-domain antibody further comprises at least four heavy chain framework regions for connecting heavy chain variable regions.

[0044] Further, the heavy chain framework region comprises part or all of an antibody heavy chain framework region selected from human, mouse, primate or camelid sources or variants thereof;

[0045] Preferably, it comprises part or all of an antibody heavy chain framework region selected from camelid origin or a variant thereof;

[0046] More preferably, it comprises part or all of an antibody heavy chain framework region selected from alpaca origin or a variant thereof.

[0047] In a second aspect, the present invention provides an antibody, wherein the antibody comprises a first single domain antibody and a second single domain antibody;

[0048] The structure of the first single domain antibody is:

[0049] FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4;

[0050] The structure of the second single domain antibody is:

[0051] FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8;

[0052] The amino acid sequences of HCDR1, HCDR2 and HCDR3 are as shown in SEQ ID NO: 1-3; or; amino acid sequences having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 amino acid differences compared with SEQ ID NO: 1-3;

[0053] The amino acid sequences of HCDR4, HCDR5 and HCDR6 are as shown in SEQ ID NOs: 4-6; or; amino acid sequences having 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid differences compared with SEQ ID NOs: 4-6;

[0054] The amino acid sequences of FR1, FR2, FR3, and FR4 are as shown in SEQ ID NOs: 7-10; or; an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 7-10;

[0055] The amino acid sequences of FR5, FR6, FR7, and FR8 are as shown in SEQ ID NOs: 11-14; or; an amino acid sequence having at least 80% sequence identity with SEQ ID NOs: 11-14;

[0056] The amino acid differences are achieved by at least one of addition, deletion, modification and / or substitution on the amino acid sequences shown in SEQ ID NOs: 1-6.

[0057] Specifically, the amino acid sequences of FR1, FR2, FR3, and FR4 are as shown in SEQ ID NOs: 7-10; or; an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID NOs: 7-10;

[0058] Specifically, the amino acid sequences of FR5, FR6, FR7, and FR8 are as shown in SEQ ID NOs: 11-14; or; amino acid sequences having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity compared to SEQ ID NOs: 11-14.

[0059] SEQ ID NO:7 is: DVQLVESGGGLVQPGGSLRLSCAAS.

[0060] SEQ ID NO: 8 is: MGWYRQLPGQEREWVAI.

[0061] SEQ ID NO:9 is: NYRDSVKGRFTLSRDVATNTIYLQMTSLKPEDTAVYYC.

[0062] SEQ ID NO: 10 is: WGQGTQVTVSS.

[0063] SEQ ID NO:11 is: EVQLVESGGGLVQPGGSLRLSCAAS.

[0064] SEQ ID NO: 12 is: MGWYRQAPGKQRELVAT.

[0065] SEQ ID NO: 13 is:

[0066] NNADSVKGRFTISRDNAKNTAYLQMNSLKPGDTAVYYC.

[0067] SEQ ID NO: 14 is: WGQGTQVTVSS.

[0068] Preferably, the HCDR1 has the amino acid sequence shown in SEQ ID NO:1, the HCDR2 has the amino acid sequence shown in SEQ ID NO:2, the HCDR3 has the amino acid sequence shown in SEQ ID NO:3, the HCDR4 has the amino acid sequence shown in SEQ ID NO:4, the HCDR5 has the amino acid sequence shown in SEQ ID NO:5, and the HCDR6 has the amino acid sequence shown in SEQ ID NO:6.

[0069] Preferably, the amino acid sequences of the first single-domain antibody and the second single-domain antibody are indirectly connected via a linker; or; directly connected.

[0070] Furthermore, the linker is (GGGGS)n, where n is selected from 1, 2, 3, 4, 5 or 6; preferably, the linker is (GGGGS)3.

[0071] Preferably, the first single domain antibody and the second single domain antibody are connected in an optional order; or; the first single domain antibody and the second single domain antibody are connected in an order from C-terminus to N-terminus; or; the first single domain antibody and the second single domain antibody are connected in an order from N-terminus to C-terminus.

[0072] Preferably, the antibody is an anti-IL-17A antibody.

[0073] In a third aspect, the present invention provides a recombinant protein, wherein the recombinant protein comprises the antibody described in any one of the first aspect or the second aspect.

[0074] Preferably, the recombinant protein further comprises a biologically active protein or a functional fragment thereof that assists its expression and / or secretion, or prolongs its half-life in vivo.

[0075] Furthermore, the bioactive polypeptide or its functional fragment is selected from at least one of serum albumin, His tag, GST tag, albumin binding polypeptide, prealbumin, carboxyl terminal peptide, elastin-like polypeptide, immunoglobulin Fc domain, MBP tag, FLAG tag and SUMO tag.

[0076] Specifically, the immunoglobulin Fc domain is derived from a human antibody, a mouse antibody, a primate antibody or a camelid antibody, or a variant thereof;

[0077] Preferably, the immunoglobulin Fc domain is derived from human IgG antibody, such as IgG1 Fc, IgG2 Fc, IgG3 Fc or IgG4 Fc, preferably IgG1 Fc.

[0078] In some embodiments, one or more amino acid modifications may be introduced into the Fc region or Fc domain of an antibody provided herein to generate an Fc region variant. The Fc region variant may comprise a human Fc region sequence (e.g., human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution, deletion and insertion) at one or more amino acid positions.

[0079] In some embodiments, the recombinant protein can be a monomer, a dimer, or a multimer.

[0080] In a fourth aspect, the present invention provides a drug conjugate, wherein the drug conjugate comprises:

[0081] (1) the antibody or the recombinant protein described in any one of the first aspect or the second aspect; and;

[0082] (2) A coupling moiety coupled to (1).

[0083] In some embodiments, the conjugated moiety comprises a detectable label, a drug, a toxin, a cytokine, a radionuclide, or an enzyme.

[0084] In a fifth aspect, the present invention provides an antibody preparation, comprising:

[0085] (1) the antibody or the recombinant protein described in any one of the first aspect or the second aspect; and;

[0086] (2) A pharmaceutically acceptable carrier.

[0087] Specifically, the pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration.

[0088] Furthermore, the pharmaceutically acceptable carrier is selected from one or more of an excipient, a buffer, an emulsifier, a stabilizer, a diluent, a binder, a preservative, and a lubricant.

[0089] In a sixth aspect, the present invention provides a kit, comprising the antibody, the recombinant protein or the antibody preparation described in any one of the first aspect or the second aspect;

[0090] Optionally, the kit further comprises a container for holding the antibody preparation.

[0091] In a seventh aspect, the present invention provides an isolated nucleic acid molecule, wherein the nucleic acid molecule encodes the antibody or the recombinant protein described in any one of the first aspect or the second aspect.

[0092] Specifically, the nucleic acid may be RNA, DNA or cDNA.

[0093] For some embodiments, the nucleic acid of the present invention may also be in the form of a vector, may be present in a vector and / or may be part of a vector, such as a plasmid, a cosmid or a YAC. The vector may be an expression vector, which typically comprises at least one nucleic acid of the present invention, which may be operably linked to one or more suitable expression control elements (e.g., promoters, enhancers, terminators, etc.).

[0094] In an eighth aspect, the present invention provides an expression vector comprising the nucleic acid molecule.

[0095] Preferably, the expression vector is selected from DNA, RNA, viral vector, plasmid, transposon, or a combination thereof.

[0096] In a ninth aspect, the present invention provides a pharmaceutical composition, comprising the antibody, the recombinant protein, the drug conjugate, the antibody preparation, the nucleic acid molecule or the expression vector described in any one of the first aspect or the second aspect;

[0097] Optionally, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

[0098] Specifically, the pharmaceutically acceptable excipients are selected from one or more of diluents, isotonic agents, buffers, emulsifiers, thickeners, sweeteners, disintegrants, preservatives, absorbents, dispersants, stabilizers, suspending agents, solvents, precipitation inhibitors, surfactants, glidants, adhesives, lubricants, hydrating agents, emulsification accelerators, colorants, flavoring agents, ion exchangers, release agents, coating agents, flavoring agents or antioxidants.

[0099] In a tenth aspect, the present invention provides a use of the antibody, the recombinant protein, the drug conjugate, the antibody preparation or the pharmaceutical composition described in any one of the first aspect or the second aspect, wherein the use is at least one of the following uses:

[0100] (1) preparing detection reagents or kits;

[0101] (2) Preparation of drugs for preventing and / or treating autoimmune diseases;

[0102] (3) Preparation of drugs for preventing and / or treating cancer.

[0103] Specifically, the autoimmune diseases include focal scleroderma, polymyositis, multiple sclerosis, systemic scleroderma, progressive systemic sclerosis, dermatomyositis, psoriasis, allergic vasculitis, idiopathic azoospermia, scleroderma, habitual miscarriage, fibromyalgia, polymyalgia rheumatica, Basedow's disease, chronic discoid lupus erythematosus, autoimmune hemolytic anemia, Behcet's disease, adult Still's disease, systemic lupus erythematosus, large vessel vasculitis, Addison's disease, rheumatoid arthritis, myasthenia gravis, Castleman's disease, idiopathic thrombocytopenic purpura, chronic atrophic gastritis, malignant rheumatoid arthritis, vasculitis, autoimmune hepatitis, alopecia areata, vitiligo, spondyloarthritis, lupus nephritis, bullous pemphigoid, pemphigus, pernicious anemia, psoriatic arthritis, idiopathic Addison's disease, inflammatory bowel disease disease, sarcoidosis, herpes gestationis, rheumatoid vasculitis, neuromyelitis optica, periarteritis nodosa, aortitis syndrome, juvenile idiopathic arthritis, mixed connective tissue disease, Sjögren's syndrome, allergic granulomatous vasculitis, IgG4-related disease, ANCA-associated vasculitis, Good-pasture syndrome, Cogan syndrome, nonalcoholic steatohepatitis, autoimmune neutropenia, linear IgA bullous dermatosis, Hashimoto's disease, slowly progressive type 1 diabetes, type 1 diabetes, RS3PE syndrome, temporal arteritis, antiphospholipid antibody syndrome, eosinophilic fasciitis, Guillain-Barré syndrome, primary biliary cirrhosis, rapidly progressive glomerulonephritis, megaloblastic anemia, autoimmune adrenocortical insufficiency, primary thyroid dysfunction

[0104] hypoplasia, pemphigoid, epidermolysis bullosa acquisita, vitiligo vulgaris, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, giant cell arteritis, amyotrophic lateral sclerosis, Harada disease, autoimmune optic neuropathy, celiac disease, ankylosing spondylitis, severe asthma, chronic urticaria transplant immunization, familial Mediterranean fever, eosinophilic chronic sinusitis, dilated cardiomyopathy, systemic mastocytosis, or inclusion body myositis;

[0105] Furthermore, the autoimmune disease is plaque psoriasis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis or lupus nephritis.

[0106] Specifically, the cancer includes oral cancer, respiratory cancer, eye cancer, skin cancer, head and neck cancer, laryngeal cancer, esophageal cancer, lymphoma, gastric cancer, bone cancer, digestive system cancer, liver cancer, liver cancer, lung cancer, kidney cancer, neuroblastoma, leukemia, glioblastoma, bile duct cancer, bladder cancer, endometrial cancer, breast cancer, peritoneal cancer, cervical cancer, bile duct cancer, choriocarcinoma, colorectal cancer, connective tissue cancer, melanoma, myeloma, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, basal cell carcinoma, rectal cancer, salivary gland cancer, sarcoma, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine cancer, urinary system cancer, B cell lymphoma, chronic lymphocytic leukemia, acute lymphoblastic leukemia, hairy cell leukemia or chronic myeloblastic leukemia.

[0107] In an eleventh aspect, the present invention provides a method for detecting IL-17A in a sample in vitro for non-diagnostic purposes, the method comprising the following steps:

[0108] (1) contacting the antibody, the recombinant protein, the drug conjugate or the antibody preparation described in any one of the first aspect or the second aspect with a sample to be tested;

[0109] (2) Detect antigen-antibody complexes and interpret the results.

[0110] In a twelfth aspect, the present invention provides a method for preventing and / or treating an autoimmune disease, the method comprising:

[0111] A therapeutically effective amount of the antibody, the recombinant protein, the drug conjugate or the antibody preparation described in any one of the first aspect or the second aspect is administered to a subject in need.

[0112] In a thirteenth aspect, the present invention provides a method for preventing and / or treating cancer, the method comprising:

[0113] A therapeutically effective amount of the antibody, the recombinant protein, the drug conjugate or the antibody preparation described in any one of the first aspect or the second aspect is administered to a subject in need.

[0114] The technical effects achieved by the present invention are:

[0115] (1) The tandem antibody 1-C11+1-F12 of the present invention has good affinity and can bind to the target protein, and its binding ability is significantly better than that of the positive antibody.

[0116] (2) The blocking effect was good. The tandem antibody 1-C11+1-F12 could block the activation of 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc by Human IL-17A protein. The blocking effect of 1-C11+1-F12 was significantly better than that of the positive control antibody.

[0117] (3) Strong stability, with high thermal denaturation Tm value and Tagg. The Tm value of 1-C11+1-F12 is 65.07 and the Tagg value is 65.19, which are better than positive antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 The SDS-PAGE results of IL-17A recombinant protein, where M is a protein marker, lane 1 is the sample before IL-17A recombinant protein purification, and lane 2 is the sample after IL-17A recombinant protein purification.

[0119] Figure 2 This is the flow cytometry test result of the alpaca yeast library.

[0120] Figure 3 The results of yeast monoclonal FACS detection are shown in Figure 2.

[0121] Figure 4 This is the SDS-PAGE result of the tandem single domain antibody 1-C11+1-F12. Figure 4 The left band in the middle is the marker, and the right band is 1-C11+1-F12.

[0122] Figure 5 The results are from the ELISA test for the affinity of tandem single domain antibodies.

[0123] Figure 6 This is the experimental result of ELISA testing the affinity of the positive control antibody.

[0124] Figure 7 FACS was used to detect the binding of IL-17A recombinant protein to reporter gene cell lines.

[0125] Figure 8 These are the experimental results of IL-17A recombinant protein activating reporter gene cell lines.

[0126] Fig. 9 These are the experimental results of the positive antibody Ixekizumab blocking IL-17A recombinant protein.

[0127] Fig.10 These are the experimental results of the blocking function test of tandem single domain antibodies.

[0128] Fig.11 This is the experimental result of positive control antibody blocking function detection.

[0129] Fig.12 The thermal stability test results of tandem single-domain antibodies.

[0130] Fig.13 This is the thermal stability test result of the positive control antibody. DETAILED DESCRIPTION

[0131] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0132] Experimental reagents of the present invention:

[0133] Agar (Sigma, CAT#A1296); peptone (Sigma, CAT#93926); yeast extract (OXOID, CAT#:LP0021); sodium chloride (Aladdin, CAT#:C111533); potassium chloride (Aladdin, CAT#:P112133); magnesium sulfate (Sinopharm, CAT#:10013018); magnesium chloride (Sinopharm, CAT#:10012818); glucose (Shengang, CAT#:GT1991); SfiI (NEB, CAT#:R0123L); T4 DNA ligase (TaKaRa, CAT#:2011A); PrimeScript TMII 1st Strand cDNA Synthesis Kit (TaKaRa, CAT#: 6210B); NuHi power mix (Xinhai Biotechnology, CAT#: NH9303); 3M sodium acetate (pH 5.2-6) (Sigma, CAT#: 126-96-5); DNA fragment recovery kit (TakaRa, CAT#: 9761); gel recovery kit (Qiagen, CAT#: 28706); Tiangen plasmid extraction kit (Tiangen, CAT#: DP117); HRP-Anti-M13 (iCarTab); PE-anti-Human IgG (eBioscience, Cat#: 12-4998-82); PE-Streptavidin (Biolegend, 405204); Rabbit anti-Llama IgG (H+L) Secondary Antibody [HRP] (Novus, CAT# NBP1-75095); SS320 competent vector (iCarTab); BL21 competent vector (Biomed, BC201-02); pComF phage display vector (iCarTab); NHS-biotin (APExBIO, CAT#: A8002); HRP-Streptavidin (Boster, CAT#: BA1088); HRP-Protein A (Boster, BA1080); Genomic DNA PurificationKit (Lifetech, CAT#K0512); Mouse-IL-17A-His (ACRO, CT8-M5240); Bright-LiteLuciferase Assay System (Vazyme, CAT#DD1204-01); NHS-biotin (APExBIO, CAT#:A8002).

[0134] Experimental consumables:

[0135] 50mL Falcon centrifuge tube (Corning, CAT#352070); electroporation cup (Bio-Rad 0.2cm); RNasefree 1.5mL EP tube (QSP, CAT#:509-GRD-Q); 200μL RNase free PCR tube (Axygen, PCR-02D-C); T125 shake flask (Corning, CAT#431143); 15mL Falcon centrifuge tube (Corning, CAT#430052); 6-well plate (Corning, CAT#3516); 96-well plate (Corning, CAT#3365); 96-well black board (F-BOTTOM (CHIMNEY WELL) BLACK).

[0136] Experimental equipment:

[0137] Electroporator (Eppendorf Multiporator); centrifuge (Thermo FRESCO-17); constant temperature incubator (Shanghai Jinghong DNP-9052); constant temperature shaking incubator (Jingqi CO-O6U); clean bench (Sujing Antai SW-CJ-1FD); PCR instrument (Applied Biosystems ABI2720); biological safety cabinet (Haier, HR40-IIA2); flow cytometer (ThermoAttune Nxt flow cytometer); Thermo 3111CO2 incubator; ForteBio OCTET R2.

[0138] The primers used in the present invention for screening, cloning VHH fragments and constructing nanobodies were designed with reference to the following documents:

[0139] Maass DR, Sepulveda J, Pernthaner A, Shoemaker CB. Alpaca (Lama pacos) as a convenient source of recombinant camelid heavy chain antibodies (VHHs). JImmunol Methods. 2007; 324(1-2):13-25.

[0140] Lin, J, Gu, Y, Xu, Y et al. Characterization and applications of nanobodies against Pseudomonas aeruginosa exotoxin a selected from single alpaca Bcells. Biotechnol Biotechnol Equip 2020;34:1028-37.

[0141] Studies on design of singledomain antibodies by AlpacaVHH phagelibrary and high throughput sequencing to construct Fab antibody purification system (http: / / hdl.handle.net / 10232 / 00030916).

[0142] Example 1 IL-17 Nanobody Screening Method

[0143] 1.1 Preparation of IL-17A (Human) recombinant protein (antigen)

[0144] The sequence information of Human IL-17A (Q16552-1) (AA Gly 24-Ala155, as shown in SEQ ID NO: 15) was retrieved from the UniProt database, a 6×His tag was added to the C-terminus, and the gene was synthesized and subcloned into the pET28a vector after prokaryotic codon optimization; after verification by Sanger sequencing, plasmid extraction was performed.

[0145] The recombinant plasmid was transformed into BL21 competent cells, induced overnight with 0.5 mM IPTG, and the bacterial solution was collected and lysed; the recombinant protein was purified using a nickel column.

[0146] The purity of the target protein was detected by SDS-PAGE, and the results showed that the purity was >90% ( Figure 1 ).

[0147] SEQ ID NO:15:

[0148] MTPGKTSLVSLLLLLSLEAIVKAGITIPRNPGCPNSEDKNFPRTVMVNLNI HNRNTNTNPKRSSDYYNRSTSPWNLHRNEDPERYPSVIWEAKCRHLGCINAD GNVDYHMNSVPIQQEILVLRREPPHCPNSFRLEKILVSVGCTCVTPIVHHVA.

[0149] 1.2 Alpaca Immunity

[0150] The prepared recombinant antigen was used to immunize two alpacas (Alpaca) by subcutaneous multi-point immunization for a total of 6 times with an interval of 21 days. Peripheral blood was collected 10 days after the last immunization, and the immune titer was detected by ELISA.

[0151] After 6 rounds of immunization, the immune titers of alpacas all reached the requirements (see Table 1).

[0152] Table 1 Immune titer test results

[0153]

[0154] 1.3 Construction of antibody yeast library

[0155] (1) PBMC isolation and VHH antibody fragment cloning:

[0156] Collect 100 mL of anticoagulated peripheral blood sample and separate PBMC cells using lymphocyte separation solution;

[0157] RNA extraction using PrimeScript TM II 1st Strand cDNA Synthesis Kit for reverse transcription and preparation of cDNA;

[0158] PCR amplification of VHH fragments.

[0159] (2) Construction of single domain antibody yeast display library

[0160] 1.4 Yeast display library selection and screening:

[0161] The prepared IL-17A antigen was used for incubation with streptavidin magnetic beads, and the yeast solution was added to the magnetic beads bound to the antigen, and the yeast display library was rotated and incubated at 4°C for 60 minutes. The constructed yeast display library was subjected to two rounds of magnetic sorting using streptavidin magnetic beads. After sorting, the yeast solution was coated on SDCAA plates, and a single clone was picked for culture. After induction of expression for 48 hours, flow analysis was performed. Incubate with Biotin-IL-17A-His for 1 hour, use PE Streptavidin as the secondary antibody, and perform flow detection after incubation.

[0162] According to the flow cytometry results ( Figure 2 ), after the second magnetic sorting, the yeast positive rate was 37.9%, and the positive clones were significantly enriched. The sorting products were directly coated on SDCAA plates, and single clones were selected for flow cytometry detection.

[0163] 1.5 FACS screening

[0164] After sorting, the yeast liquid was coated on SDCAA plates, and a single clone was picked for culture. After induction of expression for 48 hours, it was incubated with Biotin-antigen, and PE-Streptavidin was used as the secondary antibody. After incubation, flow cytometry detection was performed.

[0165] The results are as follows Figure 3 As shown, FACS detected the binding of IL17A target monoclonal to the target; the amino acid sequences of the candidate single-domain antibodies obtained by sequencing were compared, and the candidate antibodies with different amino acid sequences in the CDR region were selected to construct eukaryotic expression vectors.

[0166] 1.6 Antibody sequence identification

[0167] Enrich positive clones; select enriched single clones, perform Phage ELISA identification, and sequence the clones to obtain the nucleic acid and amino acid sequence information of the candidate single-domain antibody. Randomly select 20 monoclones for sequencing analysis, the sequence differences are large, and the library diversity is good. Based on the amino acid sequence information of the candidate single-domain antibody CDR region, the In silico method is used to analyze possible post-translational modification sites.

[0168] 1.7 Antibody expression and purification

[0169] According to the ELISA test results of the candidate antibodies, positive clones were selected, and the obtained VHH antibody sequences were synthesized separately, and subcloned in tandem with human IgG1Fc into the expression vector pcDNA3.4-hIgG1-Fc. After the vector was verified by sequencing, the Qiagen plasmid extraction kit was used to prepare the endotoxin-free plasmid for use.

[0170] Take out LVTransm transfection reagent and single-chain antibody expression vector from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out PBS buffer and warm it to room temperature. Take 2mL PBS to one well of a 6-well plate, add 130μg antibody expression vector respectively, pipette up and down to mix thoroughly, add 400μL LVTransm, and immediately pipette up and down to mix thoroughly, and let stand at room temperature for 10 minutes.

[0171] Add the above DNA / LVTransm complex to 30 mL of 293F cells and shake gently to mix thoroughly. Place the cells in a 37°C, 5% CO2 incubator at 130 rpm for 6-8 hours, add 50 mL of fresh 293 cell culture medium, and return the cells to the incubator for further culture.

[0172] After 7 days of continuous culture, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using a Protein A column.

[0173] The steps for purifying antibodies using a Protein A column are as follows:

[0174] 1) Add the sample containing the target antibody into the EP tube and gently invert the tube to mix.

[0175] 2) Incubate the EP tubes at room temperature (1-4 hours or overnight) or mix on a rotator. 100 mM PMSF may be added to prevent protein degradation.

[0176] 3) Collect the beads using a magnetic separation rack and discard the supernatant.

[0177] 4) Add 1 mL of binding / washing buffer to the EP tube and mix thoroughly. Collect the beads using a magnetic stand and discard the supernatant. Repeat the washing step three times.

[0178] 5) Add 500 μL of elution buffer to the EP tube, quickly resuspend by pipetting or vortexing, and then incubate for 5 minutes at room temperature (about 25° C.) in an inverting mixer or by gently inverting the EP tube manually.

[0179] 6) Collect the magnetic beads using a magnetic separation stand and transfer the supernatant containing the eluted antibody to a clean EP tube.

[0180] 7) Repeat steps 1) and 2) twice.

[0181] 8) Add 1 / 10 of the neutralization buffer to each 500 μl of the eluate to neutralize the pH, so as to maintain the biological activity of the antibody and avoid antibody inactivation.

[0182] 9) Binding / washing buffer: 1× PBS, pH 7.0.

[0183] Elution buffer: (1) 0.1 M glycine, pH 2-3 (2) 0.1 M NaAc-HAc, pH 3.6.

[0184] Neutralization buffer: 1 M Tris, pH 8.5.

[0185] Magnetic bead regeneration buffer: 0.1M NaOH.

[0186] Example 2 Preparation of tandem single domain antibodies

[0187] 2.1 Screening of anti-IL-17A single domain antibodies

[0188] The inventors finally obtained 14 anti-IL-17A single domain antibodies after screening with immune alpacas and yeast libraries. In the antibody blocking activity test, it was found that although some single domain antibodies can block the activation of downstream target proteins by Human IL-17A protein, the blocking effect is weaker than that of the positive antibody Ixekizumab. Therefore, the inventors connected two of the anti-IL-17A single domain antibodies in series to enhance their blocking effect. The two anti-IL-17A single domain antibodies are 1-C11 and 1-F12.

[0189] The amino acid sequence of the first single-domain antibody 1-C11 is shown in SEQ ID NO:16.

[0190] SEQ ID NO:16:

[0191] DVQLVESGGGLVQPGGSLRLSCAASESLLRLYAMGWYRQLPGQEREWV AIHTTSDTTNYRDSVKGRFTLSRDVATNTIYLQMTSLKPEDTAVYYCHVTSM RDSQNYWGQGTQVTVSS.

[0192] The nucleotide sequence encoding the single-domain antibody 1-C11 is shown in SEQ ID NO:17.

[0193] SEQ ID NO:17:

[0194] GATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGAAAGCCTCCTCAGGTTGTATGCCATGGGCTGGTACCGCCAACTTCCAGGGCAGGAGCGCGAGTGGGTCGCAATACACACTACTAGTGACACCACTAATTA TAGAGACTCCGTGAAGGGCCGATTCACGCTCTCCAGAGACGTCGCCACGAACACGATTTATCTCCAAATGACCAGCCTCAAACCTGAAGACACGGCCGTCTATTATTGTCATGTTACTTCCATGAGAGATTCACAAAACTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCG.

[0195] The amino acid sequence of the second single-domain antibody 1-F12 is shown in SEQ ID NO:18.

[0196] SEQ ID NO:18:

[0197] EVQLVESGGGLVQPGGSLRLSCAASGFSIHIYAMGWYRQAPGKQRELVA TITRGGVTNNADSVKGRFTISSRDNAKNTAYLQMNSLKPGDTAVYYCNVGGT NGGYWGQGTQVTVSS.

[0198] The nucleotide sequence encoding the single-domain antibody 1-F12 is shown in SEQ ID NO:19.

[0199] SEQ ID NO:19:

[0200] GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTGCAGCCGGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTTAGTATCCACATCTATGCCATGGGCTGGTACCGCCAGGCTCCAGGGAAGCAGCGCGAGCTGGTCGCAACTATTACTAGAGGTGGTGTAACAAA TAATGCAGACTCCGTGAAGGGGCGATTCACCATCTCCAGAGACAACGCCAAGAACACGGCGTATCTGCAAATGAACAGCCTGAAACCTGGGGACACGGCCGTCTATTACTGTAATGTAGGTGGGAACGAACGGGGCTACTGGGGCCAGGGGGACCCAGGTCACCGTCTCCTCA.

[0201] 2.2 Preparation of tandem single domain antibodies 1-C11+1-F12

[0202] The candidate antibody was constructed into a bivalent single-domain antibody in the form of VHH-(GGGGS)3-VHH-IgG1 Fc and purified using Protein A magnetic beads. The amino acid sequence of IgG1 Fc is shown in SEQ ID NO:20.

[0203] SEQ ID NO:20:

[0204] DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0205] The nucleotide sequence encoding SEQ ID NO:20 is shown as SEQ ID NO:21.

[0206] SEQ ID NO:21:

[0207] .

[0208] The specific steps are as follows:

[0209] 1) The above two anti-IL-17A single-domain antibody sequences were synthesized and subcloned in tandem with human IgG1Fc into the expression vector pcDNA3.4-hIgG1-Fc. After the vector was sequenced and verified to be correct, the Qiagen plasmid extraction kit was used to prepare the endotoxin-free plasmid for use;

[0210] 2) Take out the LVTransm transfection reagent and single-chain antibody expression vector from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out the PBS buffer and warm it to room temperature. Take 2mL PBS to one well of a 6-well plate, add 130μg of antibody expression vector respectively, pipette up and down to mix thoroughly, add 400μL LVTransm, and immediately pipette up and down to mix thoroughly, and let stand at room temperature for 10 minutes.

[0211] 3) Add the above DNA / LVTransm complex to 30 mL of 293F cells and gently shake to mix thoroughly. Place the cells in a 37°C, 5% CO2 incubator at 130 rpm for 6-8 hours, add 50 mL of fresh 293 cell culture medium, and return the cells to the incubator for continued culture.

[0212] 4) After 7 days of continuous culture, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube. The antibody was purified using a Protein A column to finally obtain the tandem single-domain antibody 1-C11+1-F12.

[0213] The SDS-PAGE results of the tandem single domain antibody 1-C11+1-F12 are as follows Figure 4 As shown, the results indicate that the molecular weight of the candidate antibody is in line with expectations and the purity is sufficient for the next experiment.

[0214] Example 3 Tandem Single Domain Antibody Affinity Detection

[0215] 1 Preparation of positive control antibody Ixekizumab

[0216] The heavy chain and light chain variable regions of ixekizumab were gene synthesized (the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 22, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 23), the heavy chain variable region was subcloned into the pcDNA3.4-hIgG4 (the amino acid sequence of IgG4 is shown in SEQ ID NO: 24) vector, and the light chain variable region was subcloned into the pcDNA3.4-hIgKc vector (the amino acid sequence of IgG KC is shown in SEQ ID NO: 25); after verification by Sanger sequencing, an endotoxin-free plasmid was prepared using a plasmid extraction kit for use.

[0217] Take out LVTransm transfection reagent and heavy chain and light chain expression vectors from the refrigerator, thaw at room temperature, and mix thoroughly by pipetting up and down. Take out PBS buffer and warm it to room temperature. Take 2mL PBS to one well of a 6-well plate, add 50μg of heavy chain and light chain expression vectors respectively, mix thoroughly by pipetting up and down, then add 300μL LVTransm, immediately mix thoroughly by pipetting up and down, and let stand at room temperature for 10 minutes.

[0218] The above DNA / LVTransm complex was added to 100 mL of 293F cells, and the mixture was thoroughly mixed by gentle shaking. The cells were placed in a 37° C., 5% CO 2 incubator and cultured at 130 RPM.

[0219] After continuous culture for 5-7 days, the culture supernatant was collected by centrifugation, filtered with a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube, and the antibody was purified using a Protein A column.

[0220] The purity of the target antibody protein was detected by SDS-PAGE, and the results showed that the protein purity was >95%.

[0221] SEQ ID NO:22:

[0222] QVQLVQSGAEVKKPGSSVKVSCKASGYSFTDYHIHWVRQAPGQGLEW MGVINPMYGTTDYNQRFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARY DYFTGTGVYWGQGTLVTVSS.

[0223] SEQ ID NO:23:

[0224] DIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGNTYLHWYLQKPGQSPQL LIYKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGQ GTKLEIK.

[0225] SEQ ID NO:24:

[0226] ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

[0227] SEQ ID NO:25:

[0228] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRGEC.

[0229] 2ELISA to detect the binding activity of Human IL-17A recombinant protein and control antibody

[0230] (1) Use sterile CBS to dilute the IL-17A recombinant protein to a final concentration of 2 μg / mL. Take a new 96-well plate, add 100 μL / well and coat overnight at 4°C;

[0231] (2) Remove the antigen coating solution and wash three times with PBST (containing 0.5% Tween);

[0232] (3) Add 200 μL / well of 3% MPBS and block at 37°C for 2 hours;

[0233] (4) After removing the blocking buffer, wash the plate three times with PBST;

[0234] (5) The positive control antibody Ixekizumab was diluted to 10 μg / ml with PBS, 5-fold diluted to 7 points, added to the ELISA plate at 100 μL / well, and incubated at room temperature for 1 hour. The control wells were PBS;

[0235] (6) Remove the liquid in the wells and wash three times with PBST;

[0236] (7) Add the secondary antibody HRP-Protein A (Boster, BA1080) at a dilution of 1:10000, add 100 μL / well to the ELISA plate, and incubate at room temperature for 1 hour;

[0237] (8) After removing the liquid in the wells, wash the plate three times with PBST;

[0238] (9) Add 100 μL / well TMB colorimetric solution;

[0239] (10) Incubate at room temperature in the dark for 15 minutes;

[0240] (11) Add 50 μL / well stop solution (2 M HCL);

[0241] (12) Use an ELISA reader to read the OD450 value in the well. The results are shown in Table 2. The positive antibody binds well to the IL-17A antigen protein and can be used for immunization.

[0242] Table 2 Detection of binding activity of Human IL17A and positive antibodies

[0243]

[0244]

[0245] 3ELISA to detect affinity of tandem single domain antibodies

[0246] The purified single domain antibody was coated on a 96-well ELISA plate at 2 μg / mL, and Biotin-IL-17A-His was added at a starting concentration of 10 μg / mL, with 7 points of 5-fold gradient dilution. ELISA was performed using HRP-Streptavdin. The results showed that the EC50 of 1-C11+1-F12 was 1.681 ( Figure 5 ), which is much lower than the positive control antibody of 10.06 ( Figure 6 ). Therefore, the tandem single-domain antibody can bind to the target protein, and the binding ability is significantly higher than that of the positive antibody.

[0247] Example 4 Detection of blocking function of tandem single domain antibodies

[0248] 1 Construction of IL-17A reporter gene cell line

[0249] According to the amino acid sequence information of IL-17RA (UniProtKB: Q96F46, SEQ ID NO: 26) and IL-17RC (UniProtKB: Q8NAC3, SEQ ID NO: 27), a lentiviral expression vector was constructed and the lentivirus was packaged, which was co-infected with 293 cells, and recombinant 293 cells that simultaneously overexpressed these two receptors were screened. NFKB-Luciferase (the amino acid sequence is shown in SEQ ID NO: 28, and the nucleotide sequence encoding it is shown in SEQ ID NO: 29) and ACT1 gene (nucleotide sequence is shown in SEQ ID NO: 30) were further stably transfected to construct the IL-17A reporter gene cell line 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc.

[0250] SEQ ID NO:26:

[0251] MGAARSPPSAVPGPLLGLLLLLLGVLAPGGASLRLLDHRALVCSQPGLNCTVKNSTCLDDSWIHPRNLTPSSPKDLQIQLHFAHTQQGDLFPVAHIEWTLQTDASILYLEGAELSVLQLNTNERLCVRFEFLSKLRHHHRRWRFTFSHFVVDPDQEYEVTVHHLPKPIPDGDPNHQSKNFLVPDCEHARMKVTTPCMSSGSLWDPNITVETLEAHQLRVSFTLWNESTHYQILLTSFPHMENHSCFEHMHHIPAPRPEEFHQRSNVTLTLRNLKGCCRHQVQIQPFFSSCLNDCLRHSATVSCPEMPDTPEPIPDYMPLWVYWFITGISILLVGSVILLIVCMTWRLAGPGSEKYSDDTKYTDGLPAADLIPPPLKPRKVWIIYSADHPLYVDVVLKFAQFLLTACGTEVALDLLEEQAISEAGVMTWVGRQKQEMVESNSKIIVLCSRGTRAKWQALLGRGAPVRLRCDHGKPVGDLFTAAMNMILPDFKRPACFGTYVVCYFSEVSCDGDVPDLFGAAPRYPLMDRFEEVYFRIQDLEMFQPGRMHRVGELSGDNYLRSPGGRQLRAALDRFRDWQVRCPDWFECENLYSADDQDAPSLDEEVFEEPLLPPGTGIVKRAPLVREPGSQACLAIDPLVGEEGGAAVAKLEPHLQPRGQPAPQPLHTLVLAAEEGALVAAVEPGPLADGAAVRLALAGEGEACPLLGSPGAGRNSVLFLPVDPEDSPLGSSTPMASPDLLPEDVREHLEGLMLSLFEQSLSCQAQGGCSRPAMVLTDPHTPYEEEQRQSVQSDQGYISRSSPQPPEGLTEMEEEEEEEQDPGKPALPLSPEDLESLRSLQRQLLFRQLQKNSGWDTMGSESEGPSA。

[0252] SEQ ID NO:27:

[0253] MPVPWFLLSLALGRSPVVLSLERLVGPQDATHCSPVSLEPWGDEERLRVQFLAQQSLSLAPVTAATARTALSGLSGADGRREERGRGKSWVCLSLGGSGNTEPQKKGLSCRLWDSDILCLPGDIVPAPGPVLAPTHLQTELVLRCQKETDCDLCLRVAVHLAVHGHWEEPEDEEKFGGAADSGVEEPRNASLQAQVVLSFQAYPTARCVLLEVQVPAALVQFGQSVGSVVYDCFEAALGSEVRIWSYTQPRYEKELNHTQQLPDCRGLEVWNSIPSCWALPWLNVSADGDNVHLVLNVSEEQHFGLSLYWNQVQGPPKPRWHKNLTGPQIITLNHTDLVPCLCIQVWPLEPDSVRTNICPFREDPRAHQNLWQAARLQLLTLQSWLLDAPCSLPAEAALCWRAPGGDPCQPLVPPLSWENVTVDKVLEFPLLKGHPNLCVQVNSSEKLQLQECLWADSLGPLKDDVLLLETRGPQDNRSLCALEPSGCTSLPSKASTRAARLGEYLLQDLQSGQCLQLWDDDLGALWACPMDKYIHKRWALVWLACLLFAAALSLILLLKKDHAKGWLRLLKQDVRSGAAARGRAALLLYSADDSGFERLVGALASALCQLPLRVAVDLWSRRELSAQGPVAWFHAQRRQTLQEGGVVVLLFSPGAVALCSEWLQDGVSGPGAHGPHDAFRASLSCVLPDFLQGRAPGSYVGACFDRLLHPDAVPALFRTVPVFTLPSQLPDFLGALQQPRAPRSGRLQERAEQVSRALQPALDSYFHPPGTPAPGRGVGPGAGPGAGDGT。

[0254] SEQ ID NO:28:

[0255] MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILLQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKIAV。

[0256] SEQ ID NO:29:

[0257]

[0258] SEQ ID NO:30:

[0259]

[0260] 2. Binding of IL-17A recombinant protein to reporter gene cell lines

[0261] The 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cell line was revived from liquid nitrogen and the cells were adjusted to the logarithmic growth phase;

[0262] The cells were divided into several portions, with the number of cells in each portion being 2×10 5 cells;

[0263] The IL-17A-His protein was incubated with the target cells, mixed thoroughly, and incubated at room temperature for 1 hour;

[0264] Centrifuge at 800×g for 3 minutes at room temperature, remove the supernatant containing the antibody, and wash the cells three times with PBS; add the secondary antibody APC-His (1:500 dilution), mix thoroughly, and incubate at room temperature in the dark for 30 minutes;

[0265] Centrifuge at 800 × g for 3 min at room temperature, remove the supernatant containing the secondary antibody, and wash the cells three times with PBS;

[0266] Resuspend the cells in 500 μL PBS and perform flow cytometry analysis.

[0267] FACS results showed that the constructed IL-17A receptor overexpressing cell line could bind to IL-17A, with a positive rate of more than 90% ( Figure 7 ).

[0268] IL-17A recombinant protein was used to activate 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc. The results showed that IL17A recombinant protein can effectively activate the expression of luciferase in the 293F-IL17Ra / IL17Rc-NFκB-Luc reporter gene cell line ( Figure 8 ).

[0269] 3Ixekizumab blocking the function of IL-17A recombinant protein

[0270] In this example, the positive antibody Ixekizumab was used to detect the effectiveness of the fluorescent reporter system. The positive control antibody Ixekizumab was added to 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc cells together with the IL-17A recombinant protein. The results showed that the positive control antibody Ixekizumab could inhibit the binding of IL17A protein to its membrane receptor and inhibit the intracellular NFκB signal, showing a dose effect ( Fig. 9 ).

[0271] 4. Tandem single domain antibody blocking function

[0272] The blocking activity of tandem single-domain antibodies was tested using the 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc reporter gene cell line. The results showed that the IC50 of 1-C11+1-F12 was 0.7807μg / mL, and the IC50 of the positive control antibody was 1.356μg / mL ( Fig.10 and Fig.11 ). Therefore, both 1-C11+1-F12 and the positive control antibody could block the activation of 293F-IL-17RA-IL-17Rc-ACT1-NFκB-Luc by Human IL-17A protein; the blocking effect of 1-C11+1-F12 was significantly better than that of the positive control antibody.

[0273] Example 5 Stability test of tandem single domain antibodies

[0274] NanoDSF technology can be used to detect fluorescence changes, detect thermal and chemical denaturation of proteins under native conditions, and accurately determine the temperature (T) at which 50% of the protein is in an unfolded state. m ) and the temperature at which aggregation begins (T agg ); thermal denaturation T m Value and T agg The higher the value, the more stable the antibody protein.

[0275] Take 100 μL of candidate antibodies and Ixekizumab (sample concentration greater than 200 μg / mL) prepared in the previous project, centrifuge at 4°C, 12000×g for 10 min, and use a capillary to absorb the sample. Prepare two capillaries for each sample as parallel controls and place them in the corresponding card slots in order to ensure that the capillaries are full of samples without bubbles for detection and analysis.

[0276] Experimental results:

[0277] The experimental results are as follows Figure 12-13 As shown, the stability of the tandem antibody 1-C11+1-F12 is stronger than that of the positive control, with Tm=65.07 and Tagg=65.19. The positive control antibody has Tm=56.1 and Tagg=61.86.

[0278] Application Example 1 An antibody preparation

[0279] The antibody preparation includes: tandem single domain antibody 1-C11+1-F12, buffer, surfactant, amino acid, tension agent and the like.

[0280] In one embodiment of the present invention, the preparation of the antibody preparation comprises: weighing each substance, adding water to dissolve and mix, and adjusting each component to the following concentration: (100-200) mg / ml tandem single domain antibody 1-C11+1-F12, (1-10) mM citrate buffer, (0.1-1% w / v) Tween 80, (100-200) mM arginine and (1-10)% sucrose, and the pH of the preparation is 5.0-8.0.

[0281] Application Example 2 A kit

[0282] The kit contains: tandem single domain antibody 1-C11+1-F12, recombinant protein, antibody preparation and / or polyclonal antibody, container for loading antibody preparation, buffer, etc.

[0283] In one embodiment of the present invention, the kit comprises: (100-200) mg / ml of tandem single domain antibody 1-C11+1-F12, and a buffer solution with a pH of 5.0-8.0.

[0284] Application Example 3 Antibody Drug Conjugate

[0285] The antibody-drug conjugate comprises: a tandem single-domain antibody 1-C11+1-F12, a recombinant protein, an antibody preparation and / or a polyclonal antibody, the drug is a physiologically active substance (such as a nucleic acid, etc.) and a linker connecting the antibody and the drug (the linker includes a maleimide linker, a Val-Cit linker, an SS linker and a DMSS linker).

[0286] In one embodiment of the present invention, the tandem single domain antibody 1-C11+1-F12 is connected to the drug via an SS linker, a (100-200) mM aqueous solution is added and mixed at room temperature, and the linker reaction is terminated to obtain an antibody-drug conjugate.

[0287] Application Example 4 Pharmaceutical Composition

[0288] The pharmaceutical composition includes tandem single-domain antibodies 1-C11+1-F12, recombinant proteins, antibody preparations, polyclonal antibodies, nucleic acid molecules, biological expression vectors and / or host cells, and also includes pharmaceutically acceptable excipients.

[0289] In one embodiment of the present invention, the preparation of the pharmaceutical composition comprises: preparing a tandem single domain antibody 1-C11+1-F12 at a concentration of (100-200) mg / ml, adding (1-20 w / v) sucrose, (10-300) mM histidine and (0.1-10)% Tween 80 to obtain the pharmaceutical composition.

Claims

1. A tandem single domain antibody against IL-17A, characterized in that: The tandem single domain antibody comprises a first single domain antibody and a second single domain antibody; The structure of the first single domain antibody is: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; The structure of the second single domain antibody is: FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8; The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR4 is shown in SEQ ID NO: 4, the amino acid sequence of the HCDR5 is shown in SEQ ID NO: 5, and the amino acid sequence of the HCDR6 is shown in SEQ ID NO:

6.

2. The tandem single domain antibody according to claim 1, characterized in that The heavy chain framework region comprises part or all of an antibody heavy chain framework region selected from human, mouse, primate or camelid sources or variants thereof.

3. The tandem single domain antibody according to claim 2, characterized in that The heavy chain framework region comprises part or all of an antibody heavy chain framework region selected from camelid origin or a variant thereof.

4. The tandem single domain antibody according to claim 3, characterized in that It comprises part or all of an antibody heavy chain framework region selected from alpaca or a variant thereof.

5. A tandem single domain antibody against IL-17A, characterized in that: The antibody comprises a first single domain antibody and a second single domain antibody; The structure of the first single domain antibody is: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; The structure of the second single domain antibody is: FR5-HCDR4-FR6-HCDR5-FR7-HCDR6-FR8; The amino acid sequence of the HCDR1 is shown in SEQ ID NO: 1, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 3, the amino acid sequence of the HCDR4 is shown in SEQ ID NO: 4, the amino acid sequence of the HCDR5 is shown in SEQ ID NO: 5, and the amino acid sequence of the HCDR6 is shown in SEQ ID NO: 6; The amino acid sequence of FR1 is shown in SEQ ID NO:7, the amino acid sequence of FR2 is shown in SEQ ID NO:8, the amino acid sequence of FR3 is shown in SEQ ID NO:9, the amino acid sequence of FR4 is shown in SEQ ID NO:10, the amino acid sequence of FR5 is shown in SEQ ID NO:11, the amino acid sequence of FR6 is shown in SEQ ID NO:12, the amino acid sequence of FR7 is shown in SEQ ID NO:13, and the amino acid sequence of FR8 is shown in SEQ ID NO:

14.

6. The antibody according to any one of claims 1 to 5, characterized in that: The amino acid sequences of the first single-domain antibody and the second single-domain antibody are indirectly connected via a linker; or; directly connected.

7. The antibody according to claim 6, characterized in that: The linker is (GGGGS) n , n is selected from 1, 2, 3, 4, 5 or 6.

8. The antibody according to claim 7, characterized in that: The linker is (GGGGS)3.

9. A recombinant protein, characterized in that: The recombinant protein comprises the antibody according to any one of claims 1 to 8; the recombinant protein also comprises a biologically active protein or a functional fragment thereof that assists its expression and / or secretion, or prolongs its half-life in vivo.

10. The recombinant protein according to claim 9, characterized in that: The bioactive protein or its functional fragment is selected from at least one of serum albumin, His tag, GST tag, albumin binding polypeptide, prealbumin, carboxyl terminal peptide, elastin-like polypeptide, immunoglobulin Fc domain, MBP tag, FLAG tag and SUMO tag.

11. The recombinant protein according to claim 10, characterized in that: The immunoglobulin Fc domain is derived from human antibody, mouse antibody, primate antibody or camelid antibody, or variants thereof.

12. The recombinant protein according to claim 11, characterized in that: The immunoglobulin Fc domain is derived from human IgG antibody, including IgG1 Fc, IgG2 Fc, IgG3 Fc or IgG4 Fc.

13. The recombinant protein according to claim 12, characterized in that: The immunoglobulin Fc domain is IgG1Fc.

14. An antibody preparation, characterized in that: The antibody preparation comprises: (1) the antibody according to any one of claims 1 to 8 or the recombinant protein according to any one of claims 9 to 13; and; (2) A pharmaceutically acceptable carrier.

15. A kit, characterized in that: The kit comprises the antibody according to any one of claims 1 to 8, the recombinant protein according to any one of claims 9 to 13, or the antibody preparation according to claim 14.

16. The kit according to claim 15, characterized in that: The kit also includes a container for holding the antibody preparation.

17. An isolated nucleic acid molecule, characterized in that: The nucleic acid molecule encodes the antibody according to any one of claims 1 to 8 or the recombinant protein according to any one of claims 9 to 13.

18. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule according to claim 17.

19. A pharmaceutical composition, characterized in that: The pharmaceutical composition comprises the antibody according to any one of claims 1 to 8, the recombinant protein according to any one of claims 9 to 13, the antibody preparation according to claim 14, the nucleic acid molecule according to claim 17 or the expression vector according to claim 18.

20. The pharmaceutical composition according to claim 19, characterized in that: The pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.

21. Use of the antibody according to any one of claims 1 to 8, the recombinant protein according to any one of claims 9 to 13, the antibody preparation according to claim 14 or the pharmaceutical composition according to any one of claims 19 to 20, characterized in that: The use is at least one of the following uses: (1) Preparing reagents or kits for detecting IL-17A; (2) Preparation of drugs for treating autoimmune diseases, wherein the autoimmune diseases are plaque psoriasis, rheumatoid arthritis, and psoriatic arthritis.

22. A method for detecting IL-17A in a sample in vitro for non-diagnostic purposes, characterized in that: The method comprises the following steps: (1) contacting the antibody according to any one of claims 1 to 8, the recombinant protein according to any one of claims 9 to 13, or the antibody preparation according to claim 14 with a sample to be tested; (2) Detect antigen-antibody complexes and interpret the results.

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